PALM MicroBeam User Manual
Contents
1. 16 For the correct settings of the power supply and the controller for the filter please consult the PALM RoboSoftware Manual For detailed information about operating the microscope with fluorescence illumination please see section 7 page 26 Depending on specific customer requirements P A L M offers two different possibilities of fluo rescence attachment Basic Fluorescence Attachment for PALM MicroBeam with Zeiss Axiovert 200 200M Simultaneous fluorescence observation and laser functions are not possible resp wanted Depend ing on the type of microscope the handling of the filter revolver for filter change and cutting position is done manually Fluorescence Hardware components HBO lamp or X Cite PALM FluorescenceShutter standard Any Basic Fluorescence Filter Set The basic fluorescence attachment is suitable for fixed samples only Advanced Fluorescence Attachment for PALM MicroBeam and PALM CombiSystem with Zeiss Axiovert 200 200M Simultaneous laser functions and fluorescence illu mination are provided Fluorescence Hardware components HBO lamp or X Cite Motorized filter wheel Selected Advanced Fluorescence Filter Sets Depending on the type of microscope motorized or manually shutter and filter wheel there is a different adjustment necessary Notice If you use a Zeiss ApoTome Advanced Fluores cence is not possible PALM MicroBeam User Manual 11052. The ablation occurs within the area of the laser focus please see figure 16a The smallest avail able cutting size can only be achieved within the center of the laser focus using as little energy as possible The catapulting is performed by placing the laser focus underneath the specimen to be catapulted please see figure 16b a ablation The laser beam is guided through the epifluores cence channel of the microscope and focused through the objective Therefore the energy and the size of the laser focus depend on the applied objective Generally the following is valid The higher the magnification and the aperture of the objective the smaller the focus of the laser and the higher the energy density Figure 16 Position of the laser focus when ablating a or catapulting b PALM MicroBeam User Manual 1105 23 How to Prepare the PALM MicroBeam for the Application 6 5 1 Laser Energy Adjustment The laser emits a fixed laser energy For precise laser micromanipulation a precise laser energy adjustment is required A neutral density filter is mounted into the laser beam path which allows to continuously adjust the laser energy to the individual requirements without beam displacement This gradually coated filter is turned by a high precision DC stepping motor which is conveniently regulated on the monitor screen by means of the PALM RoboSoft ware please refer to the PALM RoboSoftware Man u
3. PALM RoboStage 17 PALM RoboStage II 17 Preparation for application 21 Preparation for laser operation 15 Preventive measures 10 R Remaining risks 10 Electricity 10 EMC 11 Fire extinction 11 Infectious material 11 S Safety 8 Disposal 11 Electricity 10 EMC 11 Emergency case 8 Fire extinction 11 Infectious material 11 Laser safety 8 Preventive measures 10 Remaining risks 10 Safety markings 9 Staff qualification 10 Safety markings 9 Settings Fluorescence attachment 26 Simultaneous fluorescence observation 26 Staff qualification 10 Survey PALM MicroBeam 12 T Technical data 32 Electricity 32 Nitrogen Laser 32 Trouble shooting 28 FTSS Laser 28 Nitrogen Laser 29 Type plate 9 34 PALM MicroBeam User Manual 1105
4. Bo ca E 21 61 2 Switch Off the Device 0 ea RR wy ek AE e DA de dd eek 21 6 2 CCD Video Camera Adapter o a 21 PALM MicroBeam User Manual 1105 3 List of Contents 6 3 6 4 6 5 6 6 6 7 7 1 7 2 7 3 7 4 8 1 8 2 8 3 8 4 8 5 9 1 9 2 10 10 1 10 2 10 3 Check of the laser beam path in the Microscope sanaaa aaa 21 ESS laser ici oh Stee BAe A A i 22 6 4 1 How to switch On Off the Laser o o oo es 22 Laser energy and fTOCUS i siai swede tin a Aa AA a TESE aogise ed Pa E 23 6 5 1 Laser Energy AdjUSEMENE ns e ia A bet or dd ee a etd Wt As dEi 24 6 5 2 Laser Focus Adjustment 24 How to adjust laser focus and energy 1 2 25 GUULING Speed 2 ize cert cert E NR RAR 25 PALM MicroBeam with Fluorescence Illuminati0N lt lt lt lt lt lt lt lt lt lt 26 Operate the Microscope with Fluorescence Illumination naa saaa ees 26 Simultaneous Fluorescence Observation o 26 SOLIS cove cate awinn ed ee ea A ee pate ial Peo done een ee 26 7 3 1 Settings for working with motorized microscope Zeiss Axiovert 200M 26 7 3 2 Settings for working with manual microscope Zeiss Axiovert 200 0 26 Freeze Mode ar i ido pis he Wie cde wei a A Gadi Madea rch Goad ewig de eai dido wd 27 Trouble Shooting 0 2 0 c eee ee 28 ETSS aser re eee nC A A i e aS a la a dla 28 Nitrogen Laser viii WA ith edhe ehh a SAE Bee led ea
5. Tech nologies GmbH For changing camera parameters please observe the manual of the camera Notice Depending of the Camera Adapter the com puter monitor only shows a section of the area visible in the ocular There are several camera ports available to integrate up to 5 cameras Caution The camera is aligned in parallel to the stage Don t turn or remove the camera in any way otherwise the calibration of the system is destructed and the laser light might leak out from the camera port Figure 14 CCD Video Camera 20 5 10 Computer The system is equipped with a state of the art computer The computer itself is provided with a CD burner and with an image handling software for easy image selection and editing as well as archiving With the integrated network card you have the possibility to connect your computer with your local network If you want to connect it please contact your local area network administra tor Notice It is not allowed to change or modify software or hardware components on the computer P A L M Microlaser Technologies GmbH is not responsible for any damage resulting from dis obeying these points Any modification of the computer hard or software underlies to the prior written consent of P A L M In any case P A L M is not liable for any loss due to any kind of computer virus 5 11 Foot stepper The foot stepper is used to activate the laser cut ting function manually This o
6. contrast For objectives with a short working distance special Petri dishes and 0 17mm slides are nec essary 6 7 Cutting speed The laser pulse repetition rate of the N gt laser is fixed at 30 pulses per second resp 100 pulses per second for the FTSS Laser Therefore to reach a fine cutting line it is necessary to optimize the laser energy with regard to the cutting speed 25 PALM MicroBeam with Fluorescence Illumination 7 PALM MicroBeam with Fluorescence Illumination 7 1 Operate the Microscope with Fluo rescence Illumination The fluorescence attachment is mounted with clamping screws to the microscope adapter please see Figure 8 Notice Make sure that there is no filter within the beam path of the laser that could block the laser transmission Therefore the excitation filter wheel is mounted outside the laser beam path directly between the fluorescence lamp housing and the microscope adapter see Figure 8 Pos 1 7 2 Simultaneous Fluorescence Obser vation The beam splitter reflector module in the reflector turret is custom made The beam splitter must have a special coating suit able for the simultaneous reflection of the laser light and the fluorescence excitation wavelength as well as for the transmission of the emitted fluo rescence light Caution Do not use at any rate the beam splitters offered by microscope manufacturers along with the laser because such beam splitters may be damag
7. disposed according to the guidelines 11 The PALM MicroBeam Survey 5 The PALM MicroBeam Survey Modular Survey The PALM MicroBeam Device consists of the parts shown in Figure 3 microscope see section 5 4 page 15 CCD video camera see sec tion 5 9 page 20 microscope stage see sec tion 5 6 page 17 CapMover option see sec tion 5 7 page 18 5 filter wheel option 6 microscope adapter see section 5 3 page 14 7 fluorescence illumination option see section 5 5 page 16 8 Laser Interface and laser see section 5 2 page 13 Figure 3 Survey of the PALM MicroBeam 12 These parts and their function are described in detail in the following chapters coc 14 13 9 control unit see section 5 1 page 13 10 monitor see section 5 9 page 20 11 control unit filter wheel 12 power supply fluo illum 3 mouse 14 foot stepper see section 5 11 page 20 15 keyboard 16 joystick CapMover option 17 computer see section 5 10 page 20 PALM MicroBeam User Manual 1105 The PALM MicroBeam Survey 5 1 PALM ControlUnit The PALM ControlUnit is the main device which runs and controls the movement of the PALM RoboStage energy and focus unit and the laser The key switch also operates the mains for the sin gle components of your system So you can switch on off the whole system with this key switch If you want to run single components as the com pu
8. freehand graphic tool spanning at least the monitor screen choose an appropriate stage speed not too fast e start automatic cutting with the Cut function e reduce resp increase the laser energy with the Page down resp Page up key at the key board or with the slider for laser energy at the monitor screen until only a faint cut can be seen e move the laser focus up and down with the keys home and end at the keyboard or the corresponding slider for laser focus at the mon itor screen to locate smallest cutting size e reduce the laser energy and repeat the above described procedure a second or third time until the smallest available focal spot is found The perfect laser focus setting is achieved when with a minimum of laser energy a fine cutting line is visible which immediately disappears as soon as the laser focus is slightly shifted up or down The cutting edges have to show identical symme try when the object is moved into x or into y direction With increasing energy the membrane margins should still appear symmetrically Notice For each type of sample the laser focus adjust ment has to be performed individually The individually determined values can be stored in a separate file please refer to the PALM RoboSoftware Manual Notice For objectives with aperture diaphragm adjust the diaphragm to the thickness of the object Slide or the Petri dish used to ensure an opti mum presentation focusing
9. ms Adjustable via software PALM MicroBeam User Manual 1105 Index A Ambient conditions 7 ApoTome 20 Application 5 B Basic Information 5 C CCD Video camera adapter 21 CCD Video camera and monitor 20 Computer 20 Contact 6 Control apparatus 19 D Dimensions 7 Disposal 11 Electricity 10 Technical data 32 EMC 11 Emergency case 8 F Fire extinction 11 Fluorescence attachment 16 Advanced 16 Basic 16 settings 26 Fluorescence illumination 26 Simultaneous fluorescence observation 26 Foot stepper 20 Freeze mode 27 FTSS Laser 22 Trouble Shooting 28 PALM MicroBeam User Manual 1105 Infectious material 11 Installation 6 Intended use 5 L Laser Maintenance 31 Laser beam path Check 21 Laser energy 23 Adjustment 24 Laser focus 23 Adjustment 24 Laser Interface 13 Laser safety 8 Legal notes 5 M Maintenance 31 Laser 31 Microscope 31 Microscope Check laser beam path 21 Maintenance 31 Operation with fluorescence illumination 26 Nitrogen Laser 32 Trouble Shooting 29 O Operating room 7 P PALM CapMover 18 Control apparatus 19 PALM ControlUnit 13 PALM MicroBeam CCD Video camera adpater 21 CCD Video camera and monitor 20 Computer 20 Fluorescence attachment 16 Foot stepper 20 Laser Interface 13 Maintenance 31 PALM CapMover 18 33 PALM ControlUnit 13 PALM RoboMover 19 PALM RoboStage 17 Preparation for application 21 Survey 12 PALM RoboMover 19 PALM RoboStage 17
10. the dirty parts very carefully Clean dry objectives and oculars smoothly with a cotton bud or a tooth pick padded with cotton wool Only use surgical cotton from the phar macy Remove cotton fluffs and similar by blowing with a small pair of bellows such as an enema syringe Caution Moisten cleaning tissue only slightly do not soak Excessive solvent might dissolve the cementing of the lenses Do not use acetone for cleaning at any rate PALM MicroBeam User Manual 1105 31 Technical Data 10 Technical Data 10 1 Electricity Input voltage Voltage fluctuations Frequency Frequency fluctuations 10 2 Nitrogen Laser Wavelength Pulse energy Pulse duration Pulse frequency Beam divergence Laser control Pulse frequency of laser control 10 3 FTSS Laser Wavelength Pulse energy Pulse duration Pulse frequency Beam divergence Laser control Pulse frequency of laser control 32 115 230 V AC 5 50 60 Hz 2 Hz 337 nm gt 270 4 3 nsec 1 30 pulses per second 0 3 mrad Laser control can be changed by impulse packages external by earth free foot stepper or via RS232 interface automatic permanent impulses Impulse width 0 1 ms Adjustable via software 355 nm 90 y 1 nsec 100 pulses per second 3 mrad Laser control can be changed by impulse packages external by earth free foot stepper or via RS232 interface automatic permanent impulses Impulse width 0 1
11. to be gt 400kg m Minimum space needed in total 140x90cm For the aluminium plate a table measuring at least 120x90cm on which the system will be set up and with a load carrying capacity of the table of gt 90kg m is necessary Power supply 115 230VAC 16VA safety fuse Notice Please consider also vibrations raising from your routine lab equipment PALM MicroBeam User Manual 1105 7 Safety 4 Safety 4 1 What You Should Do in an Emergency Case e Switch off the device immediately by turning the key switch in the OFF position see Figure 4 and make sure that it cannot be switched on again e Unplug the multiple socket e Call the service staff in charge 4 2 Observe the User Manual This User Manual contains important information concerning the safe operation of the device Only when strictly observing the User Manual accidents can be avoided and a trouble free operation of the device can be guaranteed By observing the User Manual downtimes and repair costs will be reduced and the service life of the device increased This User Manual must be stored close to the device at a place accessible and known to the staff where it is available at any time This User Manual must be read understood and observed in all points by the responsible and oper ating staff Please observe all safety instructions and other information given in the User Manual and the attached manuals of the accessories Cauti
12. zeiss com 2 Installation Your PALM MicroBeam is only to be installed by P A L M service engineers or their representatives In case you want to move an already installed unit please contact P A L M Microlaser Technologies GmbH for assistance The PALM Laser Systems basically are mainte nance free In case of any malfunction of the device please con tact the PALM Service see section 1 4 page 6 PALM MicroBeam User Manual 1105 Operating Room 3 Operating Room In order to guarantee a correct function and com 3 1 Admissible Ambient Conditions fortable work with your new PALM MicroBeam Sys tem the following requirements have to be paid The device may be operated at the following ambi attention ent conditions The system should be installed in a room which Ambient Temperature of the air is adequately large about 6 square meters 15 C to 25 C 59 F to 77 F and well aired Air humidity 30 to 80 not condensing There should be a free space available about 90cm above the table and all shelves wall cup boards etc should be removed The PALM RoboMover extends the dimension of a PALM System considerably to the left side It exceeds the left side of the PALM MicroBeam base plate for 12 centimeters Figure 1 Dimensions Nature of the floor low vibration if not special working table recommended and even For a steel or granite table the load carrying capacity of the floor has
13. Control unit is defective external or internal control and safety systems Interlock enable key switch in position 1 are plugged Correction Let laser system cool down and switch on again Reset the control e g by the reset switch on the front panel left from LED2 Check for the reason of the high tempera ture and make sure of a cooling down Contact customer service see No 1 Check if INTERLOCK connection on Inter lock and the external control and safety systems are connected properly Wait for 3 minutes until LED3 becomes green Laser diode driver is out of order Switch off and on the power supply Try to start the laser again Contact customer service Check input voltage and power supply cable Check fuse beside the main switch Contact customer service Check external trigger pulses Disconnect external trigger and use the internal trigger to check the laser Contact customer service Check input voltage and power supply cable Check key switch position and fuse beside main switch stand alone Hardware reset can reactivate the diodes Reset the control e g by the reset switch on the front panel left from LED2 Contact customer service PALM MicroBeam User Manual 1105 Trouble Shooting 8 2 Nitrogen Laser check energy and focus settings as described in section 6 6 page 25 if necessary take factory settings check red LED on Laser unit LED i
14. LM MicroBeam especially for the micro scope the fluorescence attachment option the video camera and the laser P A L M Microlaser Technologies GmbH will not be responsible for mistakes and possible consequences caused by non observance of the user s manual Please consider also the manuals provided for your periphery devices 1 1 Intended use of PALM MicroLaser Systems The PALM MicroLaser family of products is a collec tion of state of the art tools for precise non contact laser based microdissection and microma nipulation and consists of the PALM MicroBeam the PALM MicroTweezers and as a combination of both the PALM CombiSystem It can be supple mented by different modules that can be adapted to the individual needs Two different microscope stages are available PALM RoboStage and PALM RoboStage that can be equipped with different target holders for slides petri dishes capillaries and even for work with serial sections only RoboStage ll For the positioning of different collecting ves sels there are available PALM CapMover for fast positioning of collection vessels with highest precision PALM RoboMover for work at higher troughput This unique robotic collection device enables computer driven sampling of catapulted speci men from multiple spots into a variety of col lecting devices The fluorescence attachment expands the microscope to a fluorescence microscope All functions can be controll
15. M MicroBeam Survey Inserting a 3 SlideHolder into Stage I The surface of the stage is even and not narrowed down by engines or spindles There are two 3 Sli deHolders available 1mm black anodized and 0 17 mm achromatic anodized that can be used only in combination with PALM RoboStage II The slides have to meet the demands of DIN ISO 8037 1 this means 25 26 mm width and 75 76 mm length PALM RoboSoftware 3 0 or a higher level is required Figure 11 Inserting a 3 SlideHolder into Stage e For loading start the PALM RoboSoftware and wait until the referencing of the stage has fin ished and it has reached the central position again e Press the button Loadposition and wait until the loadposition is reached e Withdraw a possibly assembled holder and insert the 3 SlideHolder into the provided opening The sharp edge has to be positioned at the rear right Due to this edge the holder can only be inserted in one direction e In order to return to the work position press the button Loadposition again The software now asks you to select the correct holder e Choose PALM Robo in the menu Setup e Select Configuration and click on 3 Slide Holder Notice The objectives have to be focused on the sam ple or lower to avoid a collision between the objective and the stage Insertion of the slides in the 3 SlideHolder Choose the appropriate slides e Push the slider the metal clamp on the lower
16. PA L M Microlaser Technologies PALM MicroBeam User Manual PALM MicroBeam Version 1105 We make it visible This document is delivered only to persons who are trained and authorized by P A L M Microlaser Technol ogies GmbH No part of this document may be reproduced or distributed in any form or by any means without the prior written consent of P A L M Microlaser Technologies GmbH The information in this document is subject to change without notice P A L M Microlaser Technologies GmbH assumes no responsibility for any errors that may appear Printed November 2005 Fifth Edition LMPC is a trademark of P A L M Microlaser Technologies GmbH PALM is a registered trademark of P A L M Microlaser Technologies GmbH Patent rights The instruments instrument components or methods described in this manual are protected by the follow ing patents Laser catapult technology Laser Pressure Catapulting LPCP Patents US 5 998 129 EP 879408 B1 and others Three dimensional laser beam positioning system Patents US 5 689 109 EP 679325 B1 and others Element List Patent US 6 930 764 Additional patents pending Regulatory Notice PALM MicroLaser Systems are intended for research use only and are not approved for medical applications in the United States and Canada P A L M Microlaser Technologies GmbH Am Neuland 9 12 82347 Bernried Germany Phone 49 0 8158 9971 0 Fax 49 0 8158 9971 249 E mail info p
17. The PALM MicroBeam Survey 5 6 PALM RoboStage P A L M has developed the PALM RoboStage and II The computer controlled PALM RoboStage is a motorized stage especially designed for high preci sion laser micromanipulation in sub micrometer range Its movement is carried out by two stepping motors at a speed of only a few u meters up to sev eral mm per sec and displayed largely vibration free between 1um and approx 60 mm The step ping motors are controlled by two encoders that are affixed below the stage The maximum traverse path is 40 mm in y direction and 60 mm in x direction for the PALM RoboStage or 76 mm in y direction and 114 mm in x direction for the PALM RoboStage II Caution Do not move the stage manually Do not touch the moving stage Avoid contaminating the guide of the stage with oil or chemicals Avoid any kind of mechanical stress e g putting heavy parts on it or leaning against as it is a precision instrument Notice Do not clean the stage with alcohol 5 6 1 PALM RoboStage 1 stepping motor Y direc 5 stepping motor X direc tion tion 2 microscope table board 6 holding frame 3 CapMover 7 snap on clip 4 lever for cap height 8 specimen slide adjustment Figure 9 PALM RoboStage Several special functions are integrated to speed up routine work In the PALM RoboStage there is a holding frame into which the specimen slides or capillaries can be clamped Optional t
18. Type Plate Caution The safety markings on the device must always be clearly visible and legible and must strictly be observed PALM Micr laser Technologies MicroBeam C Manufactured Serial No PALM MicroBeam User Manual 1105 EE EE P A L M Microlaser Technologies GmbH Am Neuland 9 12 D 82347 Bernried Phone 49 8158 9971 0 Ned www palm microlaser com Identification label Safety 4 5 Qualification of the Staff The device shall only be operated by trained and specially instructed staff having the legal minimum age and having carefully studied this User Manual Staff in the course of being taught trained instructed or being in a general education is only allowed to work with the device under permanent survey of an experienced person 4 6 Points that must strictly be ob served Caution Service Works on the laser may only be per formed by P A L M Microlaser Technologies GmbH or its representative When service is needed please contact the P A L M Microlaser Technologies GmbH Service Protective equipment may not be circum vented removed or made ineffective at any rate All requirements regarding the condition of the device the qualification and behavior of the staff set forth in the User Manual have to be met Make sure that unauthorized persons cannot use the device The device may not be operated or maintained without wearing the prescribed perso
19. al 6 5 2 Laser Focus Adjustment Precise laser focus alignment allows laser cutting with a nearly diffraction limited laser focus The coincidence of the laser focus with the optical focus of the microscope is mandatory for precise laser micromanipulation Every wavelength is diffracted differently This is important to know as the spectrum of the visible light of the microscope is at a different wavelength as the UV laser When performing laser microma nipulation at a greater distance from the specimen slide or within a thicker or strongly refracting object the offset of the laser focus position has to be compensated This is especially important for living cells The same applies when using different objectives To compensate for the so called chromatic aberra tion the laser focus position has to be changed independent from the microscope focus The Laser Focus Correction is done motorized A high precision DC stepping motor moves the cor responding lens Focus setting is conveniently con trolled and regulated on the monitor screen by means of the of the PALM RoboSoftware The dis play allows relocating selected laser focus settings Laser energy and focus is predefined by P A L M Microlaser Technologies GmbH over the factory settings for each objective please refer to the PALM RoboSoftware Manual Only the sample specific values have to be defined by the individual user These user specific values can be stored ina spe
20. alm microlaser com www palm microlaser com P A L M Microlaser Technologies GmbH 2005 All rights reserved 2 PALM MicroBeam User Manual 1105 List of Contents Content Chapter Page 1 Important Basic Information 0002 es 5 1 1 Intended use of PALM MicroLaser SystemS 0 00 ee 5 152 Legal NOtes dyin te Cee eos ee Ee ee oe ee Feo es ee Ee eee aoe rele eG 5 1 3 Conventions for this User Manual sasaaa aaa ee 6 1 4 How You Can ContactUS iaid ha ad Ree ae aie Soc eek aE aa A Ree SM Gd a a 6 2 Installation 2 a i e a a eia es 6 3 Operating ROOM 00 cee ee es 7 3 1 Admissible Ambient Conditions 0 aaa 7 4 Safety iie Oye ee aed Sik eee ate ahaa ca a er a aie ta weer 8 4 1 What You Should Do in an Emergency Case 1 2 aaa ee 8 4 2 Observe the User Manual ee 8 E Laser Satety iia ii ws bettie Aaa Bal nie aie Bn oh ek cs A ies et eat hack ew SaaS 8 4 4 Safety Markings and Type Plate o 9 4 5 Qualification of the Static a Aw A Gee aes 10 4 6 Points that must strictly be observed o 10 4 7 Pay Attention to Remaining Risks and Preventive Measures 0000 eee eee 10 4 7 1 Remaining Risks and Protective Measures Laser uaaa aa aaa ee 10 4 7 2 Remaining Risks Electricity 2 0 000 ee 10 4 7 3 Remaining Risks Infectious Material 20 aaa ee 11 4 74 Remaining Risks EMG 2 oe ee ee a a oe ee ee a ak Ge ee 11 4 7 5 Remai
21. aser function you have to turn the reflector revolver of the microscope into a position which is suitable coated for this wavelenght For basic fluorescence attach ments this is the first position only PALM MicroBeam User Manual 1105 27 Trouble Shooting 8 Trouble Shooting In general if malfunctions occur with the laser system first ascertain that all connections to power supply and laser head as well as to existing 8 1 FTSS Laser No 28 Error The laser system switches off automati cally during operation The LED1 is yellow Red LED Laser On fails to light up even though the button On Off was actuated LED3 fails to light up LED2 fails to light up even though the power switch was actuated LED3 is red but laser emission is absent No indication of LED2 and LED3 Possible Cause Out of the operating tem perature range e g the laser head is overheated Room temperature too high Internal electrical fault in laser head or in control unit see No 1 INTERLOCK signal is miss ing The LED3 fails to light up which means laser system is not ready for operation Internal electrical fault in laser head or in control device Power supply is not acti vated Control unit is defective External or internal trigger is not activated or does not work properly Control unit or booster is defective Power supply is not acti vated LEDs are deactivated
22. cial file for each user and or application Notice The factory settings regarding the laser focus and energy settings have been adjusted to the objectives supplied by P A L M Microlaser Technologies GmbH all of a comparable lens configuration Please note The factory settings apply to the standard P A L M membrane slide with out any tissue 24 Please contact P A L M Microlaser Technol ogies GmbH prior to the use of other objec tives For the catapulting and microdissection a LD 40x objective has proved successful in most of the applications For the visualization of catapulted specimen with the LD 40x objective you need a cap with a flat non frosted clear surface and a rim which is not higher than 2mm available from P A L M Microlaser Technologies GmbH Figure 18 Laser focus below specimen plane The smallest laser focus diameter can be obtained when using objectives of a high numerical aper ture When using objectives with a lower numerical aperture the minimal achievable diameter of the laser focus increases correspondingly When working with objectives having a short working distance the thickness of the object slide must be adapted to the used objective If the objective is manually adjustable please assure that the objective is adjusted to the thickness of the actually used object slide PALM MicroBeam User Manual 1105 How to Prepare the PALM MicroBeam fo
23. cope and deflected into the objective 2 fluorescence illumination lamp option 3 microscope adapter 1 filter wheel filter holder with shutter option Figure 6 Microscope adapter The especially designed beam splitter See Figure 7 Pos 2 allows to connect the fluorescence illu mination lamp and the filter wheel to the system for simultaneous usage of fluorescence illumina tion with laser application Notice Due to the high power laser for simultaneous fluorescence illumination especially designed beam splitters have to be used For more infor mation please contact P A L M Microlaser Technologies GmbH Caution Do not touch or remove the screws on the microscope adapter The alignment of the sys tem would be destroyed and if the mirror is removed invisible radiation of a laser class 3B might be guided unprotected to your eyes PALM MicroBeam User Manual 1105 The PALM MicroBeam Survey 5 4 1 display 2 special coated beam splitter 3 button for fluorescence shutter 4 illumination arm Figure 7 Survey of the microscope The high optical and mechanical quality Zeiss inverse microscope Axiovert 200 resp 200M is part of the PALM MicroBeam This microscope can also be used as a normal research microscope Please observe the Axiovert 200 resp 200M user Ss manual The laser beam is guided through the microscope adapter see section 5 3 page 14 into the epiflu orescence channel o
24. d Bee dd eee dd 29 NO VIGO MAIS ing ie i sedi cond ies Bake decks eae eddie be aig dd a aa R si Se 30 Shift between drawing and cutting aa 30 Reflex in video image 6 ee 30 Maintenance of the PALM MicroBeam 000000 cee e ee eee eee eee eee eee nee 31 Las ira wok ee ae A ae Se eos ate E aUn aE n AA E 31 MIGFOSCODE a ahah ie bee apt dd de id doo 31 9 2 1 Cleaning of the Optical Parts ee 31 9 2 2 Cleaning When Actually Dirty 0 000000 02 ee 31 Technical Data s renia e 23d eBay yak a die hw eyes ar bya tas ge eae a teva Seca E al ayes 32 Electricity a5 san tone ANET hgh a ee tate fete tang oe eb pee AR 32 Nitrogen Laser cui ook aoe cece onde wh ete tees A A ADA 32 PISS LASOR A A bn ited Shah Sele Ee ae ee E 32 PALM MicroBeam User Manual 1105 Important Basic Information 1 Important Basic Information Congratulations With your new PALM MicroBeam from Carl Zeiss you obtained the State of the Art Technology in microdissection and micromanipula tion The PALM MicroBeam is a high precision instrument which allows to perform microdissec tion in nearly every kind of research as well as in routine examination with a higher throughput To meet a very high safety standard and to use all the benefits of your PALM MicroBeam you should read this user s manual carefully and observe all instructions given in this manual Please follow also the instructions for the single components of your PA
25. ed by the PALM RoboSoftware to facilitate operation and con trol of PALM MicroLaser Systems PALM MicroBeam User Manual 1105 The lasers used are interfaced into a research microscope and focused through objectives with high numerical aperture to a minimum possible spot size The effective laser energy is concen trated on this minute focal spot only and most bio logical objects are transparent for the applied laser wavelengths It is therefore possible to work even inside a living cell without disturbing its viability Applications PALM MicroLaser Systems are deployed in a wide array of research biological and industrial applica tions Our one of a kind laser technology makes it possible for microscopic specimens to be posi tioned microsurgically processed and captured with absolute precision and unlike other systems without risk of contamination due to our patented non contact Laser Pressure Catapulting process lifting the desired objects into a vessel for further analysis only by the force of laser light It is intended for the collection of precisely defined cells living or fixed cell areas or subcellular par ticles from all kind of samples as they are used in pathology oncology neurology or are needed for proteomics and cytogenetics as well as genetic engineering or other fields of biological medical or pharmaceutical research Microprocessing of non biological matter as for example latex beads or glass is als
26. ed by the high energy of the laser If you want to work with fluorescence and use the laser simultaneously you must use a special filter set available from P A L M For further information please contact P A L M Microlaser Technologies GmbH 26 7 3 Settings 7 3 1 Settings for working with motorized microscope Zeiss Axiovert 200M All elements designed for the use under fluores cence illumination can be controlled by the PALM RoboSoftware Basic settings are preloaded and can be changed and saved by the PALM RoboSoft ware please refer to the RoboSoftware manual In order to use the fluorescence illumination you must switch on the fluorescence illumination and select the desired fluorescence filter unit With this the functions Excitation filter Reflec tor position Microscope light Timer and Shutter are performed via a single command The basic settings of these functions have been made during installation of your PALM system The PALM RoboSoftware provides you with the capabil ity of saving various combinations of these param eters and retrieving them when they are needed Note Close the shutter before switching on the fluo rescence lamp and before finishing or inter rupting your work This will prevent unintentional exposure of your sample to fluo rescence light 7 3 2 Settings for working with manual mi croscope Zeiss Axiovert 200 In order to use the fluorescence illumination you mu
27. emission starts Notice If laser emission does not occur check that the Trigger switch 6 is positioned on Ext The laser works with an output pulse energy regu lation After a total warmup period of less than 10 minutes the laser parameters will be within the specifications 22 Interlock When changing your samples on the microscope stage you should put the microscope illumination arm back thus activating the laser safety interlock The laser emission will stop immediately and only the left LED 2 will light up yellow To reactivate the laser emission press the LASER ON OFF button 5 After approx 1 second the right LED 3 will light up red Switch Off the Laser e To deactivate the laser emission press the LASER ON OFF button 5 The red LED 3 will extinguish e To finish work with the laser system turn the key switch 1 into position 0 and switch off the main power supply PALM MicroBeam User Manual 1105 How to Prepare the PALM MicroBeam for the Application 6 5 Laser energy and focus To be able to precisely cut the material and reliably catapult it the laser focus has to be set accurately to optimal values under various conditions The diameter of the cuts is dependent on the numerical aperture of the objective used the position of the laser focus with regard to the microscope focus the applied laser energy and on the absorption properties of the mate rial to be cut
28. er marker check alignment of camera the camera must be adjusted parallel to the stage movement test this by moving the stage with the arrow keys in a slow speed Calibrate new as described in the PALM RoboSoftware Manual make sure that you are using the settings for the active objective check LED s on encoder of moving stage LED is green o k LED is orange or red please contact P A L M Microlaser Technologies GmbH 8 5 Reflex in video image Incidence of light through the ocular may cause reflexes in the video image in case of reflexes close shutter at the microscope 30 PALM MicroBeam User Manual 1105 Maintenance of the PALM MicroBeam 9 Maintenance of the PALM MicroBeam Caution Before starting any maintenance works dis connect all system components from the mains 9 1 Laser See Laser Manual 9 2 Microscope For detailed actions see Microscope Manual 9 2 1 Cleaning of the Optical Parts Clean all easily accessible optical parts once a week Carefully wipe the optical parts with a cleaning tissue moistened with alcohol of 70 to 80 9 2 2 Cleaning When Actually Dirty Actual dirt e g caused by finger prints or immer sion oil must immediately be removed otherwise the optical functions will permanently be impaired Dirt on surfaces of lenses or filters burnt in by laser light can not be removed any more Slightly moisten a cleaning tissue with pure alcohol 100 or spirit and wipe
29. f the microscope It is reflected by a special coated beam splitter in the reflector turret and focused by the objective It is necessary to observe the following instruc tions otherwise the laser beam might be blocked and performing microdissection will not be possi ble The electromagnetic fluorescence shutter in the microscope has to be opened This can be done with the button FL on off When the shutter is open the sign FL appears on the display of the microscope PALM MicroBeam User Manual 1105 Microscope Zeiss Axiovert 200 200M preparation for laser operation adjustment aid in left position filters and 5 fluorescence attachment option 6 microscope adapter 7 epifluorescence channel 8 reflector turret diaphragms open A reflector 3 turret set to Laser A gt OC FL shutter open mo FL on off O HAL on off 9 adjustment aid 10 two diaphragms 11 filter slider The reflector turret has to be in the position in which the beam splitter reflects the laser beam This position is labeled with 1 Laser see indi cation plates at the front of the microscope accordingly the reflector turret has to be set on position 1 In case of installed fluorescence attachment option it is possible to use also the installed beam splitters of the fluorescence filter sets only in case of advanced filter sets The two diaphragms in the epifluorescence c
30. hannel must be open The adjustment aid for the fluorescence illumi nation has to be in the left position otherwise the laser beam will be blocked Do not insert any filters in the filterslider in the epifluorescence channel They might block the laser beam therefore put it to free passage rightmost position 15 The PALM MicroBeam Survey Notice The magnification lens is not in the laser beam path So if the magnification selector knob is pulled out there is an additional magnification by 1 6x in the light path to the video camera but not in the laser beam path So the laser cuts and catapults not the lines and points you marked on the screen If the illumination arm of the microscope is pushed back the laser will be switched off immediately Interlock This is a security device which protects you from looking into the laser beam If you use a FTSS Laser you will have to reactive the laser emission by pressing the laser ON OFF button 5 5 Fluorescence attachment The fluorescence attachment expands the micro scope to a fluorescence microscope 1 filter wheel filter holder with shutter 2 HBO lamp or X Cite 3 controller for filter wheel 4 power supply 5 especially designed beam splitter Figure 8 fluorescence attachment The filter wheel is accessed by the PALM RoboSoft ware It is mounted outside of the optical path of the laser To change the lamp please refer to microscope manual
31. here is a double slide holder available PALM MicroBeam User Manual 1105 Both transversal sides of the object slide are held by a snap on clip Notice At the longitudinal side of the holding frame there is a special rim Please make sure that the longitudinal side of the slide rests on this rim entirely This will reduce the risks that 0 17 mm slides will bend by the adhesion force of the immersion oil The PALM RoboStage will be moved by means of the PALM RoboSoftware 5 6 2 PALM RoboStage I 4 Check Point 1 stepping motor Y direc tion 5 3 SlideHolder 2 stepping motor X direc 6 specimen slide tion 3 microscope table board Figure 10 PALM RoboStage In addition RoboStage fulfills its specification in high throughput mode Up to 3 microscope slides can be investigated at the same time which is useful especially for serial sections Also different stage inserts like petridish holders for living cells capillaries and microtiter plates can be inserted by using P A L M designed stage inserts ask P A L M Microlaser Technologies GmbH for details For easy insert change a so called Load position is defined In this position the stage insert or microtiterplate can be withdrawn powerless and any kind of holder can be inserted A separate check position located on the right side of the Stage II allows for checking the cata pulted and captured samples with all collection devices 17 The PAL
32. l immersion or water immersion must be used with the respective immersion medium otherwise the objectives might be damaged Caution Do not turn the objective turret during simultaneous laser operation Never use the laser without an objective in the appropriate position Never insert reflecting objects in the laser beam path 21 How to Prepare the PALM MicroBeam for the Application 6 4 FTSS Laser If you use a PALM MicroBeam system with FTSS Laser the laser is operated via an additional con trol box Figure 15 FTSS 355 50 TRIGGER LASER 6 8 INT 4 RESET ON OFF EXT e SERVICE INTERLOCK OCHO oto Figure 15 Control Box 6 4 1 How to switch On Off the Laser Switch On the Laser e Press the red main power switch on the back side of the Laser Controller and turn the key switch 1 on the front panel into position 1 Only the left LED 2 will light up yellow indi cating that a supply voltage is present After a warm up period of approx 5 minutes the left LED 2 and the right LED 3 will light up green and the laser is ready for operation and can be activated Notice If the green LED 3 is not glowing the laser cannot be started and must be reset with the reset button 4 or by turning the key switch off and on again e To activate laser emission press the LASER ON OFF button 5 The green LED 3 will change color and lights up red After a delay of approx 1 second the laser
33. lower speed range is useful in the case of high magnification Prolonged pressing of the button will cause the arm to move outside of the objective area for example in order to take up a new cap Speed change joystick and the functions of the left button are deactivated in this position Renewed activation of the right button will cause the arm to be retrieved and the cap to be moved to the saved reference position 5 8 PALM RoboMover The PALM RoboMover is the state of the art device for modern cell harvesting and sorting of different kinds of microdissected specimen at higher throughput It enables you to automatically posi tion collecting devices above your specimen There are various collectors available containing one or several caps or tubes or even collectors for micro titerplate formate All functions are controlled by the PALM RoboSoftware For more details see the PALM RoboMover Manual Figure 13 PALM MicroBeam with PALM RoboMover 19 The PALM MicroBeam Survey 5 9 CCD Video Camera and Monitor Pictures taken by a CCD camera are transmitted directly to the computer via a frame grabber The video transferred microscope image is dis played in real time on the monitor screen which allows to simultaneously observe the microscope image with stage or manipulator control functions You can store the transferred images on the hard disk of the computer or on CD The camera is preset by P A L M Microlaser
34. meets the protection requirements according to DIN EN 61000 6 2 Regarding interference emission the device meets the protection requirements according to DIN EN 55011 Do not operate any devices emitting high fre quency radiation in the same room where the PALM MicroBeam is placed 4 7 5 Remaining Risks Fire Extinction To extinguish fire use sand or carbon dioxide Never ever use water because of the electric dan gers PALM MicroBeam User Manual 1105 4 8 Disposal 4 8 1 How to Ensure a Safe and Ecological Disposal Dispose the following materials in an environmen tally friendly way Microscope lamps with incorporated bulb or individual mercury vapor short arc lamps as hazardous waste Laser as electronic scrap Electronics as electronic scrap Cables as reusable material 4 8 2 For EU states only PALM Microlaser devices are produced according to the EU guidelines 2002 95 EG RoHS and 2002 96 EG WEEE as far as these are applicable We have installed a process for disposal and recy cling within the member states of the guidelines mentioned above Please contact your relevant Carl Zeiss Service resp sales organization to learn about the details for the disposal or recycling of your device The product must not be disposed of the domestic waste or be given to the municipal disposal collec tion In case of resale the seller is committed to point out to the buyer that the product has to be
35. nal pro tective equipment Please find the respective instructions in the relevant chapters in this manual Persons under the influence of alcohol other drugs or substances that affect the ability to react are not allowed to operate the device The device may only be used in a technically faultless condition In case of troubles or prob lems with the power supply and or damage of the electric equipment the device has to be shut off immediately Any malfunctions or damage indicated by the system or detected otherwise must immedi ately be notified and removed The device may not be modified Protective equipment e g protective filters or covers of laser beam outlets may not be dis mantled The device may only be operated if the entire protective equipment is perfectly working 10 The application conditions and performance data defined in this User Manual may not be al tered For Technical Data see section 10 page 32 4 7 Pay Attention to Remaining Risks and Preventive Measures 4 7 1 Remaining Risks and Protective Measures Laser Remaining risks The laser works with invisible light When the casing is open the laser pertains to the laser protection class 3B The high power radiation leads to dimmed sight of the eye The casing must always kept closed and locked and may only be opened by the ser vice staff of P A L M Microlaser Technologies GmbH Unintentionally mirrored reflectio
36. nd energy set tings are done by the user see chapter 6 5 also observe PALM RoboSoftware Manual Caution Do not open the housing of the Laser Interface Invisible radiation of a laser class 3B might be guided unprotected to your eyes Laser The PALM MicroBeam works with a pulsed N laser wavelength 337nm or with a frequency tripled solid state Laser FTSS wavelength 355nm of a high beam quality which is coupled via the epiflu orescence path see Figure 7 Pos 7 into a research microscope and focused through the objective Depending on the applied objective the laser focus can reach less than one micrometer in diameter In theory the high beam quality of the laser allows to focus the laser beam to its diffrac 13 The PALM MicroBeam Survey tion limit However the cutting width is dependent on correct laser focus settings optimal laser energy settings the numerical aperture of the focusing objec tive and the absorption behavior of the specimen Notice P A L M provides factory settings for each objective The fine tuning of laser focus and energy setting is done by the user please see section 6 5 page 23 and is dependent on the focusing objective and the kind of material you want to handle For cutting the stage is moved and not the laser beam itself 14 5 3 Microscope Adapter The microscope adapter connects the laser to the microscope The laser beam is guided into the micros
37. ning Risks Fire Extinction 2 0 0 00 ee 11 453 DISPOSAliama Der eS de RE A Wa a ee bee he hilt Bas A 11 4 8 1 How to Ensure a Safe and Ecological Disposal saasaa aaa ee 11 478 2 For EUistatesion Yao aig Gta ae Raye Na ee MO ee nat AAA ig uk Shek 11 5 The PALM MicroBeam Survey 0000 eee ee 12 5T PALM ControlUnit o 3 2 03 2 ese egret ee anar Gi Pee Se e E ar ae es 13 52 gt Laser interface eck tee ta eck be cd wad Boat Roce se ceed eae Sa Ok eee ba eck id oh cade es 13 5 3 cMICFOSCOPE Adapter oir da ds aa E ay Shoe A a 14 5 4 Microscope Zeiss Axiovert 200 200M preparation for laser operation 15 5 5 Fluorescence attachment 0 ooo o 16 5 0 PALM RODOStag iasi o srt de dd adidas EA ase 17 56 1 PALM RObDOStage li su i ramara data dl Ad AR AAA a a a ae 17 56 2 PALM RODOStage Ml E A A AS da de ida 17 Dele IPAEM CapMOv Gl ica ci A i a te ca ants da Ge Gh ee ae SO Aa ed 18 58 PALM RoboMove r ei arre adas bate une gid Ga ee By ode Wie eed gee ad Boe bse 19 5 9 CCD Video Camera and Monitor 0 0 0 0 20 5 10 COMPUTE io ates Mee sce cet A ase ee Pee A ined eee A a ae tees 20 SLE Foot Stepper ut aa A ii tea ee aide Raed AA hot Wastes 20 5 12 APOTOMCs ee vice AA A REM ba ea bd Ow EU AA oa EO OE 20 6 How to Prepare the PALM MicroBeam for the Application 0000 eee oo 21 6 1 How to switch On Off the Device o ooo 21 6 1 1 Switch On the Device sas rolas Ra
38. ns must be avoided by a risk conscious behavior of the staff Protective measures taken by the manufacturer On the microscope lighting arm and the con denser if it can be swiveled out of the beam paths a safety switch is installed that shuts off the laser as soon as the lighting arm is swiveled The travel paths of the laser beams as well as those of stage and CapMover are equipped with end switches to avoid damage 4 7 2 Remaining Risks Electricity Before cleaning current carrying parts make sure that the power has been switched off Even after switching off the electric energy supply life threatening voltage might occur in areas marked with an electric flash as well as in the fol lowing supply systems dangerous areas Process computer DC power unit Laser fan Stepping motor control a c network feed lines PALM MicroBeam User Manual 1105 Safety 4 7 3 Remaining Risks Infectious Material Danger of infectious particles Works on infectious materials e g viruses must be carried out under a sterile protective cover When working with infectious material the respec tively applicable safety regulations must be observed Clean the working area of the microscope after use if required The cleaning agent should neither be abrasive nor strongly basic or acid as this would attack the protective coating 4 7 4 Remaining Risks EMC Regarding interference resistance the device
39. o possible 1 2 Legal Notes P A L M Microlaser Technologies GmbH is not lia ble for damages resulting from the fact that this User Manual was not or only partly observed The warranty extinguishes in case the customer opens the laser housing or uses the device for other than the specified purposes If used parts are replaced by similar parts which are however not identical to those recommended by the manufacturer in this manual P A L M Microlaser Technologies GmbH is only liable if the product supplied has already been faulty before the parts were replaced For details concerning the manufacturers war ranty please refer to the contractual conditions Installation 1 3 Conventions for this User Manual Special markings on the left margin point out information you should observe by all means Warning This sign warns you of possible most serious irreversible injuries to persons even going as far as to death Caution This sign signalizes possible moderate personal damage and possible damage of the device Notice This sign signalizes that information is given demanding your special attention 1 4 How You Can Contact Us Business hours 9 h 15 00 h CET Mo Fr Phone 49 0 81 58 99 71 0 Fax 49 0 81 58 99 71 249 E mail Info palm microlaser com Service Address P A L M Microlaser Technolo gies GmbH Am Neuland 9 12 82347 Bernried Germany support palm microlaser com www palm microlaser com www
40. on Any use other than the ones defined in this User Manual might lead to personal injuries or material damage which P A L M Microlaser Technologies GmbH is not liable for 4 3 Laser Safety The PALM MicroBeam is a laser device of laser pro tection class 1 M with no special safety require ments if operated according to the instructions given in this User Manual The accessible laser beam is safe for the unpro tected eye when the laser casing is closed Built in is a Laser of class 3B It bears a potential risk A direct or reflected beam must not strike the unprotected eye do not look directly into the beam In service case the device has to be operated by the service staff or an authorized representative of P A L M Microlaser Technologies GmbH even with the laser casing open Warning The housing of the Laser Interface may only be removed by the service staff or an authorized representative of P A L M Microlaser Technolo gies GmbH Warning Please note wavelength is invisible for the human eye A 337nm resp A 355nm PALM MicroBeam User Manual 1105 Safety 4 4 Safety Markings and Type Plate Make sure that the following safety markings and the type plate exist and are legible Replace miss ing or illegible safety markings or type plate for USA and Japan 7 only for Europe only Complies with 21 CFR 1040 10 and 1040 11 5 E Figure 2 Safety Markings and
41. ontrolUnit and after 10 seconds switch it on again Should the LED not turn off please contact P A L M Microlaser Technologies GmbH 6 1 2 Switch Off the Device e Exit WINDOWS e Switch off the ControlUnit e Switch off the monitor 6 2 CCD Video Camera Adapter Caution Do not touch the camera If camera position is changed new calibration is necessary please refer to the PALM RoboSoftware Manual how to calibrate your system Notice Depending of the Camera Adapter the com puter monitor only shows a section of the area visible in the ocular PALM MicroBeam User Manual 1105 6 3 Check of the laser beam path in the Microscope The laser is coupled into the microscope via the epifluorescence beam path Prior to the use of the laser make sure that the aperture of the iris diaphragm in the epifluorescence beam path is completely open see Figure 7 Pos 10 that a possibly existing filter slide is set to free passage see Figure 7 Pos 11 that the reflector turret under the objective is switched into the correct position called laser see Figure 7 Pos 8 that the Fluorescence shutter in the reflector turret of the microscope is open FL appears in the display See Figure 7 Pos 8 adjustment aid is in left position see Figure 7 Pos 9 Caution Only use the objectives that are authorised by P A L M Microlaser Technologies GmbH for laser operation Objectives designed for oi
42. ption is only avail able in stage mode please refer to the PALM RoboSoftware Manual 5 12 ApoTome ApoTome the innovative insert module from Carl Zeiss allows the very fast production of extremely high quality optical sections through fluorescence labeled biological specimens with two and three dimensional visualization ApoTome offers better image quality contrast and resolution Notice If you use a Zeiss ApoTome Advanced Fluores cence is not possible Caution When the laser runs make sure that the grid of the ApoTome is not within the optical path and the position of the ApoTome must not be changed Otherwise the transmission grid may be destroyed To avoid simultaneous laser function and integra tion of the transmission grid a safety switch can be integrated into the system Ask P A L M Micro laser Technologies GmbH for details PALM MicroBeam User Manual 1105 How to Prepare the PALM MicroBeam for the Application 6 How to Prepare the PALM MicroBeam for the Application 6 1 How to switch On Off the Device 6 1 1 Switch On the Device e Set the key switch of the ControlUnit to ON and switch on the computer for details see section 5 1 page 13 The PALM RoboSoftware will be loaded auto matically If not please refer to the PALM RoboSoftware Manual how to start it Notice If the red system LED F of the control unit see Figure 4 does not turn off automatically please switch off the C
43. r the Application 6 6 How to adjust laser focus and energy As the laser beam itself is not visible only the laser effect ablation can be used for control To visualize the changes of the laser focus posi tion you must cut ablate an object whereby the laser is permanently pulsating and the object is being moved simultaneously We recommend to use an LPC object slide membrane coated for this purpose Notice Make sure that the membrane is always focused within the microscope focus otherwise the laser focus setting might be incorrect Simultaneous stage movement will create a cut ting line within the sample This line will be fuzzy or fairly thick due to yet insufficient laser focus setting A star like glass ablation around the laser focus indicates a good focussing of the glass surface With a higher energy setting cutting is possible not only within the laser focus but also above or below If the laser focus is just inside the glass smaller or bigger pieces of glass will be blown out Figure 19 Zig zag line cut with the laser with different energy and focus settings Find the correct focal plane in the following ways please refer to the PALM RoboSoftware Manual for using the graphic tools the automated laser func tions and how to adjust energy and focus settings for the laser PALM MicroBeam User Manual 1105 use a plain membrane slide without tissue specimen e draw a zig zag line with the
44. s from the power supply and ascertain the cause of the error For example check if the traverse path of the PALM CapMover is blocked You should not switch on the control apparatus until after having done this r Reference Slow Fast short 0 Oe long L Set Ref Figure 13 Control Apparatus Brief extinguishing of the LED also indicates a change of speed as well as the saving of a new reference position The joystick serves to move the PALM CapMover arm and consequently the cap or tube in two dimensions The buttons have the following functions Left button If the button is pressed briefly the PALM Cap Mover arm moves to the reference position that is currently saved By pressing the button for a sec ond time the arm moves back to the last position it was moved to If the arm has been moved from the reference position with the joystick then this position is accepted as the last targeted position If the button is pressed for a while then the cur rent position is saved as the new reference posi tion The LED will extinguish briefly as a confirmation signal Right button If the button is pressed briefly this will switch between the fast and slow speed ranges The LED will extinguish as a confirmation signal with each change of the speed range The faster speed range is suitable for examination at a low optical magnification factor whilst the PALM MicroBeam User Manual 1105 s
45. s on o k LED is off check key switch on laser unit check power connection of laser check green LED on Laser unit LED is on o k LED is off check function of safety switch in illumination arm of the microscope check position of adjusting aid for the fluorescence lamp see Figure 7 Pos 9 left position o k right position switch to left check shutter for FL see Figure 7 Pos 3 shutter open FL on display o k shutter closed open shutter press FL on off button check position of reflector turret reflector turret on position laser o k any other position except you have switch to laser fluorescence equipment with P A L M simultaneous cutting check both sliders with diaphragm see Figure 7 Pos 10 diaphragm open o k check position of filter slider see section 11 page 15 one of the three open positions o k check position of the correction ring if available of the objective according to the thickness of the slide PALM MicroBeam User Manual 1105 29 Trouble Shooting 8 3 No video image check image in oculars no image check illumination of microscope check position of main switch on camera s power supply if applicable check connection between camera and camera power supply if applicable check connection between camera and computer S VHS plug must be plugged in framegrabber board check position of switching mirror in microscope 8 4 Shift between drawing and cutting check position of las
46. side of the Holder to the left and insert the slide e Make sure that the slide fits exactly into the three edges e Release the slider carefully 18 Notice When you change from one slide to another you will have to focus manually If you use an 100x oil objective lower the stage when changing from one slide to another 5 7 PALM CapMover The motorized PALM CapMover was developed for fast positioning with highest precision and quick exchange of the collection device for sample col lection There are different collection devices and collection device holders for up to 8 caps or single tubes available This way the cap is positioned and moved via joystick to check for the catapulted specimen Please contact P A L M Microlaser Technologies GmbH for further information Caution If any vessels e g Petri Dishes instead of specimen slides are used make sure that the cap resp tube holder is in the upper position when moved in or out Otherwise the PALM CapMover may be damaged 1 cap 4 lever for cap height 2 CapMover adjustment 3 CapHolder Figure 12 CapMover PALM MicroBeam User Manual 1105 The PALM MicroBeam Survey Control Apparatus At the front of the control apparatus there are two buttons a joystick and an LED The LED essentially indicates the operational readiness In the event of an error or a circuit overload this LED will extinguish In this case dis connect the apparatu
47. st switch on the fluorescence illumination select the desired fluorescence excitation filter only necessary when using a filter wheel please refer to the PALM RoboSoftware Manual swivel in the desired reflector position in the microscope switch the microscope light on or off as neces sary activate the timer if you want to preset a time for which the excitation light should illuminate your sample refer to the PALM RoboSoftware Manual and open the shutter please refer to the PALM RoboSoftware Manual The PALM RoboSoftware controls all functions of the filterwheel and the shutter please refer to the PALM RoboSoftware Manual Settings of the microscope has do be done manu ally PALM MicroBeam User Manual 1105 PALM MicroBeam with Fluorescence Illumination 7 4 Freeze Mode A very special feature is the possibility of freez ing the fluorescence image on the screen with the PALM RoboSoftware tool Freeze Mode please refer to the PALM RoboSoftware Manual Thus bleaching effects of the sensitive samples can be avoided while defining the laser cutting lines is possible e g freehand select elements In the Freeze Mode the fluorescence is deactivated as well as all functions to move the stage After han dling the frozen picture on the screen you can leave the Freeze Mode to continue with other regions under fluorescence or carry out laser func tions Note Before you switch on a l
48. ter for example it is possible to connect these components directly to the mains The actual status of the PALM ControlUnit can be observed by LED s on the front panel OUT FO 10 02 30 04 50 06 70 08 40V O 24V O 5V O key switch Figure 4 PALM ControlUnit After switching on the system with the key switch the three diodes for the voltage ranges 40V 24V and 5V should be illuminated and the green LED 2 should flash every second Notice Shortly after switching on the PALMControlUnit the red LED F turns off automatically Other wise switch off the ControlUnit and after 10 seconds switch it on again Should the LED not turn off please contact P A L M Microlaser Technologies GmbH The type of ControlUnit may differ depending on the individual device configuration PALM MicroBeam User Manual 1105 5 2 Laser Interface The Laser Interface is a one box device which houses the laser and all the necessary optics to guide the laser into the microscope to bring the laser focus coincide with the optical focus of the microscope at the object plane and to adjust the laser energy Laser Interface Figure 5 Laser Interface This is a prerequisite for precise laser micromani pulation with highest precision Furthermore the laser position stays stable which allows to routinely work with the system without time consuming search for the laser beam focus Notice The adjustment of laser focus a
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