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Leica TCS SP5 Leica TCS SP5 X User Manual

1. Intermediate Image Edge Resel Field Resolution 1667 2344 3000 2x Oversampling 3333 4688 6000 Dx Oversrping e000 ras 00 Table 5 Table of resolution elements at 400 nm for a variety of objectives over the entire scan field It becomes apparent here that a resolution of 64 megapixels 8000x8000 pixels is appropriate for quality microscopy applications It is thus possible to truly capture all of the image information resolved by the microscope in a single image at that setting This naturally results in large data volumes which are especially undesirable for measurements with a high temporal resolution Zoom is the correct solution here When capturing data in the standard 512 x 512 format this means limiting the image to a field 10 15 times smaller to avoid loss of information Zoom factors of 10x and higher provide usable data but from rather small fields of view The information required will determine what constitutes an acceptable compromise here Such an image is initially a cutout in the center of the scan field That is not always desirable as it can be difficult to center the interesting structures with such precision However the scan field excerpt can be moved across the entire scan field to the actual points of interest a method called panning 97 Figure 73 The same image detail in a variety of pixel resolutions Please note that the printing medium may not be capable of reproducing the full deta
2. Upright microscope Inverted microscope Finish the scanning operation Finish the scanning operation Ensure that no laser radiation is present in Ensure that no laser radiation is present in the specimen area the specimen area Tilt the transmitted light arm back Exchange the specimen Exchange the specimen Insert the specimen correctly into the Insert the specimen correctly into the specimen holder specimen holder Tilt the transmitted light arm back into the working position 96 6 6 Changing Objectives A Do not change objectives during a scanning operation To change objectives proceed as follows 1 Finish the scanning operation 2 Switch off the internal lasers using the detachable key switch 3 If any external lasers are present switch them off with their detachable key switch or as described in the operating manual of the laser manufacturer 4 Rotate the objective nosepiece so that the objective to be changed is swiveled out of the beam path and points outward 5 Exchange the objective A All unoccupied positions in the objective nosepiece must be closed using the supplied caps aperture NA larger than 0 85 This does not apply to immersion objectives oil For MP systems dry objectives air objectives may not be used with a numerical water If a piezo focus is installed in your system please also observe the safety notes related to changing objectives with a piezo focu
3. ooccooccoconccccnccccnonononanononnnnanocnnnnnnncnannnos 50 5 10 9 Mil a a a aaa a e a 51 6 Safety Instructions for Operating the SySteM oonconcccnccccconocononcnnncnconancnannonns 53 6 1 Requirements Related to the Installation Storage Location ococoo 53 6 2 General Safety Instructions for Operation ccoocconnconnconncocnconnconnconnonnnonnconnnos 53 6 3 Eye Protec ia 54 6 3 1 MP System with Upright MiCroSCOpe occccccccocnccccconcncconncncononnnnnnnnnnnconnnnonons 54 6 3 2 MP System with Inverted MiCrOSCOPE coocccoccccccococcnoncncconconcnnonnnnonnnnnncnnnnnnnnos 54 6 3 3 6 4 6 5 6 6 6 7 6 8 6 9 6 10 6 10 1 7 1 7 2 7 3 9 1 9 2 9 2 1 9 2 2 9 2 3 9 3 9 3 1 9 4 10 10 1 10 1 1 10 1 2 10 1 3 10 1 4 10 2 10 2 1 10 2 2 10 2 3 10 2 4 VIS and UV Systems with Inverted or Upright Microscope occcoccooccooccoooo 54 SPECIMENA ET uoman as 55 Changing SpeciMNENS aia 56 Changing OD SCUV CS iran died co as alla 57 Changing the Transmitted Light Lamp HOUSINO ooccoocccccnccccncccnconcnconncnncnnos 58 Mirror housing ON upright MICTOSCOPE cooccocccncccccncocncocnconnnononononanonanonanoncnnnnonos 60 Changing Filter Cubes Beam Splitters or Condenser oocconcccnnccnncocnconnconnnoo 62 Piezo focus on upright MICTOSCOPE ocoocccocccoccccccocncocncocnnonnnonnnnrnnnnonoronanonancnnnnos 63 Objective Change with Piezo Focus Configuration ooccocccoccc
4. Leica MICROSYSTEMS Published by Leica Microsystems CMS GmbH Am Friedensplatz 3 D 68165 Mannheim Germany http www leica microsystems com http www confocal microscopy com Responsible for contents Leica Microsystems CMS GmbH Copyright Leica Microsystems CMS GmbH All rights reserved Table of Contents 1 1 1 1 2 2 1 2 2 2 3 2 4 2 5 2 6 2 1 2 1 1 2 1 1 1 2 1 1 2 2 1 1 3 2 1 1 4 2142 2 1 2 1 2 1 2 2 2 1 3 2 1 4 2 7 5 2 8 2 8 1 2 8 2 2 8 2 1 2 8 2 2 2 8 3 2 8 4 2 8 5 3 GOWNS chlo esse a ee sede etanegneteacee sea necendlceaces 9 COPY Omer o io io 9 About this Operating Manual ones 10 Leica TCS SPO TOS SP5 a saa 11 TCS SPD SYStEM OVOIVIOW aspius a EAA 11 TCS SP5 X SYSTOMOVCIVIOW icre id ii 12 Meded USE O RU O a a ee 12 Ao ene er cr Se ee eer eee ee ey eee 13 RONIS eran oe eee E 14 A N E Gat heen dca eens 14 SCC I GCANOMS iris 15 DIMENSIONS 1 ii EOE ee 15 TCS SP5 with inverted MiICTOSCOPEO nicin 15 TES SP5 with Upright MICCOSCO DC siria dat 15 TCS SP5 X with inverted MICTOSCOPE occcocccocccocccocccocnonnnonnnonnnonnconncnnocnonons 16 TES SPS X With UDlIGHUMICGFOSCODC sicario ious 16 Electrical Connection RequireMent S seriinin aa a 17 Electrical connection requirements of supply unit ccocccocccoccconcconconnco 17 Electrical connection requirements of achromatic light laser 17 Requirements Regarding Ambient ConditiONS ccco
5. SAFETY DATA SHEET Gem 91 155 EG 2001 58 EG Stand 2 12 2004 innovatek gt innovatek OS GmbH www innovatek de info innovatek de 1 Substance preparation and company name Trade name company Tel 08405 92590 Fax 08405 925921 innovatekProtect IP innovatek OS GmbH Stadtweg 9 85134 Stammham Emergency phone No 08405 92590 2 Composition information on ingredients Chemical nature Hazardous Compounds Ethanediol 2 ethylhexanoic acid sodium salt Ethylene glycol ethane diol Corrosion inhibitors CAS No 107 21 1 Hazard symbol Xn R phrases 22 Content w w gt 90 EC No 203 473 3 INDEX No 603 027 00 1 CAS No 19766 89 3 Hazard symbol Xn Content w w 2 3 EC No 243 283 8 R phrases 63 The wording of the hazard symbols and R phrases is specified in Chapter 16 if dangerous ingredients are mentioned 3 Hazard identification Harmful if swallowed 4 First aid measures General advice If inhaled On skin contact On contact with eyes On ingestion Note to physician 5 Fire fighting measures Suitable extinguishing media Special protective equipment Further information Remove contaminated clothing If difficulties occur after vapour aerosol has been inhaled fresh air summon physician Wash thoroughly with soap and water Wash affected eyes for at least 15 minutes under running water with eyelids held open Rinse mouth immediately and then
6. 5 10 8 Cover for Replacement Flange Front view of cover Figure 31 Cover for replacement flange If the replacement flange for transmitted light is not equipped with a functional module such as a lamp housing a cover must be placed over the opening for laser safety reasons 90 5 10 9 Mirror Housing Front view of the mirror housing 91 92 6 Safety Instructions for Operating the System 6 1 Requirements Related to the Installation Storage Location This equipment is designed for connection to a grounded earthed outlet The A grounding type plug is an important safety feature This device was designed for use in a lab and may not be set up in areas with medical devices serving as life support systems such as intensive care wards To avoid the risk of electrical shock or damage to the instrument do not disable this feature To avoid the risk of fire hazard and electrical shock do not expose the unit to rain come into contact with any electrical components The instrument must be or humidity A Do not open the cabinet Do not allow any liquid to enter the system housing or completely dry before connecting it to the power supply or turning it on 6 2 General Safety Instructions for Operation A A Do not look into the eyepieces during the scanning operation Do not look into the eyepieces when switching the beam path in the microscope Never look directly into a laser beam or a reflection o
7. h A i lg A 5 7 r T ea F F eit wh a r Y A 3 F 3 3 IA ID 8 bye os rl Li i 7 a ee 2 1 7 584 jim MES ST TT ENT gt do REN e p pr j 2 26 376 pm 27 48 5 jur z 28 5P4 pm 20 673 pm 2530 0 72 pum z 31 8 71 um Figure 79 Gallery of a z stack This thumbnail gallery is well suited for monochrome publications As mentioned earlier a three dimensional image cannot truly be displayed on a two dimensional monitor Therefore a variety of methods are available for presenting this information 10 4 5 1 Gallery The simplest of these is to display all of the sections of a series in a thumbnail gallery Figure 79 Changes from section to section can thus be analyzed and the images printed in periodicals 10 4 5 2 Movie Many publications today are available on the Internet making it possible to include movies in which these sequences can be viewed at a convenient speed These movies provide the impression of focusing directly through the specimen at the microscope Both methods are suitable for monochrome black and white and multichannel scans 108 10 4 5 3 Orthogonal Projections A further option for displaying the full range of information with losses compressed into two dimensions is to compute projections of the entire series The most common method is the so called maximum projection The brightest value along the z axis is determined for each pixel and entered into the resulting image at this point T
8. occcoccccocccccnccocncccnoconononnonnnnonanonannononenanonanoss 116 Maintaining the Scanner Cooling SySteM cooncccccncccccconncccnconcnonononnnnnncnnonoss 116 Transport and DIS DOS Alisa cada 117 Changing the Installation Location oocococcconccccnncconcconnnonnncnnccannnonnnnnnoss 117 ro A AN 117 o A teas eee 117 OS e aa E 118 ADPENA DEA ooo 124 Safety Data Sheets from Third party Manufacturers ooccconcconconnccnnccnnconnnn 124 Declaration Or CONO MI eiii 129 People s Republic Of CAM iininiinnciaai 130 1 General 1 1 Copyright The instructions contained in the following documentation reflect state of the art technology and knowledge standards We have compiled the texts and illustrations as accurately as possible Nevertheless no liability may be assumed for the accuracy of this manual s contents If you have any comments on this operating manual or on any of our other documentation we would be pleased to hear from you The information contained in this Operating manual is subject to change without prior notice All rights to this documentation are held by Leica Microsystems CMS GmbH Adaptation translation and reproduction of text or illustrations in whole or in part by print photocopy microfilm or other method including electronic systems is not allowed without express written permission from Leica Microsystems CMS GmbH Programs such as LAS and LAS AF are protected by copyright laws All righ
9. 76 101 Figure 76 Profile cut through the Convallaria majalis specimen indicating a thickness of approx 30 um 10 3 Multiparameter Fluorescence In many cases today specimens are used that contain more than one fluorescent dye Multiple dyes are achieved using hybridization of various linked fragments fluorescence in situ hybridization FISH through differently marked antibodies or with fluorescence proteins with differing spectral properties Traditional histological fluorescent dyes and autofluorescence are also usable parameters Figure 77 Figure 77 Simultaneous scan of two fluorescences in this case excited by a single laser line The depiction in the colors green and red is arbitrary 10 3 1 Illumination Specimens with multiple dyes generally require illumination with multiple colors in this case laser lines simultaneously That is not always the case however there are naturally also dyes with differing emissions that can be excited by the same wavelengths A distinctive example would be a botanical specimen with a FITC dye and blue excitation The emission of FITC would then be visible in the blue green range of the spectrum The same excitation can also be applied to chlorophyll however which would respond with emission in the deep red range 102 Figure 78 Simultaneous scan of two fluorescences in this case excited by a single laser line The depiction in the colors green and red is arbitrary Flu
10. attention to the fact that the safety notes and regulations stipulated in the operating manual must be observed for the secure and interference free operation of the system The operating manual in particular the safety notes must be observed by all people who are working with the system 25 Notes either contain additional information on a specific topic or special instructions on the handling of the product 26 4 General Safety Instructions 4 1 Laser Class for VIS and UV Systems In accordance with IEC EN 60825 1 this system is a laser product of Class 3B IIIb A Never expose eyes or skin to direct radiation The laser light can cause permanent eye damage 4 2 Laser Class for MP Systems In accordance with IEC EN 60825 1 this system is a laser product of Class 4 IV A Never expose eyes or skin to direct or indirect radiation Laser light can cause permanent eye damage and skin injuries 4 3 What does the owner operator have to observe The owner operator of this product is responsible for proper and safe operation and safe maintenance of the system and for following all applicable safety regulations The owner operator is fully liable for all consequences resulting from the use of the system for any purposes other than those listed in the operating manual or the online help A This laser product may be operated only by persons who have been instructed in the use of the system and the po
11. be used to make deductions about membrane permeability diffusion speeds and the binding behavior of molecules The capture of a time series is always integral to such measurements 10 6 Spectral Series Section 10 2 4 described how the Leica SP detector is capable of selecting emission bands over a continuously variable range Incremental shifts of the emission band can also be used as the basis for an image series The Leica SP detector was thus the first instrument with which a spectral image series could be scanned using a confocal microscope Experience has shown that its technology is the most efficient all other spectral microscopes that have arrived on the market since its introduction have significant weaknesses with regard to their signal to noise ratio 10 6 1 Data Acquisition and Utilization The scanning of a Lambda series does not differ significantly from that of a z series or a time series The emission band for the beginning and the end of the measurement must be specified as well as the number of steps for the spectrometer to cover the specified range Sections of the image are then chosen interactively for evaluation Their average intensity is then graphed as a function of the wavelength a spectrum at the selected point 10 6 2 About Spectral Resolution A recent debate has developed about which technology offers the best spectral resolution in conjunction with spectral series i e technology capable of detecting the f
12. cw Continuous wave cw Table 1 Usable lasers for TCS SP5 without MP 19 The components for laser safety are designed only for the laser variants listed A 2 8 2 Overview of Usable MP Lasers IR Lasers Furthermore the MP system may contain additional VIS UV lasers see the table for usable VIS UV lasers 2 8 2 1 Picosecond laser Luminous Luminous power at laser power in focal Pulse duration output W plane W pulsed 1 0 1 5 ps pulsed 1 0 1 5 ps Wavelength nm MaiTai ps 780 920 MaiTai ps wideband 990 MaiTai ps broadband ee Laser type Chameleon ps 690 1020 Ultra Chameleon ps 690 1040 Ultra Chameleon ps 680 1080 Ultra II Table 2 Usable MP lasers for TCS SP5 and TCS SP5 X pulsed 1 0 1 5 ps pulsed 1 0 1 5 ps pulsed 1 0 1 5 ps 20 2 8 2 2 Femtosecond laser Luminous Luminous power at laser power in focal Pulse duration output W Wavelength Laser type nm MaiTal MaiTaifs 780 920 920 lt 06 pulsed BOfs 80 pulsed 80fs ith pem pe fs o Chameleon fs 690 _ 1020 lt 4 lt 1 9 pulsed 140 fs Ultra Chameleon fs Chameleon fs 690 1080 lt 4 lt 1 9 pulsed 140 fs Ultra Il Chameleon 690 1040 lt 40 lt 1 9 pulsed 140 fs Vision Chameleon le6so 1080 lt 4 0 lt 1 9 pulsed 140 fs Vision 680 1080 pulsed 140 fs Chameleon Compact OPO 1000 1600 pulsed gt 100 fs Table 3 Usable
13. drink plenty of water seek medical attention Symptomatic treatment decontamination vital functions Administer 50 ml of pure ethanol in a drinkable concentration Water spray alcohol resistant foam dry extinguishers carbon dioxide CO2 In case of fire wear a self contained breathing apparatus The degree of risk is governed by the burning substance and the fire conditions Contaminated extinguishing water must be disposed of in accordance with local legislation 125 EEC SAFETY DATA SHEET t k Gem 91 155 EG 2001 58 EG n n ovate TE gt innovatek OS GmbH Stand www innovatek de 2 12 2004 info innovatek de 6 Accidental release measures Personal precautions Avoid excessive contact with skin and eyes In case of release of larger amounts remove contaminated clothing and wash bo dy down thoroughly with water Hand protection Pick up im mediately as product renders floors slippery Environmental pre Contain contaminated water firefighting water Do not discharge cautions product into natural waters without pretreatment biological treatment plant Methods for cleaning Bind the liquid by using suitable absorbent material saw dust up taking up sand etc and dispose of in accordance with the regulations Wash away spills thoroughly with large quantities of water In case of release of larger quantities which might flow into the draining system or waters contact appropriate authorities 7 Handling a
14. inside widths of 1 0 m for installation maintenance and transport With regard to the load bearing capacity of the floor note that the system will apply a static load of 200 kg m A Ensure that the environment is as vibration free as possible 2 1 4 Permitted Ambient Conditions Permissible temperature range for operation 18 to 25 C Temperature range for optimum optical 22 C 41 C behavior 7 Permitted relative humidity 20 80 non condensing Permitted vibrations Frequency range 5 Hz 30 Hz lt 30 um s RMS Frequency range gt 30 Hz lt 60 um s RMS Pollution degree 18 2 7 5 Waste Heat Required Cooling Performance The TCS SP5 system has a maximum power consumption of 3 2 kW VIS system or 6 2 kW MP system The TCS SP5 X has a maximum power consumption of 3 4 kW For the specifications of external lasers such as UV and MP lasers please refer to the manufacturer s documentation 2 8 Features 2 8 1 Overview of Usable VIS UV Lasers The Leica TCS SP5 features a combination of the lasers listed below Maximum Maximum Wavelength luminous power luminous power at laser output in focal plane Laser type mW Pulse duration Diode 405 Continuous wave cw Continuous wave cw Diode 405 p 0 pulsed 60 ps 458 476 488 Continuous wave DPSS 445 458 476 488 Continuous wave Continuous wave cw Continuous wave cw Continuous wave cw Continuous wave
15. light of 45 The transmissivity or reflectivity of triple dichroic filters depends on three specific wavelengths of light With a TD 488 568 647 triple dichroic filter for example the excitation light at 488 nm 568 nm and 633 nm is reflected and above these values it is transmitted The transmission values are generally around 80 and the reflection values are between 90 and 95 122 Dry Objective A microscopic objective used without immersion media Between the objective lens and the specimen is air UV Laser Laser with a wavelength lt 400 nm invisible laser radiation VIS Laser Laser of the wavelength range 400 700 nm visible laser radiation Voxel An acronym based on the words volume and pixel A voxel represents the smallest indivisible volume element in a three dimensional system In this documentation both the volume elements of the specimen and the 3D pixels are referred to as voxels Achromatic light laser Laser where 8 wavelength bands can be selected simultaneously from the wavelength range of 470 670 nm Z stack Z stacks are comprised of two dimensional images that were scanned on different focal planes and displayed as three dimensional bs Applies only to the TCS SP5 X system 123 15 Appendix 15 1 Safety Data Sheets from Third party Manufacturers The scanner is liquid cooled Following are the safety data sheets from the manufacturer Innovatek for the coolant used 124 EEC
16. scanning microscope Follow the safety instructions provided in Chapter 6 Safety Instructions for Operating the System For switching off the system refer to Chapter 8 Switching Off the System Applies only to systems with an external UV laser 69 7 2 Starting the LAS AF With the motorized stage 156504145 for DMI 6000 inverted Before the system start or start of the LAS AF the illuminator arm of the inverted microscope must be swung back because the motorized stage can be initialized and damage the condenser With the motorized stage 156504155 for DM 6000 upright 70 Before the system start or start of the LAS AF the stage must be moved downwards because during initialization it can come into contact with the objective nosepiece and damage the objectives 1 Click the LAS AF icon on the desktop to start the software Figure 49 LAS AF icon on the desktop 2 Select whether the system should be operated in resonant or non resonant mode iveecad Ausr scente LEP balde 17 Figure 50 Resonant or non resonant mode 3 Start the LAS AF by clicking the OK Figure 51 LAS AF start window You are now in the main view of the LAS AF CESEN a an a us mu rur n im e M CE ta a ED AP PP A al button DOORNDE 4 S400 L vow Figure 52 LAS AF main view Display may differ based on the system configuration 7 3 Setting Up Users The
17. see Figure 17 If the white light laser is operated as a component of the TSC SP5 X system you have to use the remote interlock jack on the supply unit see chapter 5 4 The A remote interlock plug shorting bridge must be connected to the remote interlock jack of the white light laser SP5 system you have to use the remote interlock jack on the white light laser If you operate the white light laser separately without connecting it to the TCS A see Figure 17 for connecting remote interlocks Remote interlock devices such as those connected to the room the door or other onsite safety interlock systems can also be connected to the remote interlock connector The laser beam path is interrupted if the contact is open KOHER SuperK Exti Figure 17 Remote interlock connection at the achromatic light laser Applies only to the TCS SP5 X system 36 Remote Interlock Connections on External Lasers 9 6 For external lasers please refer to the operating manual supplied by the laser manufacturer for the position of the remote interlock connection Remote interlock jack interlock connector on the scanner 5 7 The interlock jack is located on the rear side of the scanner operating voltage 12 V DC Figure 18 For laser safety reasons the inverted microscope must be connected to this connection or if 37 This ensures that the safety switch of the O 2 a o 3 lt Q O
18. unauthorized use of the laser products is located on the main switch board see Figure 10 Figure 10 Detachable key switch for the internal lasers The detachable key switch for protection against unauthorized use of the external achromatic light laser is located on the front of the achromatic light laser see Figure 11 Figure 11 Detachable key switch for the achromatic light laser 32 The key switch for protection against unauthorized use of the external UV laser is located on the front of the power supply see Figure 12 Figure 12 Key switch for the external UV laser For other external lasers please refer to the operating manual supplied by the laser manufacturer for the position of the detachable key switches 33 5 3 Emissions Warning Indicators The operational readiness of lasers located in the supply unit is signaled by an emission warning indicator Figure 13 The emission warning indicator is located above the detachable key switch and is yellow when lit The emission warning indicator of the achromatic light laser is located on the front of the achromatic light laser See Figure 14 and is red when lit As soon as the emission warning indicator of the lasers is lit it is possible from a functional standpoint that laser radiation is present in the specimen area Figure 13 Emission warning indicators on the main switch board Figure 15 Emission warning indicator on power supply of the ex
19. used to calculate the size of the specimen being observed A scanner always acquires square or rectangular excerpts of course If such a square or rectangle is exactly circumscribed by the field of vision than the diagonal dimension will correspond exactly to the field number allowing the largest possible image to be displayed on the monitor without restrictions Unlike eyes or conventional cameras scanners can simply be set to a smaller angle A further enlarged section of the field of view will then be displayed on the monitor It is thus possible to zoom into details without the need for additional optics As the scan angle can be 95 adjusted very quickly and continuously over a wide range the magnification can be increased up to around 40x simply by moving a slider As always in microscopy the total magnification must be appropriate i e within a suitable range in order to obtain good images Other scales are important for overviews and bleaching experiments As errors can easily be made when interpreting scanned data the following is an example of how an appropriate total magnification can be calculated as well as the information that is automatically provided to the user by the software Figure 72 Fields for the conventional scanner formerly 21 2 mm now 22 mm and the resonant scanner 15 1 mm A smaller scanning angle increases the magnification zoom while a scan offset shifts the image detail pan within the field of
20. yield In general however lasers deliver much more light than necessary and attenuation to 10 is generally sufficient for good images although that depends very strongly on the specimen s dye of course One can thus also excite the fluorescence on the blue side of the excitation maximum which has the advantage of providing a wider band for the collection of the emission Figure 68 91 Figure 68 Excitation spectrum of a fluorescent dye blue and emission spectrum red An excitation in the maximum Exc1 would result in only a narrow band to be collected on the emission side Em1 A significantly wider emission band Em2 is available from an excitation in the blue range at which point the intensity of the laser can be increased without detrimental effects A little experimentation is worthwhile here An acousto optical beam splitter AOBS permits all available laser lines to be conveniently added or removed without devoting attention to the beam splitter characteristics or the spacing of the lines 10 2 4 Emission Bands Once the excitation light has reached the specimen via the AOBS and the objective an emission is generated in the fluorescent molecules the light of which is shifted toward longer redder wavelengths This is known as Stokes shift and its degree depends on the fluorochrome As a rule the excitation and de excitation spectra of the fluorescent dyes overlap and the Stokes shift is the difference betwee
21. 5 10 5 6 10 6 10 6 1 10 6 2 10 7 11 11 1 11 2 The Pinnole and lts EMCCIS tardado 93 Image Detail and Raster SettingS coocccocnoconnoccnocanicncnonanonannnnaniconononoss 95 P tie eaters anh eect deeded eese 99 TONE A 101 Multiparameter FlUOrescCenCe occoocoocccccconccccconconocononnncnnnononnocononnnnnnnnnonannnnnnns 102 NUMIDA UOI aa ss 102 Beam SP ivssiaind ico 104 EMISSION ANG S li 104 Erossta la a a ie 104 Sequental Scan o e a a e Lo 105 A A tilt etd eaten E seaman abet sears 105 A A EEE E EA E 106 no ce ea 106 SECHON LNICKNESSES rir i a a aett nace sencoeenstead 106 o A E N ONE PA E E A A E AE N 107 Data VOLIMEOS esmas 107 1D GU OMS a EN 108 Sale ld dio 108 MOM tl caos 108 Orthogonal Pro eco a ee ei 109 Rotated FO SCHON ici e oia duna 110 OE E veranda A 110 SCAM DSCC ur ia 110 POINTS EA a EE a EE 111 EDO Pee Egg a esate 111 PIANOS A e E 111 Spaces TIME Space a e o teas teas a eo Lo 111 FRAP Mesas Mens cciiaiaiadi 112 Special Series iaa tia 112 Data Acquisition and Utilization oocooccocnconcocnconconiccnconicononcnonncaninnnon 112 About Spectral Resol usina add 112 COMPDINATONIAlWAMAIY SIS usas 112 Care and Maintenance iii 115 EEE e E A AE A EAE EE E E AAEE E 115 Cleaning the Optical System ooccooccccocncconncccnoconocunnonononanonoonocnnonannonaninnnns 115 11 3 11 4 12 12 1 12 2 13 14 15 15 1 15 2 15 3 Cleaning the Microscope Surface
22. DMI 6000 CS Angled rear view of right side of microscope Figure 22 Safety label for inverted microscope DMI 6000 CS 41 Angled front view of right side of microscope 29100 017 zust 00 52005 008 u t 01 Figure 23 Safety label for inverted microscope DMI 6000 CS 42 5 10 2 Upright microscope DM 5000 6000 CS Angled front view of right side of microscope Figure 24 Safety label for upright microscope DM 5000 6000 CS 43 Rear view of microscope Figure 25 Safety label for upright microscope DM 5000 6000 CS 44 5 10 3 Scan Head Angled front view of left side of scan head A ooo ol SS LA a ly Pi u E a E E mA 4 of N dl a le at i aoe al a Es a ay 7 AAN y i iJ Ye E Figure 26 Safety label for the scanner 45 5 10 4 Achromatic light laser Rear side of achromatic light laser Figure 27 Safety label on the rear side of the white light laser i Applies only to the TCS SP5 X system 46 5 10 5 External UV laser Figure 28 Safety label on external UV laser i Applies only to systems with an external UV laser 47 5 10 6 Supply Unit View of supply unit 52005 002 Figure 29 Safety label for the TCS SP 5 supply unit front side 48 5 10 7 MP beam coupling unit Angled front view of the right side of the MP beam coupling unit 52005 002 Figure 30 Safety label for the MP beam coupling unit top side 49
23. Dimensions of TCS SP5 with upright microscope 15 2 7 1 3 TCS SP5 X with inverted microscope 1400 mm 3040 mm Figure 6 Dimensions of TCS SP5 X with inverted microscope 2 7 1 4 TCS SP5 X with upright microscope 1490 mm 2900 mm 1200 mm Figure 7 Dimensions of TCS SP5 X with upright microscope 16 2 7 2 Electrical Connection Requirements The building installation must feature three separate power connections with the following fuse protection e 3x100V 120 V power supply at 20 A or e 3x200V 240 V power supply at 12 16A For the specifications of external lasers such as UV and MP lasers please refer to the manufacturer s documentation 2 7 2 1 Electrical connection requirements of supply unit Supply voltage 100 240 V AC 2 10 2 7 2 2 Electrical connection requirements of achromatic light laser Supply voltage 100 240 V AC 2 10 Applies only to the TCS SP5 X system 17 2 7 3 Requirements Regarding Ambient Conditions A Ensure that the system is not installed next to air conditioners or ventilation systems For this reason the installation location should be carefully planned A Also read the notes on protection against dust in Chapter 11 Care and Maintenance Do not expose the system to drafts Ensure that the environment is as dust free as possible Installing the system in darkened rooms is also advisable The system requires doors with
24. Leica TCS SP5 A dialogue is available to set smaller or larger diameters if required Playing with this parameter to study its effects can be very worthwhile when you have the time 8 10 2 Acquiring Optical Sections Setup Acquisition Light Path Acquisition Moda xy Figure 63 Use the Acquire arrow key to acquire data in all Leica LAS AF applications The Leica TCS SP5 contains many functions in its user interface that reflect its wide range of potential applications The functions not needed for a given application are disabled however to ensure efficiency and ease of use Select the task at hand from the row of arrow keys at the top The functions required for data acquisition the sole focus of these chapters are grouped under Acquire Figure 63 For descriptions of the individual functions please see the online help This section will describe the aspects affecting the configuration of the most important scanning parameters and special points that must be taken into consideration 10 2 1 Data Acquisition Press the Live button to begin data acquisition Figure 64 Data will be transferred continuously to video memory and displayed on the monitor Initially the data will not be stored in a manner suitable for subsequent retrieval Figure 64 The Live button starts data acquisition in all Leica LAS AF applications This is a preview mode suitable for setting up the instrument Stopping data acquisition will
25. MP lasers for TCS SP5 and TCS SP5 X A The components for laser safety are designed only for the laser variants listed 21 2 8 3 Overview of Usable VIS UV Lasers for TCS SP5 X The Leica TCS SP5 X features a combination of the lasers listed below Maximum Maximum Wavelength luminous power luminous power at laser output in focal plane Laser type mW Pulse duration Diode 405 cw Diode 405 p e pulsed 60 ps power 55 458 476 488 Continuous wave laser cw Achromatic 170 670 lt 500 lt 50 Pulsed light laser Table 4 Usable lasers for TCS SP5 X A The components for laser safety are designed only for the laser variants listed 22 2 8 4 Which Laser Class Does the Product Have Wavelength range Configuration variant 400 700 nm visible laser radiation 350 1600 nm visible and invisible laser radiation 350 700 nm UV visible and invisible laser radiation 2 8 5 Combination of lasers from Chapter 2 8 3 or 2 8 1 without lasers having wavelengths of 350 400 nm 3B IIIb 3B IIIb 4 1 Combination of lasers from Chapter 2 8 3 or 2 8 1 VIS and UV lasers Combination of lasers from chapter 2 8 1 VIS UV Laser chapter 2 8 2 IR Laser or chapter C2 8 3 VIS UV Laser Required Laser Safety Measures Please observe the laser safety measures for laser class 3B lllb VIS and UV systems or laser class 4 IV MP systems in accordance with applicab
26. Pv 6 E O E oo soc MS e In icroscope is use tegrated Location of the interlock jack 0 O N nu E ca N 00 S 8 5 O D c p lt LL 5 8 Function and Position of Safety Switches When the safety switches are released the light path of the laser beam is interrupted Figure 19 Position of the transmitted light illumination arm 1 and switching from scan mode to eyepiece 2 Inverted mentir Prevents laser light microscope DMI Ami MG OS while working on the 6000 CS a voina ONNE specimen specimen Transmitted light illuminator arm Prevents stray light if changeover Inverted the user switches mirror for the between scanning microscope DMI from confocal Motorized The deflection scanner is swung observation to out by motor eyepiece observation mode and 6000 CS eyepiece 38 5 9 Special Laser Safety Equipment 5 9 1 Laser protection tube and beam stop On inverted microscopes the safety beam guide and the beam stop serve as protection against laser radiation emission and are located between the condenser base and the transmitted light detector see Figure 20 1 Laser protection tube 2 Beam stop illustrated is the version of the beam stop for MP systems 3 Condenser base Figure 20 Inverted microscope a If you reorder a condenser base Figure 20 item 3 be aware that the condenser base is delivered without the beam stop Fig
27. a sheet reprinted in the Appendix provided by the manufacturer Innovatek regarding the coolant used A The coolant scanner cooling system must be replaced by Leica service or an authorized Leica dealer every two years In case of a coolant leak switch the power off immediately Inform Leica or a Leica approved service facility immediately A A The coolant contains an irritating substance Avoid eye and skin contact 116 12 Transport and Disposal 12 1 Changing the Installation Location Clean the laser scanning microscope thoroughly before moving it to another place Whenever any system parts are removed these also have to be cleaned MN thoroughly This applies in particular to systems that are located in biomedical research labs This is necessary to remove any possible contamination thereby preventing the transfer of dangerous substances and pathogens and avoiding hazards and dangers 12 2 Disposal K In addition to surfaces pay particular attention to fans and cooling devices as dust is particularly likely to accumulate at these locations If you have any questions related to disposal please contact the Leica branch office in your country see Chapter 13 13 Contact If you have any further questions please contact your country s Leica branch office directly The respective contact information can be found on the Internet at http www confocal microscopy com 117 14 Glossar
28. age can be reconstructed in parallel To create a two dimensional image the spot must be moved over the entire surface and the associated signal recorded on a point by point basis This is performed in a raster process similar to that of SEM instruments or the cathode ray tubes still used in computer monitors and televisions Figure 60 In a confocal microscope with point scanners the movement is realized by two mirrors mounted on so called galvanometric scanners These scanners have the same design as electric motors their rotors are fixed at their base to the housing Applying power to the scanner rotates the axis the rotation ceases at the point at which the torsional force and the electromagnetic force balance The mirror can thus be moved quickly between two angles by applying an alternating voltage 85 Figure 61 Smallest possible diffraction limited illumination spot Airy disk Below an intensity profile To scan a line the x mirror must travel once across the field of view The y mirror is then moved a small amount after which the x mirror then scans the next line The signals from the specimen are written to an image memory in synchronization and can be displayed on the monitor 10 1 4 How Is an Optical Section Created The term confocal is strictly technical and does not describe the effects of such an arrangement That will be described in greater detail here As already described in10 1 3 the illumination of th
29. age in a scientific context must always contain a scale Such a scale can simply be added to the image and adjusted in its shape color and size as required Scales were not added to the images in this document for the sake of clarity 10 2 7 Signal and Noise The gain of the capture system must be matched to the signal intensity when capturing data Signal strengths can vary by several orders of magnitude making such an adjustment necessary to ensure a good dynamic range for the scan The goal is to distribute the full range of intensity over the available range of grayscale values 256 grayscale values from O to 255 are available for images with 8 bit encoding If the gain is too low the actual signal may only correspond to 5 grayscale values causing the image to consist solely of those values If the gain is too high parts of the signal will be truncated i e they will always be assigned the grayscale value of 255 even though differences information were originally present in the signal This image information is then lost Figure 75 99 Correctly setting the zero point is also important This can be accomplished by shutting off the illumination via the AOTF and setting the signal to zero with Offset Turn the illumination back on and adjust the gain to prevent distortion This configuration work is simplified by special color tables such as Glow over Glow under a table that initially uses yellow and red for intensities in st
30. also immediately stop the scanning operation even if the image has not been fully rendered Alternatively a single image can be captured This image is then stored in the experiment and can be retrieved later or stored on any data medium Individual image scanning has the advantage of exposing the specimen only once but is less convenient if additional setup 88 work is required Once all parameters are correctly set up an image of the result may be captured Functions such as accumulation and averaging are supported The third data acquisition situation is the acquisition of a series in which the preselected parameters are changed incrementally between scanning the individual images Time series lambda series and z stacks can be created in this manner Figure 65 Z stack K 4 j A y Time series d o 7 t Y e i Fa amp amp amp Lambda series VO J0 Jo 7 Figure 65 Stack acquisition for 3D time and lambda series When using the instrument in LiveDataMode all captured images are automatically stored with the time of capture A preview mode is not available in that case Figure 66 This method is especially suitable for the observation of living objects over time while changing the medium applying electrical stimuli or executing changes triggered by light 89 Figure 66 LiveDataMode supports the continuous acquisition of data while changing setting parameters manipulating th
31. are visible We recommend focusing to ensure that the brightest signals in the field of view are really used for the adjustment This is also the right time to check whether the intensity of the excitation light is correctly set The intensity of the illumination can be increased at the AOTF to reduce image noise However it must be taken into consideration here that a higher illumination intensity is detrimental to the specimen In the case of 100 extremely sensitive specimens and in situations in which rapid changes in intensity in living specimens is of interest images with more noise can be acceptable However this compromise depends on the specimen and the application The signal to noise ratio may be influenced by a number of other factors in addition to illumination intensity the speed at which data are captured The actual speed of the scan which can be adjusted via the horizontal frequency 1 Hz 1 400 Hz conventional scanners only and the averaging method offer additional options for enhancing the signal The change of the scan speed itself leads to averaging in the pixels as data are recorded for each pixel over a longer period When averaging lines each line is scanned several times and the result of the averaging displayed In the case of image averaging an entire image is scanned and then averaged with the subsequent image scanned at the same location All processes have their advantages and disadvantages as always Tempora
32. at a time and the emissions are thus solely from that dye regardless of the spectral range in which the signals are captured This is of course the ideal state in practice other dyes may also be excited slightly nevertheless the separation is clearly better than that achieved using simultaneous scanning Generally crosstalk can be almost completely eliminated this way A further advantage of the sequential method is that the emission bands of the individual dyes can be set rather widely This improves sensitivity and is thus easier on the specimen An obvious disadvantage is that the scan takes twice as long with two dyes however the advantages listed above compensate for this 10 3 6 Unmixing As in most cases a software solution is available to deal with crosstalk whenever a physical separation is not possible However we recommend optimizing separation with the means provided by the instrument see 10 3 4 and 10 2 5 to the greatest extent possible and to use the software only in those cases in which the results are still not satisfactory The unmixing method determines the share of a dye s emissions distributed across the various scanning channels This process is applied to each of the dyes The result is a distribution matrix that can be used to redistribute the signal strengths so that they correspond to the dyes This is described for two dyes in the following figures but it is equally valid for any number of dyes The precondi
33. at cover most of the tasks related to the instrument a Upright microscope 1 Objective 2 Cover slip 3 Seal 4 Specimen slide 5 Stage focus b Inverted microscope 1 Embedding 2 Specimen 3 Immersion 4 Lens focus Figure 58 Arrangement of cover slip and specimen on an upright microscope a and inverted microscope b When using objectives with cover slip correction ensure that the cover slip i e the top side of embedded specimens is facing down Background information has also been provided to explain the reasons behind various settings These are not descriptions of the individual functions and controls of the instrument and graphical user interface but an informative tour of the essential tasks that is designed to remain valid even if future upgrades change the specific details of operating the instrument 81 The very first step of course is to place a specimen in the microscope When placing specimens in an inverted microscope ensure that fixed specimens on slides are inserted with the cover slip facing down Figure 58 Failing to do so is a frequent reason for not being able to find the specimen or focus on it in the beginning 10 1 1 The Objective Select the objective with which you want to initially examine the specimen Modum petrate maer o fm pse Table 3 Table of various immersion media When using immersion objectives ensure that an adequate quantity of immersion medium is applied betw
34. bjective to the focal point For a variable working distance the gap between the front lens of the objective and the cover slip or uncovered specimen is specified Usually objectives with large working distances have low numerical apertures while high aperture objectives have small working distances If a high aperture objective with a large working distance is desired the diameter of the objective lens has to be made correspondingly large These however are usually low correction optic systems because maintaining extreme process accuracy through a large lens diameter can only be achieved with great effort Instrument Parameter Setting An instrument parameter setting IPS consists of a file in which all hardware settings are stored that are specific to a certain recording method The designation FITC TRITC for example refers to the settings for a two channel recording with the two fluorescent dyes FITC and TRITC An instrument parameter setting enables the user to store optimum hardware settings in a file and to load them again with a simple double click Instrument parameter settings labeled with the letter L are predefined by Leica and cannot be changed User defined modifiable instrument parameter settings are stored below U in the list box 118 Curvature of Image Field The curved surface to which a microscopic image is to be clearly and distinctly mapped is described as curvature of image field It is conditional on the convex s
35. ceeeeseseeeeeseeeseseeseeeseeeeseeees 31 5 2 Detachable key SWitCH ooccocccccccncconncccnocococonnononononnnnannonnnonanonanonnanenaninnanes 32 5 3 EMISSIONS Warning Indicators sssssmnionsne een aA 34 5 4 Remote Interlock Connection on the Supply Unit ocoocccoccconnconnconicononooo 35 0 0 Remote interlock connection on the achromatic light laser 36 5 6 Remote Interlock Connections on External Lasers ccoooccccccccccccccncoccccncncnon 37 5 7 Remote interlock jack interlock connector on the SCANNET cseceeeeeeeees 37 5 8 Function and Position of Safety SwitChesS oocccccccccnccccncncncconononnncnncnnnnnnnnos 38 5 9 Special Laser Safety Equipment cccceccceecseeeeeeeeneeeseeeseeeseteeeteeeteeetaeenes 39 5 9 1 Laser protection tube and beam Stop ocooccocccccconconnconcocnnonconnnononnnononancnnnnonos 39 5 9 2 Shielding in MP Systems IR Lasers oocccoccccocccccnccccnconnccncncncnonannnnnnnoncnnos 40 5 10 Safety labels Onmeda a 41 5 10 1 Inverted microscope DMI 6000 CSO occooccncccncccocccocccococonoconoconcnnnnonnnonnnononanononos 41 9 10 2 Upright microscope DM 5000 6000 CS ooooccocccocccocccocccocccocnconnconnconocnnnonnonanonons 43 5 10 3 Scan e ala ido ds 45 5 10 4 Achromatic ORI TE 46 5 10 5 ExXtemal UV WAGON nina a a E 47 5 10 6 Supra td 48 5 10 7 MP beam coupling UNitosssissnsrmin e a T 49 5 10 8 Cover for Replacement Flange
36. cimen via the eyepieces For more information on selecting fluorescence filter cubes please refer to the Leica fluorescence brochure or contact your Leica partner For a selection of filter cubes see Table 4 below As specimen fluorescence can fade quickly always close the shutter of the mercury lamp when you are not looking into the microscope To switch to scan mode press the appropriate keys on the microscope or use the switching function in the software The switching function may vary according to the motorization of the microscope Please consult help for more information 83 ereere ersess eo ferao op freseo CA BP 335 425 e aeara oo fess CO CC CA rscran BP 546 14 ys ereo feo ACT Table 4 Selection of filter cubes for Leica research microscopes and associated filter specifications 84 10 1 3 Why Scan Specimens must be illuminated over the smallest possible area to achieve a true confocal image this is essential to attaining truly thin optical sections This has been achieved when the illumination spot is diffraction limited i e it cannot be made physically smaller The diameter of such a diffraction limited spot corresponds to dg 1 22 e NA with e representing the excitation wavelength and NA the numerical aperture of the objective used Figure 61 Figure 60 Illustration of the raster scan Two mirrors move the illumination spot in x and y directions across the specimen in rows so that the entire im
37. default user name for the system is TCS User No default password is set system administrator This will create individual directories that can be viewed by the respective user only Since the LCS AF software is based on the user It is recommended to set up a separate user ID for each user set up by the 12 administration of the operating system separate files are created for managing user specific profiles of the LCS AF software 1 Log on as administrator To do so use the username ID Administrator and the password Admin 2 Open the User Manager Select Start Programs Administrative Tools User Manager 3 Define a new user Enter at least the following information in the open dialog window e User ID e Password must be re entered in the next line for confirmation purposes 4 Select the following two check boxes e User must change password at next logon this allows the new user to define his or her own password at logon e Password never expires this allows a defined password to be valid until either it is changed in the User Manager or the user is deleted 5 Select the Profiles option in the bottom section of the dialog In the Local path field enter the following path for storing the user specific file d users username username is a wildcard which must be replaced by the currently defined user name Factory installed hard disk drives are provided with two partitions C and D Set up t
38. designations of the laser class are to be changed in the text from 3B to lllb and Class 4 to IV 13 2 5 Patents The Leica TCS SP5 product is protected by the US patents 5 886 784 5 903 688 6 137 627 6 222 961 6 285 019 6 311 574 6 355 919 6 423 960 6 433 814 6 444 971 6 466 381 6 510 001 6 614 526 6 654 165 6 657 187 6 678 443 6 687 035 6 738 190 6 754 003 6 801 359 6 831 780 6 850 358 6 867 899 Further patents are pending The Leica TCS SP5 X protect by the US patents 5 886 784 5 903 688 6 137 627 6 222 961 6 285 019 6 311 574 6 355 919 6 423 960 6 433 814 6 444 971 6 466 381 6 510 001 6 567 164 6 611 643 6 614 526 6 654 165 6 654 166 6 657 187 6 678 443 6 687 035 6 710 918 6 738 190 6 754 003 6 796 699 6 801 359 6 806 953 6 831 780 6 850 358 6 867 899 6 888 674 6 898 367 6 958 858 6 961 124 7 005 654 7 092 086 7 110 645 7 123 408 Further patents are pending 2 6 Serial Number The serial number of your system is located on the rear side of the scanner 00 00 90 668 Figure 3 Rear side of scanner label with serial number 14 2 7 Specifications 2 1 1 Dimensions 2 7 1 1 TCS SP5 with inverted microscope 1400 mm 600 mm 730 mm f Figure 4 Dimensions of TCS SP5 with inverted microscope 2 7 1 2 TCS SP5 with upright microscope 1490 mm 2400 mm 1200 mm Figure 5
39. e specimen is focused on the smallest possible spot hence the term focal The confocal design also involves an observation point The sensitivity distribution of the detector is reduced to a point by focusing light from the specimen on a very small opening Known as the pinhole This pinhole cuts off all information not coming from the focal plane Figure 62 86 in focus 3 Pinhole pinhole Figure 62 Creating an optical section using an incident light process Light not originating from the focal plane is cut off by a spatial filter here a pinhole Only information from the focal plane can reach the detector The diaphragm thus acts as a spatial filter but only when used with the correct i e point shaped illumination As a rule the optical section becomes thinner when the size of the pinhole is reduced This effect is reduced near the wavelength of the light used and at a pinhole diameter of zero one would theoretically receive the thinnest optical section for the wavelength and numerical aperture used A range apparently exists at 1 Airy which does not yet offer the thinnest optical sections but which is nevertheless very close to the theoretical limit As the intensity of the passing light increases roughly in proportion to the square of the pinhole diameter it is advisable not to close the pinhole too far to avoid excessive image noise A value of 1 Airy is a very good compromise and is selected automatically by the
40. e specimen or performing bleaching sequences between the individual scans The clock continues running throughout the experiment and intensity changes in regions of interest can be rendered graphically online The setting parameters for scanning a simple optical section are described and discussed below These settings are identical for all work with the instrument Preconfigured parameter sets have been stored in the software for typical specimen situations You may also store and recall custom parameter sets The description below is based on the assumption that you are using a specimen similar to the included standard specimen The standard specimen is a Convallaria majalis rhizome section with a histological fluorescent dye The specimen can be used for a wide range of fundamental problems and has the advantage that it practically does not bleach 10 2 2 Illumination Laser lines suitable for the excitation of fluorescence may be selected as illumination The intensity of the laser line can be adjusted continuously using the line s slider Moving the slider all the way down disables the line Using this slider the intensity is adjusted continuously via an acousto optical tunable filter AOTF The intensity at which a sufficiently noise free image of the specimen can be obtained must be determined to reduce deterioration of the specimen Factors affecting this are the fluorescent dye the line used the density of the dye in the specimen the locat
41. ected empirically Figure 69 The reflected excitation light also appears in the image as soon as the emission band crosses under the excitation line While this is naturally undesirable for fluorescence it does provide a very simple way of creating a reflection image The narrowest band is 5 nm and such a 5 nm band would generally be set under the excitation line for reflectometry applications To suppress interference from reflected excitation light it generally suffices to set the start of the emission band to around 3 to 5 nm to the red side of the excitation line Naturally this depends strongly on the reflective properties of the specimen It is usually also necessary to maintain a greater distance when focusing close to the glass surface for this reason That especially applies to specimens embedded in aqueous media The further the refractive index of the embedding material deviates from 1 52 the more likely distracting reflections become Greater caution would also be required for specimens containing a high number of liposomes for example 10 2 5 The Pinhole and lts Effects The reason for deploying a confocal microscope is its ability to create optically thin sections without further mechanical processing of the specimen The essential component of the instrument that creates these sections is a small diaphragm in front of the detector the so called pinhole as already described in 10 1 4 Ideally the diameter of this pinhole w
42. een the front lens of the objective and the specimen Immersion oil glycerol 80 or water may be used as immersion media Table 3 Apply the immersion medium generously but be sure that it does not flow into the stand of inverted microscopes 10 1 2 Conventional Microscopy To view the specimen conventionally through the eyepieces ensure that VIS operating mode is selected SCAN is for use with the laser scanning operation image process Select a suitable position and focus on the specimen 82 1 Filter cube 2 Specimen 3 Objective lens 4 Shutter 5 Lamp 6 Eyepiece Figure 59 Incident light fluorescence scheme light from a mercury lamp is collimated selected spectrally via an excitation filter and applied to the specimen via a color splitter mirror A shutter permits the specimen to be darkened The emission longer wavelength than the excitation is visible through the color splitter mirror and emission filter via the eyepiece The excitation filter color splitter mirror and emission filter are grouped in a filter cube Optical sections are created using the transmitted light method Your specimen must therefore reflect or fluoresce Fluorescent specimens are most common In many cases specimens with multiple dyes will be examined Reflective specimens can also provide interesting results however The filter cubes Figure 59 that correspond to the fluorescence must be positioned within the beam path when viewing the spe
43. electronic components DL BEBE mechanical parts optical components cables light sources RMZAEA ROME RUI PARAMS IT 11363 2006 WEA E MIRES F Indicates that the concentration of the hazardous substance in all materials in the parts is below the relevant threshold of the SJ T 11363 2006 standard gt ETUAE REPO E DERIAOR MHRPAEABBESJ T 11363 2006 PF ERMEHORBSR Indicates that the concentration of the hazardous substance of at least one of all materials in the parts is above the relevant threshold of the SJ T 11363 2006 standard Note The actual product may or may not include in all the part types listed above 130 131 Leica Microsystems CMS GmbH Phone 49 0 621 7028 0 Am Friedensplatz 3 Fax 49 0 621 7028 1028 D 68165 Mannheim Germany http www leica microsystems com Copyright Leica Microsystems CMS GmbH All rights reserved Order No 156500002 VO9
44. eps to indicate the signal strengths The grayscale value zero is always shown as green value 255 as blue Both values can thus be identified immediately The zero point is set correctly when around half the pixels are zero i e green with the light switched off To be safe the offset can be set one or two grayscale values higher to ensure that the lower signal values are not truncated The loss of dynamic range is negligible approx 0 4 per grayscale value at 8 bits Figure 75 At the top left you see an 8 bit image 256 gray scales At the right the same specimen with a considerably smaller dynamic range Around 6 gray scales can be made out in the false color image at the bottom That corresponds to less than 3 bits The electronic deviations from the zero point will generally be negligible nevertheless occasional testing is advisable The actual significance of adjusting the offset value is to compensate for nonspecific or self fluorescence in the specimen at the time of the scan Simply set the offset value in such a manner that the background fluorescence is no longer visible Please note that this may also truncate signals containing image information Such settings must always be verified by a careful examination of the results The amplification of the signal must be performed after the offset correction This operation is quite simple with the described color table adjust the high voltage at the PMT until no more blue pixels
45. ew the available logical operators 1 Select the desired operator 76 2 After the operator enter the second search term you would like to associate with the first search term Pinhole and sections Pinhole or sections Pinhole near sections Pinhole not sections This phrase finds help topics containing both the word pinhole and the word sections This phrase finds help topics containing either the word pinhole the word sections or both This phrase finds help topics containing the word pinhole and the word sections if they are located within a specific search radius This method also looks for words that are similar in spelling to the words specified in the phrase This phrase finds help topics containing the word pinhole but not containing the word sections 17 9 3 Structure of the graphical user interface 9 3 1 General Structure of the Graphical User Interface The user interface of the LAS AF is divided in five areas Figure 57 LAS AF user interface 1 Menu bar The various menus for calling up functions are available here 2 Arrow symbols Operating steps with the individual functions These operating steps mirror the typical sequence of scan acquisition and subsequent image processing The functions are grouped correspondingly into these operating steps e Configuration e Acquire e Process e Quantify e Application 78 3 Tab area Each operating step arrow symb
46. ey are intended or not using the microscopes within the specifications of Leica Microsystems CMS GmbH In such cases the Declaration of Conformity shall be invalid 2 4 Conformity This device has been tested and meets the requirements of the following standards IEC EN 61010 1 Safety requirements for electrical equipment for measurement control and laboratory use IEC EN 60825 1 Safety of laser products Part 1 Equipment classification requirements and user s guide Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Class A IEC EN 61326 This is a Class A instrument for use in buildings that do not include domestic premises and buildings not directly connected to a low voltage power supply network that supplies buildings used for domestic purposes Electromagnetic Compatibility EMC is Part 3 2 Limits Limits for harmonic currents Electromagnetic Compatibility EMC IEC EN 61000 3 3 Part 3 3 Limits Limitation of voltage fluctuations and flicker in low voltage supply systems The declaration of conformity for the system is located in the appendix of this operating manual For use in the USA U S laser products Food and Drug Administration FDA CDRH 21 CFR 1040 10 Complies with FDA performance standards for laser products except for deviations pursuant to laser notice No 50 dated 26 July 2001 For the USA area of validity of the CDRH FDA the
47. f laser lines for excitation in order to obtain sufficient room to adjust the emission bands This parameter can also be used for balancing if a dye is very 104 dominant the selection of a different excitation line can reduce the intensity of the dye and thus improve the separation against the other dye while increasing the spacing to the other excitation permitting larger emission bands and thus enhancing sensitivity Every improvement in this regard permits a reduction of excitation energy which in turn reduces bleaching A further option for the reducing crosstalk is selecting suitable emission bands The emission characteristics of the dyes used can be displayed in the user interface and a lot can be gained if the emission bands are restricted to ranges that do not overlap at least in the graphic on the monitor Naturally the stored characteristics are not necessarily identical to the actual emissions as many factors e g pH value polarity metabolic products can affect the spectrum However in this case it is also possible to change and optimize the settings during data acquisition 10 3 5 Sequential Scanning Another way to reduce crosstalk is to scan the information for the various dyes sequentially instead of simultaneously This has two advantages Whenever different laser lines are used for excitation and this is generally the case sequential scanning provides significantly improved separation as only one dye is excited
48. f the laser beam Avoid all contact with the laser beam A A Never deactivate the laser protection devices Please read the chapter Laser Protection Devices to familiarize yourself with the safety devices of the system 53 Do not introduce any reflective objects into the laser beam path A A 6 3 Eye Protection Be sure to follow the included operating instructions for the microscope 6 3 1 MP System with Upright Microscope Wearing safety goggles order number 156502570 is compulsory Appropriate de safety goggles for IR laser radiation are provided with the system when delivered nd These safety gogales do not offer any protection against visible laser radiation visible spectrum During the scanning operation all persons present in the room must wear safety O Nowe goggles The IR laser beam can be deflected or scattered by the specimen or objects moved into the specimen area Therefore it is not possible to completely eliminate hazards to the eye from IR laser radiation The supplied safety goggles only provide safe protection against the infrared lasers supplied by Leica Microsystems CMS GmbH 6 3 2 MP System with Inverted Microscope It is not necessary to wear eye protection If the device is used as prescribed and the safety instructions are observed the limit of the laser radiation is maintained so that eyes are not endangered 6 3 3 VIS and UV Systems with Inverted or Uprigh
49. hape of the lens and makes itself apparent as an error due to the short focal lengths of microscope objectives The object image is not in focus both in the center and at the periphery at the same time Objectives that are corrected for curvature of image field are called flat field objectives Refractive Index The factor by which the light velocity in an optical medium is less than in a vacuum Chromatic Aberration An optical image aberration caused by the varying refraction of light rays of different wavelengths on a lens Thus light rays of shorter wavelengths have a greater focal length than light rays of longer wavelengths Dichroic Dichroic filters are interference filters at an angle of incidence of light of 45 The transmissivity or reflectivity of dichroic filters depends on a specific wavelength of light For example with a short pass filter RSP 510 reflection short pass excitation light below 510 nm is reflected light above this value is transmitted The transmission values are generally between 80 and 90 and the reflection values between 90 and 95 Digital Phase true Filter A digital filter consists of a computing rule used to modify image data Filters are always applied to remove unwanted image components A phase true filter ensures that quantifiable image values do not change through filtering and remain a requirement for standardized measuring methods e g characterization of surfaces in accordance with ISO Doub
50. he TCS workstation Figure 56 item 5 the scanner Figure 56 item 4 and the laser Figure 56 item 3 Figure 56 Main switch board 1 emission warning indicator 2 detachable key switch 3 switch for laser 4 switch for scanner 5 switch for workstation 8 Switch off the microscope and any activated fluorescence lamps A If your system features external lasers IR UV or others switch them off in accordance with the respective operating manual from the manufacturer 74 9 Introduction to LAS AF 9 1 General The LAS AF software is used to control all system functions and acts as the link to the individual hardware components The experiment concept of the software allows for managing the logically interconnected data together The experiment is displayed as a tree structure in the software and features export functions to open individual images JPEG TIFF or animations AVI in an external application 9 2 Online Help 9 2 1 Structure of the Online Help The online help is divided into 4 main chapters Books Contents General Contains legal notices and general information on the LAS AF LAS AF Online Help Contains general information for the LAS AF online help Contains detailed descriptions of the dialogs in the LAS AF user interface Dialog descriptions Contains background information on LAS AF and Additional information application related topics such as_ digital image processing a
51. he result is an image consisting solely of the sharply focused values but distributed over the entire distance of the image in the z direction The operation also increases the depth of focus over the entire height of the z stack Such projections are therefore called extended depth of focus images This method is also suitable for multichannel scans Coloring each section differently for example by mapping the colors of the rainbow to the z axis permits the z positions of structures to be identified immediately in this projection This is only possible with one channel of course as the color is used for the height This representation is known as height color coded extended depth of focus Figure 80 Figure 80 Color coded relief of the series shown above The SFP simulated fluorescence projection method uses a more complex approach to achieve impressive images with shadow projections The quantification must always be checked with care when using this method however 109 10 4 5 4 Rotated Projections Figure 81 Stereo image of the same 3D data Although some practice is required this is nevertheless a worthwhile exercise for any confocal microscope user The methods described in 10 4 5 3 initially assume that the projection will be performed along the visual axis Because the data in the computer exist in a spatially homogeneous state however projections from any direction are possible In the simplest case two pr
52. he user directory on partition D 8 Switching Off the System A 1 Save your image data On the menu bar select File Save as to save the data record The switch off sequence must be followed If the switch off sequence listed below is not followed the lasers could be damaged 2 Close the LAS AF On the menu bar select File Exit Exit the LAS AF 3 On the main switch board switch off the lasers in the supply unit using the detachable key switch Figure 56 item 2 The emission warning indicator Figure 56 item 1 goes out 4 Switch off the achromatic light laser with the detachable key switch see Figure 53 on the front of the achromatic light laser The emission warning indicator goes out 8 Figure 53 Detachable key switch for the achromatic light laser 5 Switch off the external UV laser with the key switch see Figure 54 The emission warning indicator goes out Figure 54 Key switch for the external UV laser Applies only to the TCS SP5 X system Applies only to systems with an external UV laser 73 6 Shut down the computer On the toolbar select Start Shutdown to shut down the TCS workstation 8 se 9 8 wa a E 3 a a w ee a O amw a D 3 D fa lo 3 ne Gan Q a E 3 Sh EN amp 5 z 7 g e zZ w E o v Figure 55 Shutting down the computer 7 Next turn off the switches on the main switch board for t
53. ica Service 63 Figure 41 Spacer on objective than those without piezo focus A spacer Figure 41 is installed on all other Please note that the focus position of an objective with piezo focus is 13 mm lower A objectives to ensure the same focal plane 6 10 1 Objective Change with Piezo Focus Configuration Do not change objectives automatically The automatic motion may damage the cable of the piezo focus In addition to the regular procedure see chapter 6 6 the stage must be lowered as much as possible and the slide or specimen must be removed from the stage MN before changing the objective on the piezo focus The slide or objective may otherwise be damaged or destroyed by the initialization of the piezo focus when starting the system software z When replacing the objective on the piezo focus you must perform a teach in for the new objective in LAS Please see the instructions on this topic in the microscope operating manual 64 7 Starting Up the System 7 1 Switching On the System With the motorized stage 156504145 for DMI 6000 inverted Before the system start or start of the LAS AF the illuminator arm of the inverted microscope must be swung back because the motorized stage can be initialized and damage the condenser With the motorized stage 156504155 for DM 6000 upright Before the system start or start of the LAS AF the stage must be moved downwards because during initialization it can co
54. ices that are listed in the operating manual Otherwise please contact your local Leica service agency or Leica Microsystems CMS GmbH Leica Microsystems CMS GmbH shall not be liable for damages resulting from nonobservance of the above information The above information does not in any way implicitly or explicitly modify the warranty and liability clauses contained in the general terms and conditions of Leica Microsystems CMS GmbH 28 4 4 Safety Instructions for the User Read and observe the safety instructions in the operating manual and the safety labels located on the system Failure to observe the safety instructions may lead to serious injuries and to significant damages to the system and loss of data This laser product may be operated only by persons who have been instructed in The instrument is a Class 3B or 4 laser product depending on the laser used A the use of the system and the potential hazards of laser radiation Before carry out operating steps with the system for the first time first read the 9 corresponding description of the function in the online help 1 For an overview of the individual functions refer to the table of contents of the online help As it is impossible to anticipate every potential hazard please be careful and apply common sense when operating this product Observe all safety precautions relevant to Class 3B lllb lasers and Class 4 IV lasers for MP systems Do not deviate from the opera
55. il of high resolutions You may therefore have difficulty detecting the differences between the top two images despite the enormous differences in the optical resolution This must also be taken into consideration in publications The simplest solution is to combine both methods in the so called Zoom In function Simply select a square on the monitor that encloses the structures of interest and the instrument will automatically select the appropriate pan and zoom values This function is very fast and thus easy on the specimen An Undo Zoom function returns you to your starting point for quickly concentrating on a different cell in the field of view for example The size of the grid spacing used can be found in the image properties The spacing of the elements in x y and z can be found under Voxel Size At Zoom 1 the images calculated above would have a grid spacing between 200 nm and 300 nm Larger gaps would lead to a loss of resolution when using an objective with an aperture of 0 4 Figure 73 Rectangular formats are important for higher image scanning rates An additional parameter is required here the rotation of the scan field As field rotation is performed optically in the Leica TCS SP5 rotation by 100 does not have any effect on the speed and possible grid formats Figure 74 98 Figure 74 Zoom Pan and Rotation combined in one example Finally it must be pointed out in this section that a good microscopic im
56. inest differences in the spectrum The TCS SP5 supports the adjustment of emission bands in 1 nm steps which corresponds to a formal resolution of one nanometer The optical spectral resolution is dependent on the wavelength however and amounts to roughly 0 5 nm in the blue and 2 nm in the red range This resolution is far better than required in practice in typical specimens that are in a liquid or gel state at room temperature fluorescent emissions are never sharper than roughly 20 nm 10 7 Combinatorial Analysis Many of the methods described above can be combined and deliver new insights in biology with both fixed and living specimens The term multidimensional microscopy has been coined to describe this form of combinatorial analysis However a certain inflation in this regard has become apparent recently Stitching together a large number of dimensions 112 measuring parameters does not in itself make a good experiment and it is definitely not conducive to sound results The synthesis of a broad range of measurements is often difficult and always requires a solid intellectual overview to avoid data graveyards and incorrect conclusions 113 114 11 Care and Maintenance 11 1 General Information about maintenance and care of the microscope is located in the operating manual of the microscope The instructions and additional information relating to the components of the confocal system are summarized below A Whe
57. int gt 120 C DIN ISO 2592 Lower explosion limit 3 0 vol Upper explosion limit 15 0 vol 126 EEC SAFETY DATA SHEET e k Gem 91 155 EG 2001 58 EG mn ovate ED innovatek OS GmbH Stand www innovatek de 2 12 2004 info innovatek de 10 Stability and reactivity Substances to avoid Strong oxidizing agents Hazardous reactions No hazardous reactions when stored and handled according to instructions Hazardous decomposition No hazardous decomposition products if stored and handled products as prescribed indicated 11 Toxicological data LD50 oral rat gt 2000 mg kg LD50 dermal rabbit gt 2000 mg kg literature data Primary skin irritation rabbit non irritant Information on Ethylene glycol Further information Developmental toxicity was observed after oral ingestion of high doses in studies with rats and mice but this effect was not seen in a study with rabbits Experiences in humans Lethal dose if swallowed approx 1 5 g kg body weight Lethal dose approx 90 110 g for adults and correspondingly less for children Smaller doses can result in consciousness is affec ted kidney damage damage to the central nervous system Additional information The statements are based on the properties of the individual components There is no reason to fear a risk of damage to the developing embryo or fetus when the MAK value is ad hered to The whole of the information available provides no indication of a carcinoge
58. ion and width of the selected emission band the scanning speed and the diameter of the emission pinhole 90 100 m 488 nm Figure 67 Selecting the illumination intensity 1 via acousto optical tunable filters AOTF 2 and selecting the emission band in the SP detector 3 If you select the FITC parameter set the 488 nm argon line and a suitable band between 490 nm and 550 nm is set The entire beam path is represented graphically on the user interface A spectral band with the settings for the emission bands is located on the emission side The laser line is visible at the appropriate location in the spectrum as soon as a line is activated When viewing the specimen through the microscope the light in the selected color will become lighter or darker according to the position of the slider If the laser scanning microscope is used as prescribed and the safety instructions are observed there are no dangers to the user s eyes Always keep your eyes at a safe distance of at least 20 cm from the opening of the objective Read the safety instructions in this user manual carefully If all of the other settings are in order darker and lighter images will be visible on the monitor when moving the slider for the illumination 10 2 3 Beam Splitting The simplest case would involve the selection of a laser line roughly at the maximum of the excitation spectrum of a given fluorescent dye This would achieve the best
59. l correlation is important for moving objects calling for a slower scan On the other hand triplet phenomena call for longer times between averages i e for the averaging of entire images Averaging lines represents a compromise here The averaging of complete images is the gentlest method but has the disadvantage of not immediately showing the quality of the results this can be evaluated easier with the other methods On the other hand the averaging operation can also be aborted manually when averaging images once the quality impression is adequate Thus there is no general rule of thumb for all application situations Choosing the best method is a matter of experimentation and experience 10 2 8 Profile Cuts So far we have always assumed that images are scanned parallel to the focal plane That is both correct and appropriate for conventional microscopy However a confocal point scanning system offers interesting new options for capturing data For example profile cuts through the specimen can be made by always moving the light spot along the same line and instead of making an incremental y movement moving it between the lines of the focal plane using the fast precise SuperZ galvanometer stage for example This is similar to slicing through a cake permitting impressions to be gained online about the contents of the specimen Camera based systems including confocal systems can only compute such profiles out of complete stacks Figure
60. le Dichroic Double dichroic filters are interference filters at an angle of incidence of light of 45 The transmissivity or reflectivity of double dichroic filters depends on two specific wavelengths of light With a DD 488 568 double dichroic filter for example the excitation light at 488 nm and 568 nm is reflected and above these values it is transmitted The transmission values are generally around 80 and the reflection values are between 90 and 95 Experiment A file with Leica specific data format lei that consists of one ore more individual images or image series Images recorded with different scan parameters or resulting images from image processing can be combined here Fluorescent Dye A dye used for analysis that reacts with the emission of light of other wavelengths upon excitation with light energy Stokes shift e g fluorescein rhodamine eosin DPA 119 Fluorescence Microscopy A light optical contrast process for displaying fluorescent structures Auto fluorescent specimens have what is known as primary fluorescence They do not need to be enriched with additional fluorescent substances Secondary fluorescent substances on the other hand have to be treated with appropriate dyes or dyes called fluorochromes Specific dyeing methods allow the precise localization of the dyed structure elements of an object Fluorescence microscopy provides both the potential for morphological examinations and the ability to carry
61. le national and federal regulations The owner operator is responsible for observing the laser safety regulations 23 24 3 Safety Instructions and their Meanings DANGER This kind of warning alerts you of an operating procedure practice condition or instruction in the operating manual that must be strictly observed and followed as otherwise you expose yourself to the risk of fatal injury WARNING LASER RADIATION A laser warning points out an operation a process a condition or an instruction that must be observed strictly to prevent serious eye injuries to the persons using the system WARNING ELECTRICAL VOLTAGE A high voltage warning points out an operation a process a condition or an instruction that must be observed strictly to prevent possible injury or death of the persons using the system WARNING HARMFUL SUBSTANCES A harmful substances warning points out a substance that can be harmful to your health CAUTION A safety instruction points out an operation a process a condition or an instruction that must be observed strictly to prevent severe damage to the system or loss of data This mandatory sign draws your attention to the fact that suitable eye protection gear must be worn when commissioning and operating the system Failure to heed this warning may lead to serious and irreversible eye injuries WEARING PROTECTIVE EYEWEAR OBSERVE INSTRUCTIONS IN OPERATING MANUAL This mandatory sign draws your
62. ling set the spacing between the scans to around one half to one third of the optical section thickness Practically speaking this is between 0 7 and 0 2 um Therefore between 1 and 5 sections are scanned per micrometer in z largely depending on the aperture of the objective used 10 4 4 Data Volumes Another factor that must be considered when scanning a series is that it may result in very large volumes of data that in some cases may not be suitable for processing or which can only be processed very slowly A normal image with 512 x 512 pixels one channel and a standard 8 bit grayscale resolution weighs in at 0 25MB One hundred such images i e a 20 um thick specimen at high resolution take up some 25 MB which just a couple of years ago was an unwieldy amount of data If 5 channels are scanned and the image format is 1000 x 1000 pixel then this stack already occupies 500 MB thereby almost filling up a regular CD With 16 bit grayscale and 8000 x 8000 pixel the data record measures 64 GB which most of today s computers will not be able to handle easily A critical assessment of the data capture parameters to be used is definitely called for here 107 10 4 5 Depictions 7 0 000 pun a 251 093 jim a 25213 i z 3 207 jim s 254 396 jim z 5455j 1o z 6 594 jim 7 693 im Z 9 0917 um z 10 930 pm z 1 2 004 pm 2 14 188 ppm 514 287 pm ooo 2215 386 pm 2 16 485 pm fi i a A i i E Mp g Fi i A A h ae pa A g ee N
63. mber of lines per image When limiting the scan to a band shaped image of 16 lines a resonant scanner can scan up to 200 images per second 5 ms This standard scan process generally at 512 x 512 pixels will also be used for long term experiments in which the image of the specimen is scanned repeatedly over the course of hours or days for example when recording the development of embryos or cell cultures In these cases mechanical and photonic limitations play a subordinate role however the system must be extremely stable free of drift and climate controlled 10 5 5 Spaces Time Space The three dimensional development of structures in biology is naturally of great interest The broad application field of 4D microscopy has established itself here This is realized by recording a series of z stacks and processing them into 3D movies This is a field in which many innovations and exciting results can be expected in the future 111 10 5 6 FRAP Measurements A completely different field of application for laser scanning microscopy involves dynamic studies in which a system is subjected to interference to disturb its equilibrium and studied as the restoration of its equilibrium progresses The FRAP method fluorescence recovery after photobleaching is very well known in this regard Here a part of the specimen is photobleached using strong illumination in order then to measure the recovered fluorescence from the area Such experiments can
64. me into contact with the objective nosepiece and damage the objectives 1 Switch on the workstation PC switch at the main switch board Figure 42 Switching on the workstation You do not have to start the operating system it starts automatically when you switch on the computer Wait until the boot process is completed 2 Log on to the computer After you simultaneously press the Ctrl Alt and Del keys the logon information dialog box appears 65 Use your personal user ID if one has been set up This ensures that the user specific settings are saved and maintained for this user only If the system 1 administrator has not yet assigned a personal user ID log on as TCS User A password is not required Q After logging on with your own user ID you may change your password by 1 pressing the keys Ctrl Alt and Del at the same time Then click Change password The Change password dialog box opens 3 Check whether the microscope is switched on lf the readiness indicator Figure 43 item 1 on the electronic box is lit the microscope is operating If the readiness indicator is not lit activate the toggle switch Figure 432 of the electronic box Figure 43 Switching on the microscope 66 Switch on the scanner on the main switch board Figure 44 Turning on the scanner Switch on the lasers on the main switch board Figure 45 Switching on the lasers The power supplies and fan of the system ha
65. ms are implemented in optically conjugated locations of the beam path to achieve this They function as point light source excitation diaphragm and point detector detection diaphragm The optical resolution diameter of the detection pinhole the wavelength and the numerical aperture of the selected objective determine the axial range of an optical section optical resolution Short pass Filter Reflection short pass filters are interference filters that transmit short wave light while reflecting long wave light An optical short pass filter is characterized by the reading of the wavelength edge at which the filter changes from transmission to reflection 50 threshold 120 Lambda Series Stack of individual images of a single optical plane that were each detected at a specific wavelength Reflection Long pass Filter Reflection long pass filters are interference filters that reflect short wave light but are transparent for long wave light An optical long pass filter is characterized by the reading of the wavelength edge at which the filter changes from reflection to transmission 50 threshold Empty Magnification A magnification without any additional gain of information The term empty magnification applies whenever distances are displayed that are smaller than the optical resolution Magnifications with a larger scale than that of the empty magnification do not provide any additional information about the specimen rather they onl
66. n not in use the system should be covered with a plastic foil part of delivery or a piece of cotton cloth The system should be operated in a room which is kept as dust and grease free as possible Dust caps should always be placed over the objective nosepiece positions when no objective is in place A You must be particularly careful if your work involves the use of acids lyes or other aggressive chemicals Make sure to keep such substances away from optical or mechanical components Protect the microscope from dust and grease Exercise care in the use of aggressive chemicals 11 2 Cleaning the Optical System The optical system of the microscope must be kept clean at all times Under no circumstances should users touch the optical components with their fingers or anything which may carry dust or grease Remove dust by using a fine dry hair pencil If this method fails use a piece of lint free cloth moistened with distilled water Stubborn dirt can be removed from glass surfaces by means of pure alcohol or chloroform If an objective lens is accidentally contaminated by unsuitable immersion oil or by the specimen please contact your local Leica branch office for advice on which solvents to use for cleaning purposes Take this seriously because some solvents may dissolve the glue which holds the lens in place A The immersion oil should be removed from oil immersion lenses immediately after it is applied Do not
67. n the excitation maximum to the emission maximum It is of course advantageous for good separation and yield if the Stokes shift is very high Typical dyes have a Stokes shift between 10 nm and 30 nm But also in cases where the Stokes shift is above 100 nm for example with natural chlorophyll an excellent dye for curious experimenters is produced The emission characteristics of dyes can be displayed on the spectral band graphic of the user interface It is therefore very easy to choose where an emission band should begin and end If an emission curve has not been stored it is possible to record and save such a curve directly using the system An adjustable bar below the spectral band has been assigned to each confocal detector The limits at the left and right of these bars indicate the limits for the selected emission band 92 Excl Exc2 Figure 69 SP detector setting options for two fluorescences with different excitations above or for fluorescence and reflection with one excitation below It is possible to move the entire bar back and forth to adjust the average frequency or move the limits independently Using the excitation lines and displayed emission characteristics for orientation and adapting the emission band using the Leica SP detection system is thus very convenient This is also possible during live acquisition of images The effects of settings on the images are immediately apparent and suitable values can thus be sel
68. nd dye separation 19 9 2 2 Accessing the Online Help The online help can be accessed in three ways In the respective context context sensitive Via the Help menu With the key combination CTRL F1 In the respective context context sensitive Click the small question mark located in the top right corner of every dialog window Online help opens directly to the description for the corresponding function Via the Help menu Click the Help menu on the menu bar The menu drops down and reveals search related options including the following Contents This dialog field contains the table of contents in the form of a directory tree that can be expanded or collapsed Double click an entry in the table of contents to display the corresponding information Index Enter the term to be searched for The online help displays the keyword that is the closest match to the specified term Select a keyword View the corresponding content pages by double clicking the key word or selecting it and then clicking the Display button Search Enter the term or definition you want to look up and click the LIST TOPICS button A hierarchically structured list of topics is displayed About Opens the User Configuration dialog box where you can for example select the language in which the online help is shown 9 2 3 Full text Search with Logically Connected Search Terms Click the triangle to the right of the input field on the Search tab to vi
69. nd storage Handling Ensure thorough ventilation of stores and work areas Protection against Take precautionary measures against static discharges fire and explosion If exposed to fire keep containers cool by spraying with water Storage Product is hygroscopic Containers should be stored tightly sealed in a dry place Storage in galvanized containers is not recommended 8 Exposure controls and personal protection Components with workplace control parameters 107 21 1 Ethylene glycol MAK value D 26 mg m 3 10 ppm TRGS 900 DE Top limit category 1 There is no reason to fear a risk of damage to the developing embryo when the MAK value is adhered to Skin resorption hazard wear suitable gloves see below Personal protective equipment Respiratory Protection Do not inhale gases vapours aerosols Hand protection Chemical resistant protective gloves EN 374 Recommended nitrile rubber protective index 6 Manufacturers directions for use should be observed because of great diversity of types Eye protection Safety glasses with side shields frame goggles EN 166 General safety and The usual precautions for the handling of chemicals must hygiene measures be observed 9 Physical and chemical properties Form Liquid Colour Colourless Odour Product specific pH value 500 g l 20 C 7 0 9 0 Solidification temperature lt 18 C DIN ISO 3016 Boiling point range gt 165 C ASTM D 1120 Flash po
70. ng the period of observation While mechanical and data bottlenecks can be resolved in principle and great progress has been made in this regard in recent years limitations related to light are a hurdle that cannot be overcome Little light leads to a poor signal to noise ratio and thus to poor resolution and poor image quality It is therefore necessary to verify the parameters that truly require measurement A central difference between various measurements is the dimensionality that attempts to compensate for mechanical limits 10 5 2 Points The highest temporal resolution can be achieved when the mechanical elements of the scanner do not move at all This amounts to measuring the changes in light intensity at a fixed preselected point in the Leica TCS SP5 with a temporal resolution of 40 MHz corresponding to 25 ns Naturally that particular spot in the specimen can be expected to bleach within a very short time 10 5 3 Lines Less fast but nevertheless suitable for many highly dynamic processes is the restriction to images consisting of a single line The data can be displayed as an xt image with one dimension being the location the selected line and time as the second dimension An 8 kHz resonant scanner thus supports a resolution of 16 kHz 63s in bidirectional mode 10 5 4 Planes The standard scenario is the capture of xy images as a t series In this case the temporal resolution depends on the speed of the scanner and the nu
71. nic effect 12 Ecological information Ecotoxicity Toxicity to fish Leuciscus idus LC50 96 h gt 100 mg l Aquatic invertebrates daphnia magna EC5O0 48 h gt 100 mg l Aquatic plants algae EC50 72 h gt 100 mg l Microorganisms Effect on activated sludge Inhibition of de gradation activity in activated sludge is not to be anticipated during correct introduction of low concentrations Persistence and Elimination information Degradability Test method OECD 301A new version Method of analysis DOC reduction Degree of elimination gt 70 Evaluation readily biodegradable Additional information Other ecotoxicological advice Do not release untreated into na tural waters The product has not been tested The statement was derived from the properties of the individual components 13 Disposal considerations innovatekProtect must be dumped or incinerated in accordance with local regulations 127 a EEC SAFETY DATA SHEET Gem 91 155 EG 2001 58 EG inn ovatek ED innovatek OS GmbH Stand www innovatek de 2 12 2004 info innovatek de Contaminated Uncontaminated packs can be reused Packs that cannot packaging be cleaned should be disposed of in the same manner as the contents 14 Transport information Not classified as hazardous under transport regulations ADR RID ADNR IMDG GGVSee ICAO IATA 15 Regulatory information Regulations of the European union Labelling National legislation Regulations Direc
72. nt are not altered as the laser intensity and gain at the PMT naturally affect these coefficients In the spectral dye separation method the emission spectra of the individual dyes known from literature or determined by measurements directly at the instrument are used to calculate the relative intensity of the dyes This method is especially suited for situations in which the dyes do not significantly change their emission in situ and in which the related data is well known 10 4 3D Series Altering the position of the focus between two scans permits a whole series of optical sections to be captured that represent the structure in a 3D data record Naturally such a three dimensional image cannot be observed directly but it contains spatial information related to the observed structures and in the case of multiple dyes their local connections 10 4 1 Z stack To capture such a 3D series z stack set the upper and lower limits simply by moving to the top of the specimen marking the location and then moving to the bottom and marking it Next determine the number of sections to be scanned between the two positions the rest will be handled automatically by the instrument 10 4 2 Section Thicknesses As described in sections 10 1 4 and 10 2 5 the thickness of the optical section depends on the wavelength the numerical aperture of the objective and of course the diameter of the pinhole The relationship of these parameters is e
73. occcoccccccncconicocnconcnononcns 18 Rermitted Ambient GONGITIONS isis data liada iia 18 Waste Heat Required Cooling Performance occcoccccccncccnccccnconncnncncnnnnnnnnnnnnss 19 A A saa oie ace AE 19 Overview of Usable VIS UV Lasers ooncccnnccocncccnicocncocnoconnnoonconcnonanonanonnons 19 Overview of Usable MP Lasers IR Lasers occoocccconnccccccccncconicocnconnnnanonnnos 20 PICOSCCONC ASEM o a ab 20 Femtosecona laser ni ti ti 21 Overview of Usable VIS UV Lasers for TCS SPD Xicooocccnccccocnccocococnconcnnonnnnons 22 Which Laser Class Does the Product Have coocccccccccccccocncoccccncncocnnoncnnnanos 23 Required Laser Safety Measures oocccoccccocncocccoccnconnoconononnnnnnnonnnconnncnnnnnnnnnos 23 Safety Instructions and their Meanings ccocccocccccnconccnonnccncnnonanonnncnannonanonanos 25 4 General Safety Instructions ccsscssecesccenscnnscnnscneccneccnecenscenscesecesseessneeaas 27 4 1 Laser Class for VIS and UV SyStemS ccooccccccccocnoconococncconncncnonononannnnanonannnnnnos 27 4 2 L ser Class Tor MP Systems eakmiocintdie ua aN 27 4 3 What does the owner operator have to observe sscssssissresrerrererrerrrrerrrrenns 27 4 4 Safety Instructions for the USEF cui 29 4 5 Operational Reabren E EN 29 4 6 Maximum Current Load of the Multiple Socket Outlet at the Supply Unit 30 5 Safety DEVICES isis 31 5 1 Disconnecting the Power SUDpply ccccscccceeceeee
74. ocncocnconocononos 64 Starting Up the System ii ee ee eee 65 SWITCHING OM ING gt Si A etree eek siglo lel hetetes 65 Staning dne LAS AR td iaa 70 Seta UD US eS a a tie 72 Switching OM CIS Sy SOM ais 73 introduction to LAS AF ii 75 cn e o E EE 75 One He Dusan 75 Stucture othe Ohne Heladio 75 Accessing the Online Help occccocccccnccccccccnoconococnnconnnnononanonannnnannonnnonancnanonos 76 Full text Search with Logically Connected Search TerMS occcoccccccccccccnconncoo 76 Structure of the graphical user interface oocococccccncconccccnoconnnocnnnanonanncnanonons 78 General Structure of the Graphical User Interface c ooccoonconiconionionnconono 78 KEY COMDINAIO Sii ab bn acid 79 Introduction to Confocal Work oocooocconncconnccccconnncccnnoncnonanccnnnnonanonanenanrnannnnnnnnnn 81 Prepara lO Musas 81 ME OD ec ii 82 Conventional MIGhOSGODY sarie tio a 82 WAY OCAR oO OR PP a a esses 85 How Is an Optical Section Created ooccooccconcccncccncocnnocnnornnonncncnonnnonnnonnnons 86 Acquiring Optical Sections ccccscessssscssecusecurecusscesscesncerscassasenereperseerscersusenes 88 Data ACUSE ie do E 88 A r a E antes 90 Beam A do e 91 EMISSION BA OS usura a ideada 92 10 2 5 10 2 6 10 2 7 10 2 8 10 3 10 3 1 10 3 2 10 3 3 10 3 4 10 3 5 10 3 6 10 4 10 4 1 10 4 2 10 4 3 10 4 4 10 4 5 10 4 5 1 10 4 5 2 10 4 5 3 10 4 5 4 10 5 10 5 1 10 5 2 10 5 3 10 5 4 10 5
75. ojections from slightly different angles can be displayed next to one another and superimposed by unaided fusion or squinting We then mentally generate a three dimensional image in the same way as we would of any other object viewed with both eyes Figure 81 If only one channel is used it is possible to display both views in different colors and view them through spectacles containing filters for those specific colors red green anaglyph This is simpler for most users but cannot be applied to multiparameter data Like the sections themselves series of projections can be observed with increasing angles and presented as movies 3D movies of this type are today the most common and convincing means of displaying three dimensional data 10 5 Time Series A confocal scanning microscope records images like a camera It can therefore also be used to record a time series essentially a z stack without altering z Such time lapse experiments are an important tool in physiology and developmental biology whenever interest is focused on dynamic processes 10 5 1 Scan Speed Temporal resolution is an important parameter in dynamic processes especially those related to kinetic studies of cellular biophysical processes Unfortunately restrictions are 110 imposed here by a number of factors such as the mechanical speed of the scanner the bandwidth of the data line and the simple volume of photons that can be expected from the specimen duri
76. ol has various tabs in which the settings for the experiment can be configured Experiments Directory tree of opened files dl Setup Hardware settings for the current experiment Acquisition Parameter settings for the scan acquisition Experiments Directory tree of opened files Tools Directory tree with all the functions available in the respective a oe step Quantify Experiments Experiments Directory tree of opened files tree Experiments Directory tree of opened files opened files A Tools Tab with the functions available in this operating step E Graphs Graphical display of values measured in regions of interest ROI Statistics Display of statistical values that were determined in the plotted regions of interest ROI 4 Working area This area provides the Beam Path Settings dialog window in which the control elements for setting the scanning parameters are located 5 Viewer display window Displays the scanned images In the standard setting the Viewer display window consists of the image window in the center and the buttons for image editing 5a and channel display 5b 9 4 Key Combinations To speed up recurring software functions special key combinations have been defined CTRL N Opens a new experiment CTRL O Starts the Open dialog window for opening an existing file 79 80 10 Introduction to Confocal Work 10 1 Preparation The following sections describe a number of basic procedures th
77. on the type of additional correction for chromatic aberration Prechirp lasers Prechirp lasers are MP lasers with dispersion compensation that compensates for the pulse distribution by optical components ROI Abbreviation for Region of Interest A ROI delimits an area for which a measurement analysis is to be performed On top of that an ROI can also designate the area of a specimen to be scanned ROI scan Signal to noise Ratio The ratio of signals detected in the specimen to the unwanted signals that are caused randomly by various optic and electronic components which are also recorded by the detector Spherical Aberration An optical image aberration conditional on the varying distance of paraxial light rays of the same wavelength from the optical axis Light rays that travel through outer lens zones have shorter focal lengths than rays that travel through the lens center optical axis Stokes Shift The Stokes shift is a central term in fluorescence microscopy If fluorescent molecules are excited with light of a specific wavelength they radiate light of another larger wavelength This difference between excitation light and fluorescent light is referred to as Stokes shift Without Stokes shift separating the high intensity excitation light from the low intensity fluorescence signals in a fluorescence microscope would not be possible Triple Dichroic Triple dichroic filters are interference filters at an angle of incidence of
78. open objectives for cleaning 115 First remove the immersion oil using a clean cloth Once most of the oil has been removed with a clean tissue a piece of lens tissue should be placed over the immersion end of the lens Apply a drop of the recommended solvent and gently draw the tissue across the lens surface Repeat this procedure until the lens is completely clean Use a clean piece of lens tissue each time 11 3 Cleaning the Microscope Surface Use a lint free linen or leather cloth moistened with alcohol to clean the surfaces of the microscope housing or the scanner varnished parts A All LEICA components and systems are carefully manufactured using the latest production methods If you encounter problems in spite of our efforts do not try to fix the devices or the accessories yourself but contact your Leica representative A This is necessary to remove any existing contamination so as to prevent the risk of putting others in danger In addition to surfaces pay particular attention to fans and cooling devices as dust is particularly likely to accumulate at these locations Never use acetone xylene or nitro thinners as they attack the varnish Whenever the confocal system is moved it must first be thoroughly cleaned This applies in particular to systems that are located in biomedical research labs 114 Maintaining the Scanner Cooling System The scanner of the system is liquid cooled Observe the safety dat
79. orescence and reflection images can also be rendered at the same time Using another excitation this merely requires observing a second emission band below the laser line Under normal circumstances however dyes will be used that require different excitation wavelengths A variety of lasers are usually installed in the instrument for this purpose To activate a second excitation line simply set the desired slider for the second wavelength as described in 10 2 2 for simple excitation Additional excitation wavelengths can be added just as easily It is frequently helpful for the bleaching experiments described below to activate multiple Ar lines even if you are not capturing a signal or are using only one channel This provides additional intensity Experimenting a bit with laser combinations is always beneficial It frequently becomes apparent that one does not need all of the lines initially selected for the dyes or a different line turns out to be a better compromise Default configurations for illumination beam splitting and emission band settings can be selected from a list for most typical dye combinations 103 10 3 2 Beam Splitting Beam splitting is very easy to describe in AOBS systems there is no need to give it any thought The AOBS automatically switches a narrow band for the selected lines to ensure that the excitation is applied to the specimen Such bands have a width of around 2 nm Everything else is available to capture
80. orescence signal is emitted If the intensity of the excitation is too high however the color molecules can change via intercrossing from a singlet state to a triplet state Due to the significantly longer life of triplet states phosphorescence these excited molecules can react with triplet oxide and be lost for further fluorescence excitation 121 Phase Visualization The principle of phase visualization as used by Leica is an optimized alternative method to ratiometric displays The main area of application is measuring ion concentrations in physiology In contrast with ratiometric procedures phase visualization obtains more information on the specimen In addition this method allows for adapting the display of physiological data to the dynamics of the human eye For detailed information on phase visualization please contact Leica Microsystems CMS GmbH directly Pixel An acronym based on the words picture and element A pixel represents the smallest indivisible image element in a two dimensional system In this documentation both the sampling points of the specimen and the image points are referred to as pixels Flat field Objective Describes a correction class for objectives The image curvature aberration is corrected for objectives of this type Correcting this error requires lenses with stronger concave surfaces and thicker middles Three types of plane objectives planachromatic planapochromatic and plan fluorite are based
81. ould be infinitely small but this would no longer allow light to pass making it impossible to create an image However the effect would be lost if the pinhole were too wide as the image would contain excessive blurred portions of the specimen from above and below the focal plane 93 The relationship of the thickness of the optical section to the diameter of the pinhole is linear for large diameters and approaches a limit value at smaller diameters being roughly constant near zero Figure 70 The limit value is dependent on the wavelength of the light and the numerical aperture As the section thickness changes little when initially opening the pinhole but the passing light increases in proportion to the square of the pinhole diameter it is advisable not to use too small a diameter Figure 70 Relation of optical section thickness y axis to pinhole diameter x axis A good compromise is the point where the diffraction limitation constant dependence transitions to geometric limitation linear dependence When depicted in the specimen plane at this point the pinhole has roughly the size of the diffraction limited light disk of a focused beam This is known as the Airy diameter The Airy diameter can easily be calculated from the aperture and wavelength Setting the pinhole to roughly the size of the diffraction limited spot thus results in sharp optical sections with a good signal to noise ratio S N Figure 71 Naturally
82. out dynamic examinations on a molecular level Fluorite Objectives Describes a correction class for objectives Fluorite objectives are semi apochromatic i e objectives whose degree of correction falls between achromatic and apochromatic Frame A frame corresponds to the scan of a single optical section For example if a single optical section is acquired four times to average the data and to eliminate noise then frames are created for this optical section Immersion Objective A microscopic objective developed with the requirements for applying immersion media The use of incorrect or no immersion medium with an immersion objective can lead to resolution loss and impairment of the correction IR Laser Laser with a wavelength gt 700 nm invisible laser radiation infrared Confocal Microscopy Techniques Methods for examining microstructures that are derived from the classical contrast methods bright field interference contrast phase contrast polarization in conjunction with a confocal system These procedures each define a certain configuration of optical elements filter cubes ICT prisms phase rings In addition some of them are dependent upon the selected objective Confocality While the optical design of conventional microscopes allows the uniform detection of focused and unfocused image components the confocal principle suppresses the structures found outside of the focal plane of the microscope objective Diaphrag
83. pped with a mirror housing note the following e Ifthe mirror housing is removed you must the close off the port on the microscope Figure 36 using the cover Figure 37 e The interlock jack on the mirror housing see Figure 38 item 1 must be connected to the scan head at all times e The unused output on the mirror housing must be covered with the cover provided see Figure 38 item 3 60 When installing the cover Figure 38 item 3 ensure that the button Figure 38 item 2 is pressed by the cover Figure 38 Mirror housing on upright microscope 61 6 9 Changing Filter Cubes Beam Splitters or Condenser A Do not change any filter cubes or beam splitters during a scanning operation In LAS AF set the operating voltage of all external detectors to O V and disable MN them using the checkbox lf the detectors are not de energized they could be damaged by the infiltration of ambient light To change filter cubes or beam splitters proceed as follows Upright microscope Inverted microscope Finish the scanning operation Finish the scanning operation In LAS AF set the operating voltage of all In LAS AF set the operating voltage of all external detectors to 0 V external detectors to 0 V Remove the cover of the fluorescence pa Pull out the fluorescence module see operating manual for microscope Remove the filter cube beam splitter Remove the filter cube beam splitter Insert the desired Inse
84. rience No assurances concerning the characteristics of our product are hereby furnished 128 15 2 Declaration of conformity Leica MICROSYSTEMS EC Declaration of Conformity Manufacturer Leica Microsystems CMS GmbH Address Am Friedensplatz 3 Germany 68165 Mannheim Product TCS SP5 Confocal Laser Scanning Microscope TCS SP5X Confocal Laser Scanning Microscope We declare that the product described herein complies with the following European Directives 2004 108 EG Directive on Electromagnetic compatibility 2006 95 EG Directive on Low voltage equipment The product conforms to the standards EN 61326 1 2006 Electrical equipment for measurement control and laboratory use EMC requirements Part 1 General requirements Emission Class A EN 61010 1 2001 Safety requirements for electrical equipment for measurement control and laboratory use Part 1 General requirements EN 60825 1 1994 A2 2001 A1 2002 Safety of laser products Part 1 Equipment classification requirements and users guide Manager Research amp Development Mannheim Germany hala MOS p Lx Alas February 04 2008 Dr Lucius Remer 129 15 3 People s Republic of China Administrative Measures on the Control of Pollution Caused by Electronic Information Products AS SR tmk Hazardous substances HZ EF Name of the part ta AK fa aai SAREE LR oR b Ge Cd Q PBB PBLE pp OGOLEIER printed circuit boards HF cae
85. rt the desired filter cube beam splitter filter cube beam splitter Reattach the cover to the front of the Reinsert the fluorescence module fluorescence module Never disconnect a fiber optic cable Never remove the scanner from the microscope during operation Before removing the scanner the system must be completely switched off Do not use an S70 microscope condenser The large working distance and the low numerical aperture of the S70 microscope condenser could pose a hazard due to laser radiation Therefore only S1 and S28 Leica microscope condensers should be used 62 6 10 Piezo focus on upright microscope Figure 39 Piezo focus on objective nosepiece If a piezo focus is installed on your system please also observe the following safety notes Before switching the system on or launching the LAS AF software ensure that there is no slide or specimen on the stage and that the stage is in its lowest MN possible position The slide or objective may otherwise be damaged or destroyed by the initialization of the piezo focus when starting the system software The objective can be moved by 150 um in either direction The total travel is 300 um Piezo focus controller display Upper position 350 um Middle position 200 um Lowest position 50 um XZ scan range 250 um Figure 40 Piezo focus controller Do not make any adjustments to the piezo focus controller as it has already been optimally set up by Le
86. s in 6 10 1 57 6 7 Changing the Transmitted Light Lamp Housing If no transmitted light lamp housing is connected to protect from the potential escape of laser radiation the opening Figure 35 or Figure 36 must be securely sealed with the cover Figure 34 that accompanies the system Figure 34 Cover A To prevent the emission of laser radiation do not switch the lasers on without a lamp housing or cover on the microscope Figure 35 Port for connecting the transmitted light lamp housing on the inverted microscope 98 Figure 36 Port for connecting the transmitted light lamp housing or mirror housing on the upright microscope If your microscope features a transmitted light lamp housing that you would like to replace proceed as follows 1 2 3 4 5 Switch off the lasers Disconnect the lamp housing from the power supply Remove the lamp housing Modify the lamp housing as needed After finishing the tasks screw the new lamp housing back onto the microscope 99 6 8 Mirror housing on upright microscope If a mirror housing is not connected to the upright microscope the opening must be tightly covered using the cap provided with the system to prevent any laser radiation from escaping Figure 37 Figure 37 Cover To prevent the emission of laser radiation do not switch the lasers on without a mirror housing or cover on the microscope A If your upright microscope is equi
87. scription of software functions was intentionally omitted from this operating manual Instead reference is made to the online help of the LAS AF in which you can find up to date explanations and instructions for the corresponding software functions Please read the chapter Introduction to the LAS AF in this operating manual to familiarize yourself with its setup and operation Additional information about specific functions can then be found in the online help 10 2 Leica TCS SP5 TCS SP5 X 2 1 TCS SP5 system overview Figure 1 TCS SP5 system components overview TCS SP5 Scanner Main switch board TCS workstation Supply unit Control panel Microscope On1RWN 11 2 2 TCS SP5 X system overview Figure 2 TCS SP5 X system components overview 1 2 3 4 5 6 T 2 3 Intended Use TCS SP5 X scanner Main switch board TCS workstation Supply unit Control panel Microscope Achromatic light laser The system was designed for confocal scanning laser scanning images of fluorescence marked living and fixed specimens as well as for quantitative measurements in the area of life science This system is intended for use in a lab Applications of in vitro diagnostics in accordance with MPG German Medical Devices Act are excluded from proper intended use 12 The manufacturer assumes no liability for damage caused by or any risks arising from use of the microscopes for purposes other than those for which th
88. sides but with very low amplitude The red side in particular can be a problem Crosstalk or bleed through refers to the fact that the emission of a dye not only contributes to the signal in one channel but in other detection channels as well This should of course be avoided as it leads to the display of incorrect images and falsifies the determination of correlations The reliability of separation and thus the avoidance of crosstalk is therefore an important issue Several parameters can be considered for this purpose illumination intensity laser selection sequential capture emission bands and unmixing methods Initially we will be covering illumination and emission parameters Crosstalk is frequently caused by strong differences in the concentration of the fluorochromes used Even illumination will then result in a very good signal from the more highly concentrated dye yet it is very likely that the signal will also bleed into other channels This can be compensated by setting the various laser intensities in such a manner that dyes with weak concentrations are excited with higher intensities while the higher concentrations receive less intense excitation Balancing in this manner already eliminates a significant crosstalk problem Thanks to the continuously adjustable intensity via AOTF the results can be monitored directly on the display and can thus be adjusted online with suitable feedback It may be useful to try a variety o
89. t Microscope It is not necessary to wear eye protection If the device is used as prescribed and the safety instructions are observed the limit of the laser radiation is maintained so that eyes are not endangered 54 6 4 Specimen Area The light of all employed VIS lasers used wavelength range 400 700 nm visible spectrum and UV lasers wavelength range lt 400 nm invisible is fed through a fiber optic cable and therefore completely shielded until it leaves the microscope objective and reaches the specimen The beam divergence depending on the objective used is up to 1 16 rad Figure 33 Specimen area of upright and inverted microscope During the scanning operation the laser radiation is accessible after exiting the objective in the specimen area of the laser scanning microscope This circumstance demands special attention and caution If the laser radiation comes in contact with the eyes it may cause serious eye injuries For this reason A special caution is absolutely necessary as soon as one or more of the laser emission warning indicators are lit If the system is used as prescribed and the safety instructions are observed during operation there are no dangers to the operator Always keep your eyes at a safe distance of at least 20 cm from the opening of the objective 99 6 5 Changing Specimens A Never change specimens during a scanning operation To change specimens proceed as follows
90. tential hazards of laser radiation measures according to IEC EN 60825 1 and the corresponding national The owner operator is responsible for performing and monitoring suitable safety A regulations 21 All safety devices safety locks and safety systems of the laser product must be in an operational state A Deactivating or damaging these safety devices or any intervention in any of these safety devices may lead to serious eye injuries physical injuries or property damage In these cases Leica Microsystems CMS GmbH shall not assume any liability The owner operator is responsible for naming a laser safety officer or a laser A protection advisor according to the standard IEC EN 60825 1 Safety of laser products Part 1 Classification of systems requirements and user guidelines and the respective national regulations Repairs and servicing may only be performed by authorized Leica Microsystems CMS GmbH service personnel The owner operator is fully liable for all consequences resulting from the use of the system if it is opened improperly serviced or repaired by persons other than authorized Leica service representatives If repairs or service measures are performed that require opening parts of the housing only trained Leica service technicians may occupy the room in which the system is located Do not connect any external equipment or other components Connect to the product only those electrical dev
91. ternal UV laser 34 Immediately disconnect the system from the power supply if any of the following occur e The emission warning indicator is not lit after being switched on using the detachable key switch A e The indicator continues to be lit after being switched off using the keyswitch e Scanning of the specimen is not activated after being switched on properly laser radiation in the specimen area Contact Leica Service immediately For other external lasers please refer to the operating manual supplied by the laser manufacturer for the position of the emission warning indicator 5 4 Remote Interlock Connection on the Supply Unit The remote interlock jack is located on the rear side of the supply unit 12 V DC operating voltage Figure 16 The remote interlock plug which contains a shorting bridge is connected to this jack Remote interlock devices such as those connected to the room the door or other onsite safety interlock systems can also be connected to the remote interlock connector The laser beam path is interrupted if the contact is open The overall length of the cable between the two connecting pins of the remote interlock connector must not exceed 10 m Figure 16 Remote Interlock Connection on the Supply Unit 35 5 5 Remote interlock connection on the achromatic light laser The remote interlock connection is located on the rear side of the achromatic light laser 12 V DC operating voltage
92. the emission A suitable beam splitter must be selected when using instruments with traditional beam splitters In this regard it is important to know that not only single but also double and triple beam splitters are available DD and TD for double dichroic and triple dichroic Lines in close proximity to one another cannot be served with dichroic splitters For example no usable splitters are available for the simultaneous use of 594 nm and 633 nm HeNe lines In these cases an AOBS is a significant advantage thanks to the very small bands approx 1 to 2 nm both lines can be used for excitation while capturing an emission band of 35 nm in between with the SP detector 10 3 3 Emission Bands Naturally the same boundary conditions apply for the emission bands as described in 10 2 4 with the difference that two laser lines limit the band for all dyes except the reddest and that precautions must be taken to ensure that the excitation light does not reach the detector In addition the suppression of crosstalk can have a strong effect on the choice of band limits The following section will cover this in greater detail Setting the bands is described in Section 10 2 10 3 4 Crosstalk The emission spectra of dyes including those that are responsible for autofluorescence typically have a rather simple characteristic with a maximum emission and a blue flank that drops more steeply than the red side The emission extends quite far on both
93. the instrument can calculate and set this value automatically The objective used is known to fully automatic instruments and can be set when working with manual systems The excitation lines used are also known to the system 94 Figure 71 Optical sections with a variety of pinhole diameters 63x 1 4 objective Pinhole diameters from top left to bottom right 4 AE 2 AE 1 AE 0 5 AE 0 25 AE The strong loss of light can be seen clearly with the small diameters as can the pronounced background in the images with very large diameters A pinhole diameter of 1 Airy is therefore the default setting Switching objectives also automatically adjusts the diameter of the pinhole accordingly A larger pinhole may be selected simply by adjusting a slider on the user interface for specimens with weak fluorescence or high sensitivity against exposure to light Of course smaller pinhole diameters may also be selected for very bright specimens With reflecting specimens in particular the pinhole may be reduced to 0 2 or even 0 1 Airy units AU for the thinnest possible sections 10 2 6 Image Detail and Raster Settings Depending on the objective used conventional microscopes show a circular cutout of the specimen The diameter of the circle multiplied by the magnification of the objective is the field number FOV The field number is therefore a microscope value which is independent of the objective and which by reversing the operation can be
94. ting and maintenance instructions provided herein The failure to observe these instructions shall be exclusively at the operator s own risk and may void the warranty 4 5 Operational Reliability This instrument must not be used together with life support systems such as those found in intensive care wards This instrument may only be used with a grounded AC power supply A Contact with liquids or the entry of liquids into the housing must be avoided A 29 4 6 Maximum Current Load of the Multiple Socket Outlet at the Supply Unit The total power consumption of all loads connected to the multiple socket outlet Figure 8 must not exceed 800 VA The terminals are intended for e TCS workstation e Monitor 1 e Monitor 2 e Microscope Figure 8 Multiple socket outlet rear side of supply unit 30 5 Safety Devices 5 1 Disconnecting the Power Supply The main circuit breaker is located on the right rear side of the supply unit It is used to de energize the complete system using a single switch Figure 9 The main circuit breaker functions as a switch and as an overcurrent fuse The main circuit breaker is not to be used as the regular on off switch for the system The supply unit must be set up so that the main circuit breaker is freely accessible at all times Figure 9 Supply unit with main circuit breaker 31 5 2 Detachable key Switch The detachable key switch for protection against
95. tion is that the number of channels used is at least the same as the number of dyes The shares can then be correctly redistributed with the simple methods of linear equation systems The actual objective for effective unmixing is to determine the required coefficients of the matrix This is also covered by a variety of methods available in the Leica software It is advisable to experiment a bit to determine the best method for the task at hand Since all 105 measurement data contains certain error and noise components there is no perfect recipe for the ultimate truth The simplest approach for the user is to determine the coefficients on the basis of the Statistical data of the scanned images In this process the coefficients of the scatter diagrams of both channels are determined using statistical methods Hard and soft separation methods are available leaving the degree of separation at the user s discretion If the coefficients are known from other experiments the data can be entered into a matrix manually This method is also suitable for trial and error work when manually compensating for background interference or autofluorescence for example The method that delivers the most accurate results is channel dye separation In it the distribution of dyes in the various channels is determined directly using individual dye reference data When using this method it is important to ensure that the parameter settings of the instrume
96. tive 1999 45 EEC Preparation Directive Hazard Symbol Xn Harmful R phrases 22 Harmful if swallowed S phrases Keep out of reach of children 24 25 Avoid contact with skin and eyes 46 If swallowed seek medical advice immediately and show this container or label Hazard determinant component for labelling Ethanediol Other regulations none 16 Further Information Full text of hazard symbols and R phrases if mentioned as hazardous components in chapter 2 Xn Harmful 22 Harmful if swallowed 63 Possible risk of harm to the unborn child Vertical lines in the left hand margin indicate an amendment from the previous version This safety data sheet is intended to provide information and recommendations as to 1 how to handle chemical substances and preparations in accordance with the essential require ments of safety precautions and physical toxicological and ecological data 2 how to handle store use and transport them safely No liability for damage occured in connection with the use of this information or with the use application adaption or processing of the products here described will be accepted An ex ception will be made in the case that our legal representatives should come to be held re sponsible and liable by reason of intent or gross negligence No liability will be accepted for damage indirectly incured We provide this information and data according to our present level of knowledge and expe
97. ts reserved Reproduction adaptation or translation of these programs is prohibited without prior written permission from Leica Microsystems CMS GmbH The term Windows may be used in the following text without further identification It is a registered trademark of the Microsoft Corporation Otherwise no inference with regard to the free usability of product names may be drawn from the use of those names All other brand names and product names in this document are brands service marks trademarks or registered trademarks of the respective manufacturers Made in Germany Copyright Leica Microsystems CMS GmbH All rights reserved 1 2 About this Operating Manual Whenever this manual refers to the system or provides no specific information for one of the systems then the notes instructions and information applies to TCS SP5 and TCS SP5 X The main focus of this operating manual is directed to the safety notes that must be strictly adhered to when working with the Leica TCS SP5 and Leica TCS SP5 X In addition this operating manual provides a rough overview of the operating principle of laser scanning microscopes It presents you with the first steps for activating and commissioning the system and provides important information about the Leica Application Suite Advanced Fluorescence LAS AF software The system is delivered with the latest version of the licensed LAS AF To maintain information on the most current level the de
98. ure 20 item 2 The existing beam stop Figure 20 item 2 must always be reinstalled Please consult the microscope s operating manual provided When using a condenser base with filter holder always make sure that unused filter holders are swung out of the beam path and that the laser protection tube covers the beam path When equipping multiple filter holders with filters do so from bottom to top so that the laser protection tube can cover the beam path to the greatest possible extent Do not swing in the filters during the scanning operation 39 5 9 2 Shielding in MP Systems IR Lasers The light of all employed VIS lasers wavelength range 400 700 nm visible spectrum and UV lasers wavelength range lt 400 nm invisible is fed through a fiber optic cable and therefore completely shielded until it leaves the microscope objective and reaches the specimen For systems with infrared laser wavelength range gt 700 nm the beam is passed through a safety beam guide and if necessary also passed through a fiber optic cable Figure 21 This shields the laser beam until it leaves the microscope objective and reaches the specimen Figure 21 Safety beam guide 1 and IR laser 2 40 5 10 Safety labels on the system The corresponding safety labels are selected dependent on the laser configuration VIS UV MP and attached in the following locations either in the English or German language 5 10 1 Inverted microscope
99. ve been started 67 The power supply of the achromatic light laser is started if the main power switch on the rear side of the achromatic light laser is set to On 6 To switch on the lasers in the supply unit activate the detachable key switch on the main switch board see Figure 46 Figure 46 Activating the detachable key switch 7 To switch on the achromatic light laser activate the detachable key switch at the front of the achromatic light laser see Figure 47 Figure 47 Detachable key switch for the achromatic light laser From this time on laser radiation may be present in the specimen area of the laser A scanning microscope Follow the safety instructions provided in Chapter 6 Safety Instructions for Operating the System Applies only to the TCS SP5 X system 68 If the room temperature exceeds 40 C the white light laser switches off An error report appears in the display of the white light laser The white light laser cannot be switched on again until the room cools off 8 To switch on the external UV laser activate the key switch on the front of the power supply see Shocks to the white light laser can cause an error message in the display of the white light laser Switch the white light laser off then on again after 10 seconds Figure 48 Key switch for the external UV laser From this time on laser radiation may be present in the specimen area of the laser A
100. view The edge length of the displayed field of a conventional scanner corresponds to 15 mm without magnification by the objective 1x scale Field numbers of 21 2 and 22 respectively are thus also fully utilized Figure 72 That is suitable for most good research microscopes How many elements are now actually resolved optically in this dimension That depends on the numerical aperture of the objective and the wavelength According to Ernst Abbe s formula two points can still be distinguished if the distance between them is not smaller than d e 2 NA A line can thus contain a maximum of 15 mm d resolution elements also known as resels When using an actual objective such as a plane apochromat 10x 0 4 the edge length corresponds to 1 5 mm 15 mm 10 and d 0 625 um when using blue green light with a wavelength of 500 nm Such an image would thus contain 1500 0 625 2400 optical resolution elements along each edge x and y direction Rendering this resolution in a digital pixel image would require working with twice the resolution to prevent losses Nyquist theorem That would be an image with 4800 x 4800 pixels Some purists require 3x oversampling i e 7200 x 7200 pixels or 52 megapixels Image formats for x and y can be adjusted independently and in very fine steps with the Leica TCS SP5 supporting image capture sizes of up to 64 megapixels 8000 x 8000 pixels Table 5 Magnification 63 ft 96 Optical Resolution 400 nm
101. xpressed by the formula described there The aperture should be as high as possible to obtain truly good thin sections Confocal microscopes use objectives with large apertures for this reason The wavelength of the emission will generally be between 450 nm and 600 nm so 500 nm would be a suitable value for a rough estimate Choosing the pinhole diameter 1 Airy will result in section 106 thicknesses between 0 5 um and 2 5 um for apertures from 0 7 to 1 4 These are typical values in practice In product documentation especially in advertising materials the thickness is often stated for sections in reflection at pinhole diameter zero Although this value is much smaller and thus looks better it is not relevant for practical applications in fluorescence microscopy 10 4 3 Distances The thickness of the optical sections is important when capturing z stacks If the spacing between the scans is too large greater than the thickness of the section this will result in gaps in the data record and a loss of information A reconstruction then can no longer be calculated correctly On the other hand there is little point in taking as many sections as possible as a very tight spacing will result in reduced differences between the individual sections and an unnecessarily high data volume This relates to the z axis in the same way as empty magnification in a conventional microscope For a dense scan result with neither gaps nor superfluous oversamp
102. y Achromatic Describes a correction class for objectives The chromatic aberration for two wavelengths is corrected for objectives of this type Usually an objective of this type is corrected to a wavelength below 500 nm and above 600 nm Furthermore the sine condition for one wavelength is met The curvature of image field is not corrected Airy Disc The Airy disc refers to the inner light circle Surrounded by alternating dark and light diffraction rings of the diffraction image of a point light source The diffraction discs of two adjacent object points overlap partially or completely thus limiting the spatial resolution Capacity Aliasing An image aberration caused by a sampling frequency that is too low in relation to the signal frequency AOTF The acousto optical tunable filter is an optic transparent crystal that can be used to infinitely vary the intensity and wavelength of radiated light The crystal generates an internal ultrasonic wave field the wavelength of which can be configured to any value Radiated light is diffracted perpendicular to the ultrasonic wave field as through a grid Apochromatic Describes a correction class for objectives The chromatic aberration for three wavelengths is corrected for objectives of this type usually 450 nm 550 nm and 650 nm and the sine condition for at least two colors is met The curvature of image field is not corrected Working Distance The distance from the front lens of an o
103. y diminish the focus and the contrast MP Laser Multi photon the designation for infrared IR lasers with a high photon density generated by pulsed lasers Neutral Density Filter Neutral density filters are semi reflective glass plates They are used to distribute the light path independent of wavelength The incident light is partially reflected and partially transmitted Neutral density filters are usually placed at angle of less than 45 in the beam path The ratings of a neutral density filter are based on its reflectivity to transmissivity ratio For example for a neutral density filter RT 30 70 30 of the excitation light is reflected and 70 is transmitted Numerical Aperture Aperture is the sine of the aperture angle under which light enters the front lens of a microscope objective Symbol NA In addition to the luminous intensity the aperture also affects the resolution capacity of the objective optics Since different media can be located between specimen and objective e g the embedding medium of the specimen the numerical aperture NA n sin is generally used as the unit of measure for the luminous intensity and the resolution capacity Optical Bleaching The destruction of fluorescent dyes known as fluorochromes by intense lighting In fluorescence microscopy fluorochromes are excited with laser light to a high state of energy the singlet state When the excited molecules return to their normal energy state a flu

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