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MantraSnap Software Manual

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1. B a MantreSnap DAPI 3 So Figure 38 You can configure Mantra Snap to simply save your images or you can configure it to automatically launch the inForm Advanced Image Analysis Software program perform a batch process using inForm or even launch a custom Windows bat file In the Post Processing settings editor seen in Figure 38 above select Launch inForm Analysis Go to the Mantra Snap editor click Autoexpose or manually set your exposures in the Exposures editor return to the Mantra Snap editor and Snap a picture of your stained sample slide Then Snap a picture of your unstained slide For purposes of instruction we shall presume the slide contains several multiplexed stains DAPI counterstain Cy3 cytokeratin CD4 FITC CD8 Alexa Fluor 594 CD20 Cy5 Shortly after the images have been acquired the inForm program window will appear as shown in Figure 39 below a Mantra Snap kuga fort Masoods lin Sevi ona m E Owe matee xo Mdoe Soera Laron Soe Eam aan s i i i i CETATEA Pepen rape Figure 39 If you have chosen to open your image with inForm after Snapping a picture with Mantra Snap the inForm program window will open with your image s in the display area The inForm program will automatically detect the type of image IM3 fluorescence As shown in Figure 41 select Spectral Library Source inForm from the drop down menu in order to
2. Before using the illuminator please read all warnings and operating safety instructions in this User s Manual Keep this manual in a safe place for future reference Do not use the illuminator for purposes other than its intended use Doing so could cause damage to the unit and or personal injury and may void the warranty Do not expose the illuminator to water or moisture Do not expose the illuminator to extreme heat or cold Do not expose the illuminator to open flames Do not allow objects to fall on or liquids to spill on the illuminator Use only the AC power supply cord set provided with the illuminator If the correct cord set for the location was not provided please contact PerkinElmer for a replacement Do not use power supply cords with inadequate ratings Connect the AC power cord only to the designated power sources as marked on the illuminator e Make sure the electrical cord is located so that it is not subject to damage Always make sure the illuminator is disconnected from power before installing the bulb connecting components or cleaning the exterior surfaces Do not in any way attempt to tamper with or alter the illuminator doing so voids the warranty and may damage the system This product does not contain consumer serviceable components other than the replaceable lamp and the fuses Contact PerkinElmer for any other required service or repairs Ensure that the cooling vents in the controller case are not blocked
3. Warnings Hg LAMP CONTAINS MERCURY Handle maintain and dispose of the lamp in accordance with local Disposal Laws Before replacing a fuse DISCONNECT THE ILLUMINATOR FROM THE POWER SUPPLY e Eye damage may result from directly viewing the light produced by the lamp used in the illuminator Always make sure the light guide is properly attached to the illuminator and inserted into the collimator and that the collimator is firmly attached to the microscope before turning on the power to the unit The illuminator has built in protection features to avoid unintentional exposure to UV radiation Do not attempt to defeat the protection features Caution e Never look into the emitting end of a light guide The light could severely damage the cornea and retina of the eye if the light is observed directly Appropriate eye shielding must be used at all times clothing should be used to protect exposed skin e Never place the end of an emitting light guide near skin as this may result in burning and damage to the skin Never place the end of an emitting light guide near a flammable substrate as sufficient power is emitted from the light guide to ignite flammable substances When turned on the illuminator should be attended at all times by a qualified operator Do not leave 10 Mantra Snap the illumination lamp on and unattended longer than the amount of time required to complete operation 1 6 Table of Symbols The table below contains
4. in at least one of the following places within a NOTICE text file distributed as part of the Derivative Works within the Source form or documentation if provided along with the Derivative Works or within a display generated by the Derivative Works if and wherever such third party notices normally appear The contents of the NOTICE file are for informational purposes only and do not modify the License You may add Your own attribution notices within Derivative Works that You distribute alongside or as an addendum to the NOTICE text from the Work provided that such additional attribution notices cannot be construed as modifying the License You may add Your own copyright statement to Your modifications and may provide additional or different license terms and conditions for use reproduction or distribution of Your modifications or for any such Derivative Works as a whole provided Your use reproduction and distribution of the Work otherwise complies with the conditions stated in this License 5 Submission of Contributions Unless You explicitly state otherwise any Contribution intentionally submitted for inclusion in the Work by You to the Licensor shall be under the terms and conditions of this License without any additional terms or conditions Notwithstanding the above nothing herein shall supersede or modify the terms of any separate license agreement you may have executed with Licensor regarding such Contributions 6 Trademarks This
5. multispectral color acquisition can complete a full cycle When I take pictures of slides that have been prepared using a near infrared stain such as Alexa Fluor 750 or Cy7 is the Mantra system capturing images in the near infrared part of the spectrum I thought that the wavelength range of the tunable filter inside the camera module only tunes from 440 to 720 nm When taking pictures of near infrared stains such as Cy7 or Alexa Fluor 750 the Mantra only needs to take an image at one wavelength 770 nm Unlike in the visible wavelength range there is little autofluorescence in the near infrared wavelength range and the acquisition of many wavelengths in order to spectrally separate the true signal from background is not necessary In addition near infrared stains are distinct enough from visible wavelength stains that there is no difficulty separating them When I am not using my Mantra system should I switch it off Yes in addition to saving electricity switching off the Mantra will prevent overheating if the ambient temperature of your laboratory is relatively high or if you use a dust cover over the system See the Mantra operating environment specifications 9 Keep in mind that the Fluorescence illuminator needs at least 30 minutes to arrive at its operating temperature and at least 30 minutes to cool down after you have finished using it WARNING Do not place a cover over the Mantra instrument or the fluorescence illuminat
6. objective s name not its nominal magnification To change the magnification delete the entry and create a new entry for the objective To delete an objective from the system first remove it from any objective position Next highlight the objective entry in the list and click Delete Choose Yes to confirm you want to delete the objective from the system Use the Objective Positions boxes 1 7 to arrange the objective entries so they match the locations of actual objectives mounted in the turret Note that as you assign objectives to their positions the system prevents you from assigning the same objective to more than one turret position To change an objective s location remove it from the current location and then select the new location The Load Factory Objectives button automatically populates the Available Objectives box with the default factory objectives Click the Next button or the Epi Filters button to continue with system setup Or close the window if you are finished setting up or making changes to the hardware Appendix A Mantra Hardware Setup 51 Epi Filters If you are instructed to add or remove an meee epi fluorescence filter cube or cubes on your Mantra system you can account for that change from the Epi Filters section of the setup program amp Epi Filters Turet Size Load Factory Fiters_ The Turret Size defaults to 8 and is not editable The Mantra hardware features an eae
7. stage controller 7 trinocular 7 Illuminator Mantra Snap setup 62 Image Window 18 sL Lamp adjustment 23 License agreement 67 Manual about 12 Menu About 20 Exposures 20 Help 20 License Info 20 Load Protocol 19 Log 20 Mantra Snap 19 New Protocol 19 Post Processing 20 References 19 Save Protocol 19 Snap Rules 20 Upgrade License 20 What s New 20 Objectives 51 R References 22 Requirements environmental 9 power 8 S Setting button 18 Setting menu 19 Settings area 18 Software EULA 67 PerkinElmer Inc 68 Elm Street Hopkinton MA 01748 USA Phone 800 762 4000 or 1 203 925 4602 Fax 1 203 944 4904 Email global techsupport perkinelmer com To contact a local PerkinElmer representative outside the United States go to http www perkinelmer com j gt PerkinElmer Microsoftis a trademark of Microsoft Corporation in the United States and other countries Copyright 2012 2013 PerkinElmer Inc All rights reserved
8. OR SILICON GRAPHICS BE LIABLE FOR ANY SPECIAL INCIDENTAL INDIRECT OR CONSEQUENTIAL DAMAGES OF ANY KIND OR ANY DAMAGES WHATSOEVER RESULTING FROM LOSS OF USE DATA OR PROFITS WHETHER OR NOT ADVISED OF THE POSSIBILITY OF DAMAGE AND ON ANY THEORY OF LIABILITY ARISING OUT OF OR IN CONNECTION WITH THE USE OR PERFORM ANCE OF THIS SOFTWARE 11 This software uses the Vigra 1 6 0 library http hci iwr uni heidelberg de vigra os Mantra Snap This software includes machine executable object code generated by a source language processor from the Vigra libraries covered by the VIGRA license These libraries have not been modified by CRI and all rights are reserved by the copyright holder This software is provided as is without express or implied warranty and with no claim as to its suitability for any purpose The Vigra library is Copyright 2008 Ulrich Kothe Heidelberg Collaboratory for Image Processing The VIGRA License identical to the MIT X11 License Permission is hereby granted free of charge to any person obtaining a copy of this software and associated documentation files the Software to deal in the Software without restriction including without limitation the rights to use copy modify merge publish distribute sublicense and or sell copies of the Software and to permit persons to whom the Software is furnished to do so subject to the following conditions The above copyright notice and this permission notice
9. Practice Exercise This section will guide you through taking brightfield images using the Mantra imaging system The section that follows this one describes taking fluorescence images Skip to that section if you want to take fluorescence images instead o Configuring Mantra for Brightfield Imaging 2 o Taking a Brightfield Imagel 241 o Processing a Brightfield Image in inForm 2 2 4 1 Configuring Mantra for Brightfield Imaging Make sure that the Mantra hardware is properly configured and powered upl 14 Then turn on the computer and wait until Windows has finished booting up Launch the Mantra Snap software by double clicking on the icon on the desktop or by going to C Program Files PerkinElmer Mantra 1 0 0 MantraSnap exe In the Mantra Snap program load the default brightfield protocol by clicking on the Settings button 19 in the upper right of the Mantra Snap program window and selecting Load Protocol from the drop down menu You will be automatically taken to C Users Public PerkinEImer Mantra P rotocols Select Brightfield mpr IMPORTANT Do not move or rename the Protocols folder You may create folders within but do not move or rename this root folder for loading and saving protocols You can save new protocols but only within the Protocols folder or within folders located inside the Protocols folder Once the default Brightfield protocol is loaded you will be automatically placed into brightfield mode and prompte
10. Settings button in the upper right of the Mantra Snap program window and select Load Protocol You will be automatically taken to C Users Public PerkinElmer Mantra Protocols Select DAPI mpr IMPORTANT Do not move or rename the Protocols folder You may create folders within but do not Imaging With Mantra 31 move or rename this root folder for loading and saving protocols You can save new protocols but only within the Protocols folder or within folders located inside the Protocols folder Once the fluorescence protocol has been loaded you will be automatically placed into fluorescence mode and prompted to take your reference images if this is the first time you have used the system The presence of a blue LED dot located above the letters FL on the lower front control panel indicates that you are in fluorescence mode NOTE The Mantra Snap program s fluorescence mode has a dark gray background The darker theme reduces light scatter from the computer monitor when the imaging system is being used ina dark ened laboratory area This also makes it easier to differentiate between the fluorescence mode and the brightfield mode wth its bright white theme If it is not already in the proper position my you will be directed to slide the Beam Block shown in Figure 26 over the brightfield illuminator located below the condenser The brightfield illuminator is not needed for fluorescence imaging and the shield cover reduces the amo
11. and only the names of filters you create not the default predefined filters provided with the Mantra system Highlight the filter entry and click Edit While you can select a predefined filter and click the Edit button to view its parameters you cannot edit them You are prevented from editing the parameters of existing filters because they may have already been used in one or more experiments If you were to edit these filters the new settings would no longer agree with the settings that were in effect during the experiment If you discover an error in the information of a recently added filter you must add the filter again and then delete the filter with the error Note that deleted filters are permanently deleted and cannot be recovered Delete Filter To delete a filter from the system first un mount the filter from its epi filter position Then highlight the filter entry in the list and click Delete You can only delete filters you have created you cannot delete any of the predefined filters Choose Yes to confirm you want to delete the filter from the system Use the Epi Filter Positions boxes 1 8 to arrange the epi filter entries so that they match the locations of actual epi fluorescence filters mounted in the turret which are as follows e Position 1 BF brightfield no filter cube present e Position 2 DAPI 52 Mantra Snap e Position 3 FITC e Position 4 CY3 e Position 5 TXRED Texas Red e Positio
12. changing the bulb or switching the unit back on Failure to do so is likely to result in damage to the bulb When to Change the Bulb The bulb is installed with a timer chip that counts the hours the bulb has been lit Once the bulb reaches the recommended lifetime of 2000 hours an audible reminder sounds each time the illuminator is switched on See Installing or Replacing the Bulbl 62 e It is recommended that the bulb be changed at this point e To silence the alarm press the Alarm Reset button located at the left of the display panel on the front of the illuminator Routine Maintenance The internal dust filters require cleaning every 12 months of use 1 Make sure the illuminator is turned OFF and disconnect the power supply Wait 30 minutes to allow the bulb to cool after turning the unit off 2 Remove the three screws that fasten the rear cover 3 Lift the rear cover off Appendix C Mantra Fluorescence Illuminator Setup and Maintenance os 4 Remove the filters 5 Wash the filters in warm soapy water 6 Rinse the filters in water and allow them to dry 7 Reinstall the filters 8 Reinstall the rear cover ensuring the isolator switches are beneath the cover 9 Replace and tighten the screws If you need to clean the exterior surfaces of the illuminator switch the power switch Off and unplug the power cable before wiping the exterior surfaces with isopropyl alcohol Alarms Warnings and Actions Th
13. chose the spectra in your sample This presumes that you do know the fluorophores present in your sample of course Figure 40 Select the fluorophores that are present in your sample from those in the Synthetic category in the Select Fluors window Based on our example choose the following synthetic stains Imaging With Mantra a from the Select Fluors window that pops up Alexa Fluor 594 Cy3 Cy5 DAPI and FITC Click OK You can enter markers to be associated with each fluorophore by clicking the Edit Markers button Simply enter the marker names into each text box and click OK Next click on the Select Autofluorescence sition With the eyedropper pointer draw in the unstained tissue to select the autofluorescence spectra and then re select the multiplexed tissue image Click the Prepare Image button The inForm program allows you to view your unmixed images in a variety of ways including fluorescence images as if they were actually brightfield IHC images You can even view each fluorophore as a color stain over a DAPI counterstain as shown in Figure 41 Of course you can view your images in black amp white or color on a dark background The autofluorescence is a separate color dark gray in fluorescence and pink eosin when viewing your image as if it were brightfield IHC Fle Edt VWews Tools Licence Help 9O Waging Nomaini Egen Tad e 7 be ae p il Figure 41 inForm ca
14. coarse and fine adjustment rings around a focus knob as well as the vertically oriented X bottom and Y top stage control knobs shown in Figure 57 Under normal circumstances you should not have to adjust the height of the microscope stage If the stage height is inadvertently changed contact PerkinElmer to obtain instructions on setting the stage to the proper height Similarly the condenser knobs and rings should not have to be adjusted after initial setup Figure 57 The right side of the Mantra instrument However if they are inadvertently shown here with the knob for focus adjustment changed you can follow the on the right To the left of the knob are the stage instructions in the Mantra Hardware controller knobs for X and Y axis control of the Setup Program to perform a specimen under the objectives Koehler alignment that will help you make the correct adjustments for optimum imaging quality eo Mantra Snap 6 Appendix B Installing the Mantra Software The Mantra software suite which includes Mantra Snap and two seats of inForm advanced image analysis software s Tissue Finder is pre installed and activated at the factory If directed to do so you can perform a software installation using software installer for Mantra Suite of software that includes Mantra Snap and inForm The installer program will instruct you to unplug the USB connection on the Mantra interface panel if it is not already unplugg
15. executable code is distributed then the accompany ing documentation must state that this software is based in part on the work of the Independent JPEG Group 3 Permission for use of this software is granted only if the user accepts full responsibility for any undesirable consequences the authors accept NO LIABILITY for damages of any kind These conditions apply to any software derived from or based on the IJG code not just to the unmodified library If you use our work you ought to acknowledge us Permission is NOT granted for the use of any IJG author s name or company name in advertising or publicity relating to this software or products derived from it This software may be referred to only as the Independent JPEG Group s software We specifically permit and encourage the use of this software as the basis of commercial products provided that all warranty or liability claims are assumed by the product vendor 18 This software uses the zlib1 1 2 7 library http www zlib net This software makes use of the zlib1 library This library has not been modified by CRI and all rights are reserved by the copyright holder This software is provided as is without express or implied warranty and with no claim as to its suitability for any purpose The library is covered by the zlib1 License The zlib 1 license is reproduced below C 1995 2012 Jean loup Gailly and Mark Adler This software is provided as is without any express or im
16. following the directions in the Mantra Snap program Good reference images ensure the high quality images from your slides You do not have to take reference images every time you use the Mantra system but you certainly must take them the first time you use it aa Mantra Snap If you have already taken references you can view them using the references editor References are stored for two trinocular positions Camera Only and Shared Adjust your trinocular to one of those two positions then click View References to see the reference images for that position If you would like to retake your references click Take References Click Done when you are finished or use the menu to select another editor Figure 29 Fluorescence References Snap Rules Select Snap Rules from the drop down menu items under the Settings button This setting area lets you select which fluorophores you are using and the order in which each fluorophore is imaged Snap Rules do not appear when you are in brightfield mode Imaging With Mantra 35 You can click on the drop down menu next to each number and choose a band Click Add Band to add an additional band or click on the red X to the right of an existing band to delete the band You need at least one band to take pictures and should only use a band once Images will be acquired in the order each band appears top to bottom So if you have a preferred order add your b
17. left If an eyepiece equipped with a diopter adjustment ring is inserted into the side equipped with another diopter adjustment ring of the observation tube perform the above procedure without moving the diopter adjustment ring of the observation tube from the 0 position Step 3 The Mantra eyepieces are equipped with rubber eyeshades that can be folded down for users with eyeglasses or folded up to prevent extraneous light from entering the eyepieces and eyes Appendix A Mantra Hardware Setup so Mantra Manual Controls The left side of the Mantra instrument features a small lever located next to the tension adjustment ring that adjusts the amount of force necessary to rotate the larger coarse adjustment ring on the focusing knob The lever is used to set the upper limit of the stage position to prevent inadvertent contact by the slide and objective The center of the knob is used for fine focus adjustments Also on the left side are the Snap and Shutter buttons located on a handgrip attached to the side of the Mantra microscope body shown in Figure 56 Figure 56 The left side of the Mantra instrument shown here with the knob for focus adjustment and the small lever behind the knob to set the upper limit of the stage position on the left To the right of the knob is the handgrip with the Snap button on top and the Shutter button on the side The right side of the Mantra instrument features additional
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19. on the stage locate a portion of Fim the slide that you wish to image You may use the Stage Control knob shown in Figure 34 to move the stage or handle the slide directly The knob is normally located to the right of the stage next to one of the large focusing knobs You can have the stage control knob as well as the main focusing knob moved to the opposite side Contact PerkinElmer s Service and Support team 111 for details on performing the procedure Figure 34 Use the Stage Control knob located by default on the right side of the Mantra instrument to move the microscope stage along the X and Y axes Set the level of fluorescence illumination by adjusting the knob labeled Light Output on the front of the Mantra Fluorescence Illuminator to 25 This should be sufficient for most fluorophores If you find that the level is too low for sufficient signal from the emitted fluorescent light turn up the level However if you find that your images are washed out the peak levels are clipped turn down the level NOTE PerkinElmer recommends setting the light output level from the Mantra Fluorescence Illuminator sufficient to allow your fluorphores to be visible in the eyepieces or on the monitor being mindful that levels that are very high increase the chance that your tissue may become photobleached where the emitted signal weakens over time wth repeated or prolonged exposure to the excitation light Excessively high illumination
20. one broadband white light source fluorescence imaging uses one or more excitation imaging Band wavelengths and corresponding epi DAPI fluorescence filter cubes located in the epi FITC or AF488 fluorescence filter turret between the trinocular and the objectives Light passing through each epi fluorescence filter cube or the included quad band filter cube hits the specimen on the slide and results in emitted fluorescence light which is rapidly captured by the solid state LCTF and camera sensor Texas Red or AF594 The Mantra Snap editor will show the current objective and highlight the current band and Figure 37 Mantra Snap Exposures exposure time If the current band is not an imaging band it will be listed below the table IMPORTANT Since many tissue specimens are subject to the phenomena called photobleaching Mantra provides a shutter mechanism that can be activated by pressing the Shutter button on the handgrip on the left side of the Mantra instrument to protect the specimen from exposure to the excitation light when the slide is not actually being observed or imaged Focus the image using the focus control knob on the Mantra microscope body You can see the slide in real time on the monitor The green bar of the Focus Indicator in the lower left of the Mantra Snap program grows longer as the image displayed is sharper and becomes shorter as the image displayed is less sharp The green bar may not extend to t
21. see an improvement in image quality as compared with a poorly aligned system 5 2 Mantra Microscopy Hardware Adjustment The Mantra camera adapter is adjusted by the PerkinElmer technician during installation to maintain proper focus on the specimen when the image is displayed on the screen as well as when you are looking through the eyepieces even with different objectives This means that your setup is parfocal although there may be some small variation If you notice that the focus is significantly different contact PerkinElmer Technical Support 14 In order to optimize your comfort while using the Mantra instrument hardware there are adjustments available to suit your own needs and preferences while using the microscopy controls Each user can make adjustments to the following settings without negatively affecting the actual functionality of the Mantra workstation itself ss Mantra Snap Mantra Eyepieces While looking through the eyepieces with 50 or 100 of the light sent to the i eyepieces adjust the interpupillary distance between the two eyepieces until you achieve binocular vision with both your left and right fields of view coinciding completely J The Trinocular Optical Path Switch can be pulled or pushed into the trinocular in order to send the light to the eyepieces or both the eyepieces and the camera i Step 1 PES Gs fia Step 2 Set the objective to 20X or 40X of your Mantra
22. shall be included in all copies or substantial portions of the Software THE SOFTWARE IS PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM DAMAGES OR OTHER LIABILITY WHETHER IN AN ACTION OF CONTRACT TORT OR OTHERWISE ARISING FROM OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE 12 This software uses the Boost 1 53 0 library http www boost org This software includes machine executable object code generated by a source language processor from the Boost libraries covered by the Boost license These libraries have not been modified by CRI and all rights are reserved by the copyright holder This software is provided as is without express or implied warranty and with no claim as to its suitability for any purpose The Boost license is reproduced below Boost Software License Version 1 0 August 17th 2003 Permission is hereby granted free of charge to any person or organization obtaining a copy of the software and accompany ing documentation covered by this license the Software to use reproduce display distribute execute and transmit the Software and to prepare derivative works of the Software and to permit third parties to whom the Software is furnished to do so all subject
23. sharp the image in the Image Window as Mantra Snap appears Settings Menu New Protocol New Protocol Load Protocol Load Protocol Save Protocol Save Protocol Mantra Snap Mantra Snap References References Post Processing Snap Rules Exposures License Info Post Processing Upgrade License What s New License Info Help Upgrade License Log What s New Help About Log Figure 8 Mantra Snap 9 drop down menu settings About for brighttfield Figure 9 Mantra Snap drop down menu settings for fluorescence Drop down menu settings as shown in Figures 8 and 9 vary slightly for brightfield and fluorescence imaging New Protocol creates a new protocol for which you will need to choose settings CTRL N will bring up the new protocol dialog where you can choose to create a brightfield or fluorescence protocol Load Protocol allows you to load an existing protocol e g the default brightfield or fluorescence protocols provided by the factory or a protocol that you have created or modified Existing protocols can be opened modified and then saved under different names to create customized new protocols CTRL O will bring up the load protocol dialog Save Protocol saves a protocol that you have created or modified CTRL S will bring up the save protocol dialog Mantra Snap the main settings window for taking pictures using the Mantra system
24. that the following conditions are met 1 Redistributions of source code must retain the above copyright notice this list of conditions and the following disclaimer 2 The origin of this software must not be misrepresented you must not claim that you wrote the original software If you use this software in a product an acknowledgment in the product documentation would be appreciated but is not required 3 Altered source versions must be plainly marked as such and must not be misrepresented as being the original software 4 The name of the author may not be used to endorse or promote products derived from this software without specific prior written permission THIS SOFTWARE IS PROVIDED BY THE AUTHOR AS IS AND ANY EXPRESS OR IMPLIED WARRANTIES INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED IN NO EVENT SHALL THE AUTHOR BE LIABLE FOR ANY DIRECT INDIRECT INCIDENTAL SPECIAL EXEMPLARY OR CONSEQUENTIAL DAMAGES INCLUDING BUT NOT LIMITED TO PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES LOSS OF USE DATA OR PROFITS OR BUSINESS INTERRUPTION HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY WHETHER IN CONTRACT STRICT LIABILITY OR TORT INCLUDING NEGLIGENCE OR OTHERWISE ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE Julian Seward Cambridge UK jseward bzip org bzip 2 libbzip2 version 1 0 4 of 20 Dece
25. the Mantra instrument If it is not follow the instructions below 1 Position the illuminator on the bench so that none of the fan vents are obstructed 2 Unpack the liquid light guide from the foil packaging and remove both plastic caps from the light guide 3 Unscrew the connector on the front of the illuminator and fully insert the light guide Push the light guide in until it stops 4 Tighten the connector until tight to fully secure the light guide to the illuminator 5 Loosen the screw on the back of the collimating lens and push the light guide firmly into the hole Make sure it reaches the end stop and then tighten the screw 6 Attach the collimator to the Microscope s fluorescence illumination port 7 Switch the illuminator on and adjust the collimator for light evenness a Loosen the silver lock ring b Unscrew the end of the collimator c When light is even screw the silver lock ring forward locking the collimator end into place NOTE The liquid light guide has a limited lifetime regardless of whether it is in storage or in use However the lifetime may vary depending on climate conditions Cold and humid environments tend to extend lifetime hot and or dry envronments tend to shorten it Even though UV performance does not markedly degrade during usage it is recommended that the light guide be replaced before its expected lifetime expires The formation of bubbles in the liquid degrade performance and optical o
26. using the well known Koehler method of alignment Unless you make changes to your hardware you should not have to launch the setup program You could periodically launch the program in order to perform Koehler alignment if you believe you re your condenser and field stop settings might have been inadvertently changed Information is provided here for reference purposes The Mantra Hardware Setup program can be launched from Programs PerkinElmer Mantra 1 0 0 so Mantra Snap Objectives If you are instructed by PerkinElmer to add tiene tardnan sae or remove an objective on your Mantra system you can account for that change in w Q l i the Objectives section of the setup program are The Turret Size defaults to 7 and is not editable The Mantra hardware features a fore arse i seven position turret and the software ane requires the presence of 7 positions All oom positions need not be populated though Use the Available Objectives box to create 3 7 Emoy or edit entries for each of the objectives mounted in the objectives turret Figure 47 Adding or deleting objectives from within the Mantra Hardware Setup program To add entries click the Add button Enter a short descriptive name and choose its nominal magnification Click OK when finished The new entry is added to the list To edit the name of any objective highlight the objective entry and click Edit You can only edit the
27. wavelength would be 650nm For any subsequent bands you create the peak wavelength is 20nm above the band s starting wavelength LCTF The Mantra system always uses a broad bandwidth Liquid Crystal Tunable Filter so this entry is display only and cannot be changed sa Mantra Snap COM Ports If instructed you can change the Mantra ordre Set communications ports used by the Mantra system a ar 4 eC COM Ports Use this panel to select which COM port connects to each device Once a COM port is assigned to a device it is unavailable for res s z RS232 Port om s selection for the other devices To reassign e AAA a COM port remowe it from the current device and then assign it to the new device E The RS232 port provides communication to the microscope hardware and the USB port provides communication to the PerkinElmer electronics controller Clicking on the Validate COM Ports button tests the selected ports to confirm proper operation e g each COM port is controlling the proper hardware so that you do not receive error messages upon Mantra Snap software program startup Figure 50 The communications setting can be adjusted from within the Mantra Hardware Setup program NOTE Under most circumstances the RS232 port will be assigned COM1 Appendix A Mantra Hardware Setup 55 Koehler Alignment Finally you can follow step by step instructions to properly align the Mantr
28. 2 1 Hardware Setup The Mantra system is normally installed and configured for the first time by a PerkinElmer technician Training and Service packages beyond initial set up and training are available at nominal cost In this section we will simply confirm that the Mantra hardware is set up properly before use 1 Check that the Power and USB cables shown in Figure 3 far left are plugged into the connectors on the back of the Mantra Imaging Modulel 6 2 Check that the USB cable for the computer from the Interface Panell 7 identified in Figure 2 on the left side of the Mantra instrument is plugged into the computer 3 Check that the Power cable from the Interface Panel is plugged into a surge protected power outlet lt A A T Z m a i E a gt 7 Figure 3 Power and USB cables plugged into the back of the Mantra imaging module far left the location of the Mantra Power switch outlined in red middle left objectives in their numbered turret positions middle right and epi fluorescence filter cubes in their designated numbered locations far right If the Mantra system is not already on switch the power switch shown in Figure 3 middle left located on the lower left of the black panel on the left side of the main instrument body to the ON position The default objectives in the motorized objective turret 7 shown in Figure 3 middle right are 4X 10X and 20X A 40X objective is optional The epi fluorescence exc
29. 35 Mantra Snap Exposures Select the Exposures drop down menu item from the Settings button to go to the Exposures settings area Exposures will be displayed for your current objective If you have loaded a previously saved protocol your saved exposures will be displayed If you haven t assigned exposures for this objective and band a default exposure is DAPI 10 used FITC or AF488 Select the first Imaging Band e g DAPI and locate the brightest area on the sample before clicking Autoexpose z The system will automatically change to the correct epi filter Texas Red or AF594 21059 the shutter will be opened if it was previously closed and the system will calculate the optimal exposure time After autoexposure is complete the shutter will be returned to its previous position You can also manually select an exposure time in milliseconds by typing directly into the box in the Exposure column to the right of each of your imaging bands Exposure times must be between 1 and 2000 milliseconds If you d like to set exposures for a different objective use the Objective Rocker Switch on the hardware to select a new objective Figure 31 The Exposures editor lets you If you d like to set exposures for a different imaging band you Uloexpose each band for each objective or can either select the Imaging Band or use the Epi Filter manually set the exposure time in rocker switch The system will automatically hig
30. ARE DISCLAIMED IN NO EVENT SHALL THE COPYRIGHT HOLDERS OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT INDIRECT INCIDENTAL SPECIAL EXEM PLARY OR CONSEQUENTIAL DAMAGES INCLUDING BUT NOT LIMITED TO PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES LOSS OF USE DATA OR PROFITS OR BUSINESS INTERRUPTION HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY WHETHER IN CONTRACT STRICT LIABILITY OR TORT INCLUDING NEGLIGENCE OR OTHERWISE ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAM AGE Software EULA The BSD for libjpeg license is reproduced below The authors make NO WARRANTY or representation either express or implied with respect to this software its quality accuracy merchantability or fitness for a particular purpose This software is provided AS IS and you its user assume the entire risk as to its quality and accuracy This software is copyright C 1991 2010 Thomas G Lane Guido Vollbeding All Rights Reserved except as specified below Permission is hereby granted to use copy modify and distribute this software or portions thereof for any purpose without fee subject to these conditions 1 If any part of the source code for this software is distributed then this README file must be included with this copyright and no warranty notice unaltered and any additions deletions or changes to the original files must be clearly indicated in accompany ing documentation 2 If only
31. Contributor has been advised of the possibility of such damages 9 Accepting Warranty or Additional Liability While redistributing the Work or Derivative Works thereof You may choose to offer and charge a fee for acceptance of support warranty indemnity or other liability obligations and or rights consistent with this License However in accepting such obligations You may act only on Your own behalf and on Your sole responsibility not on behalf of any other Contributor and only if You agree to indemnify defend and hold each Contributor harmless for any liability incurred by or claims asserted against such Contributor by reason of your accepting any such warranty or additional liability 16 This software uses the bzip2 program and libbizip2 libraries http www bzip org This software makes use of the bzip2 program and associated libbzip2 libraries The program and libraries have not been modified by CRI and all rights are reserved by the copyright holder This software is provided as is without express or implied warranty and with no claim as to its suitability for any purpose The libraries are covered by the following license The bzip2 license is reproduced below This program bzip2 the associated library libbzip2 and all documentation are copyright C 1996 2006 Julian R Mantra Snap Seward All rights reserved Redistribution and use in source and binary forms with or without modification are permitted provided
32. From here you can load protocols specify a location to save your images specify a base filename and starting number for your image files and take pictures using the software Snap button or the Snap button on the Mantra instrument itself References allows you take reference images and review them if necessary Imaging With Mantra 9 Snap Rules Fluorescence mode only lets you choose which fluorophores you want to image and the order in which the images are taken of each one Exposures Fluorescence mode only lets you set the exposure in milliseconds that you wish to use for each fluorophore Post Processing lets you decide if you would like your images automatically transferred to the included inForm advanced image analysis program or a Windows batch process file run License Info displays your Mantra Snap software licensing information Upgrade License allows you to upgrade to additional functionality i e pay for additional features Free incremental upgrades are handled using the standard installation procedure and do not use this What s New find out which features in this software version are new and obtain a description of each new feature Help Access to the online help feature including this document e Log access to the log file that could be useful for record keeping or troubleshooting About information about this software release version 20 Mantra Snap 2 4 Taking Brightfield Images A
33. INTERRUPTED OR ERROR FREE OR THAT DEFECTS IN THE SOFTWARE WILL BE CORRECTED YOU ASSUME ALL RISK ASSOCIATED WITH THE USE RESULTS AND PERFORM ANCE OF THE SOFTWARE Software EULA 7 Limitation of Liability IN NO EVENT SHALL CRI ITS AFFILIATES OR SUPPLIERS OR THEIR RESPECTIVE EMPLOYEES OFFICERS OR AGENTS BE LIABLE FOR ANY DAMAGES ARISING OUT OF THE USE OR INABILITY TO USE THE SOFTWARE INCLUDING WITHOUT LIMITATION INCIDENTAL SPECIAL CONSEQUENTIAL PUNITIVE EXEMPLARY INDIRECT OR DIRECT DAMAGES INCLUDING WITHOUT LIMITATION DAMAGES FOR LOSS OF PROFITS LOSS OF DATA RE RUN TIME INACCURATE INPUT WORK DELAYS BUSINESS INTERRUPTION OR ANY OTHER COMMERCIAL DAMAGES OR LOSSES WHETHER IN AN ACTION IN CONTRACT TORT INCLUDING NEGLIGENCE AND STRICT LIABILITY OR OTHERWISE AND EVEN IF CRI HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES AND REGARDLESS OF WHETHER ANY REMEDY FAILS OF ITS ESSENTIAL PURPOSE CRI s AGGREGATE LIABILITY ARISING OUT OF OR RELATING TO THIS AGREEMENT FOR ANY AND ALL DAMAGES THAT YOU MIGHT INCUR REGARDLESS OF THE FORM OF ACTION GIVING RISE TO SUCH LIABILITY WHETHER IN CONTRACT TORT OR OTHERWISE SHALL NOT EXCEED ONE HUNDRED DOLLARS 100 Some jurisdictions do not allow the exclusion or limitation of liability for consequential or incidental damages In such jurisdictions our liability is limited to the greatest extent permitted by law or the amount you paid for your purchase whichever is less 8 U S Governmen
34. License does not grant permission to use the trade names trademarks service marks or product names of the Licensor except as required for reasonable and customary use in describing the origin of the Work and reproducing the content of the NOTICE file 7 Disclaimer of Warranty Unless required by applicable law or agreed to in writing Licensor provides the Work and each Contributor provides its Contributions on an AS IS BASIS WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND either express or implied including without limitation any warranties or conditions of TITLE NON INFRINGEMENT MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE You are solely responsible for determining the appropriateness of using or redistributing the Work and assume any risks associated with Your exercise of permissions under this License 8 Limitation of Liability In no event and under no legal theory whether in tort including negligence contract or otherwise unless required by applicable law such as deliberate and grossly negligent acts or agreed to in writing shall any Contributor be liable to You for damages including any direct indirect special incidental or consequential damages of any character arising as a result of this License or out of the use or inability to use the Work including but not limited to damages for loss of goodwill work stoppage computer failure or malfunction or any and all other commercial damages or losses even if such
35. Snap Software User s Manual for Mantra Snap 1 0 pP PerkinElmer Notice This manual is published by PerkinElmer Inc The information in this document is subject to change without notice and should not be construed as a commitment by PerkinElmer Inc PerkinElmer assumes no responsibility for any errors that may appear in this document This manual is believed to be complete and accurate at the time of publication In no event shall PerkinElmer be liable for incidental or consequential damages in connection with or arising from the use of this manual This manual describes Mantra Snap version 1 0 software For more information contact PerkinElmer Inc 68 Elm Street Hopkinton MA 01748 USA Phone 800 762 4000 or 1 203 925 4602 Fax 1 203 944 4904 Email global techsupport perkinelmer com Web site http Avww perkinelmer com This software covered by US Patent 5 953 087 and 7 655 898 Table of Contents Part Welcome to Mantra 5 1 Hardware OVE MEW ecseri aea lak deegentahd ab danccincinha dns duh cevgatbha deel dandst nclndad euch dud daspandebdnal duce Sncpdatends duane 6 2 System Specifications 0 eee eeee cece ee cece cece cece eee N AEEA EEE aa KEE AEAN EEE EENE EEE EEEN 8 3 SYSTSMT REQUIFEMENIS eaii RE TERE EE OEO 8 4 Operator and Equipment Safely scuis iernii n EEE EE EAE OENE EAE NEEE 9 5 Important Safety Information for the Fluorescence IllUMinatol c cceeeeeeeeeeceeeeeeeeeee
36. a optical imaging system using the well known Koehler alignment method Follow the steps as they appear when you click Next You can always go back if you are unsure if you correctly followed the instructions IMPORTANT If you perform Koehler alignment you should re take your brightfield references Figure 51 The Koehler alignment technique can ensure optimum quality of your images with good uniformity across the image excellent contrast and maximum efficiency of your light source Step 1 Select a brightfield slide with good detail that you can focus on Place it on the microscope stage and focus imple Setup 1 Select a brightfield specimen slide place it on the microscope stage and focus on a specimen region Figure 52 Step 1 of the Koehler alignment procedure requires a brightfield slide with good detail that you can properly focus upon 56 Mantra Snap Step 2 Use the Objective rocker switch on isasBainesny your Mantra system to select the highest magnification 20X or 40X depending on your A configuration Close down the field stop by amp D rotating the ring located around the brightfield illuminator highlighted in yellow in 00 e Adustment Figure 53 Then adjust the height of the a een on Dieci lawon the sample condenser by turning one or both of the Se eee ester ete semen condenser height adjustment knobs sc ape ne il ah Ppl highlighted in red in Figure 53 until
37. accord e a l utilisateur Contact PerkinElmer at global techsupport perkinelmer com for more information 12 Mantra Snap 2 Imaging With Mantra The best way to learn how to use the Mantra workstation is to follow this portion of the user s manual Imaging With Mantral 13 will guide you through taking a brightfield image and taking a fluorescence image Your Mantra system comes with inForm PerkinElmer s advanced image analysis program The Mantra Snap program can be configured to automatically send each image to inForm inForm can be set up to open your images and even to automatically process them using a predetermined algorithm or image processing routine can be done from within the Mantra Snap program You may wish to have the inForm user s manual available because if you would like to try using this automatic transfer feature the following exercises can be continued in the inForm users manual in Appendix B Auto Processing Data Received from Mantra Figure The Mantra system comes with the Mantra Snap software program for image acquisition and th inForm Tissue Finder advanced image analysis software program for trainable image analysis that can be easily automated o Hardware Setup 14 o Installing and Starting the Mantra Snap Softwarel 15 o Understanding the Mantra Snap Work Areal 1 o Taking Brightfield Images A Practice Exercisel 21 o Taking Fluorescence Images A Practice Exercisel 3 Imaging With Mantra
38. ademarks tools know how and processes in and to the Software The Software contains confidential information and trade secrets of CRI You i acknowledge and agree not to contest CRI s rights in the Software and ii agree not to disclose any confidential information of CRI regarding the Software or that is otherwise disclosed to you in connection with this Agreement You recognize that the covenants contained in this License are reasonable and necessary to protect the legitimate interests of CRI that CRI would not have entered into this Agreement in the absence of such covenants and that y our breach or threatened breach of such covenants shall cause CRI irreparable harm and significant injury the amount of which shall be extremely difficult to estimate and ascertain thus making any remedy at law or in damages inadequate Therefore you agree that CRI shall be entitled without the necessity of posting of any bond or security to the issuance of injunctive relief by any court of competent jurisdiction enjoining any breach or threatened breach of such covenants and for any other relief such court deems appropriate This right shall be in addition to any other remedy available to CRI at law or in equity 4 Termination The license granted in Section 1 above is effective until terminated This Agreement is conditioned upon your continued compliance with the terms and conditions hereof and will terminate automatically without notice from CRI if you f
39. ail to comply with any term or condition of the Agreement Furthermore CRI may terminate this Agreement at any time upon thirty 30 days notice Upon termination of this Agreement you shall immediately destroy all copies of the Software including all accompany ing documentation and any other confidential and proprietary information you have received during or in connection with this Agreement 5 Limited Warranty CRI warrants that the media on which the Software is provided will be free from defects in materials and faulty workmanship under normal use for a period of ninety 90 days from the date of delivery Your exclusive remedy under this Section 6 shall be at CRI s option a refund of the price paid for the Software or replacement of the media on which the Software was provided so long as that media has been returned to CRI under a CRI issued return authorization CRI shall have no responsibility to replace media damaged by accident abuse or misapplication 6 No Other Warranties EXCEPT FOR THE LIMITED WARRANTY STATED IMMEDIATELY ABOVE THE SOFTWARE IS PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND AND CRI EXPRESSLY DISCLAIMS ANY AND ALL IMPLIED WARRANTIES INCLUDING WITHOUT LIMITATION ANY WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT OF THIRD PARTY RIGHTS CRI DOES NOT REPRESENT OR WARRANT THAT THE RESULTS OR THE USE OF THE SOFTWARE WILL BE CORRECT ACCURATE OR RELIABLE OR THAT THE SOFTWARE WILL OPERATE UN
40. am window is open the new images will be processed just like the first image that you worked on Of course if you switch to a different slide that was prepared with different staining or stains your results will not be consistent with the first slide 2 5 Taking Fluorescence Images A Practice Exercise This section will guide you through taking fluorescence images using the Mantra imaging system Go to the previous section if you wish to take brightfield IHC images instead o Configuring Mantra for Fluorescence Imaging 37 o Taking a Fluorescence Imagel 32 o Processing a Fluorescence Image in inForm 4 2 5 1 Configuring Mantra for Fluorescence Imaging Make sure that the Mantra hardware including the fluorescence illuminator is properly configured and powered up 141 Then turn on the computer and wait until Windows has finished booting up Launch the Mantra Snap software by double clicking on the icon on the desktop or by going to C Program Files PerkinElmer Mantra 1 0 0 MantraSnap exe lf you are working in a laboratory with subdued lighting you can change the wallpaper on your computer monitor to a darker theme I gt Go to C Program Files PerkinElmer Mantra 1 0 0 biasi right click on PKI _AltBackground jpg and choose Figure 25 You can select the alternative darker Set as Desktop Background wallpaper for fluorescence imaging in a lab with subdued lighting Load the default fluorescence protocol by clicking on the
41. ands in that order You can also adjust the order afterwards by changing each selected band via the drop down menu selector The following fluorescence bands are loaded at the factory e DAPI e FITC or AF488 e Cy3 or TRITC e Texas Red or AF594 e Cy5 e Cy7 optional i Figure 30 The Snap Rules editor NOTE the bands that appear in the drop down menus have allows you to select which been previously defined in the Hardware Setup program fluorescence bands to acquire described in Appendix A 50 You cannot add bands that do not already appear in the Hardware Setup program PerkinElmer recommends that you have the Use Saturation Protection checkbox selected This will only use auto exposure when necessary protecting you from unintentional overexposure and clipping potentially valuable signal As a safeguard against inadvertently taking an image when the trinocular optical path switch is not in the desired position e g no light or not enough light going to the camera you can select among the following options in Prior to acquisition the trinoc must be at e Camera Only e Shared e Either Camera Only or Shared Mantra Snap will warn you if the optical path switch is ina position that you do not desire In order to ensure that your data is quantitative your trinocular should always be set to the same position for acquisition Click Done when you are finished or use the menu to select another editor
42. are provides rapid and intuitive image capture and works with the controls on the Mantra instrument itself The system can be configured so that the images you take are automatically and seamlessly transferred to the inForm software for segmentation and phenotyping in the background while you continue to take more images o Hardware Overview 6 o System Specifications 8 o System RequirementsI 8 o Operator and Equipment Safety 9 o Important Safety Information for the Fluorescence Illuminator 14 o Table of Symbols o Technical Supporti o About This Manual 12 o CE Testing and Certification 141 Welcome to Mantra 5 1 1 Hardware Overview The Mantra Quantitative Pathology Imaging System consists of the Mantra instrument and Mantra Snap and inForm software You can take pictures by using the Mantra Snap software or by using the controls on the instrument while looking into the eyepieces You can even configure the Mantra Snap software to automatically send your images to the included inForm Tissue Finder software for processing and analysis while you continue to take more pictures Mantra Imaging Module Mantra Eyepieces i Mantra Widescreen Monitor Be Mantra Trinocular Mantra Power Switch Mantra Computer Mantra r aa Filter Turret Mantra Stage Controller A R Mantra Focus Knob Mantra Interface Panel Mantra Front Panel Controls amp Snap Shutter But
43. com This software makes use of the CommandLine libraries These libraries have not been modified by CRI and all rights are reserved by the copyright holder This software is provided as is without express or implied warranty and with no claim as to its suitability for any purpose The libraries are covered by the MIT License The MIT License is reproduced below The MIT License MIT Copyright c 2005 2012 Giacomo Stelluti Scala Permission is hereby granted free of charge to any person obtaining a copy of this software and associated documentation files the Software to deal in the Software without restriction including without limitation the rights to use copy modify merge publish distribute sublicense and or sell copies of the Software and to permit persons to whom the Software is furnished to do so subject to the following conditions The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software THE SOFTWARE IS PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM DAMAGES OR OTHER LIABILITY WHETHER IN AN ACTION OF CONTRACT TORT OR OTHERWISE ARISING FROM OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE 15 This
44. ctive in brightfield e Non mechanical sensing of trinocular selection Integrated ergonomic controls on microscope base amera type Camera spectral range e 440 720nm for brightfield imaging and visible fluorescent dyes e 770nm for near infrared NIR dyes such as Cy7 Alexa Fluor 750 and similar bjectives 4X 0 16 NA 10X 0 40 NA 20X 0 75 NA 40X 0 95 NA optional 0 25 micron optional 40X 0 50 micron 20X 1 0 micron 10X 2 5 micron 4 Epi fluorescence filter cubes DAPI FITC TRITC Cy5 Cy7 optional Quad band for DAPI FITC TRITC Cy5 optional for eyepiece visualization Fluorescence illumination Metal hallide 200W lamp with liquid light guide 2000 hour bulb life Brightfield illumination High output color balanced white LED Physical Dimensions Mantra Workstation W x H x 163 x 76 x 56 cm including Mantra fluorescence illuminator monitor D computer keyboard mouse 145 x 76 x 56 cm as above without computer omputer minimum specifications General Quad core 16 GB RAM i5 processors mini tower style perating system nterfaces Monitor oftware cquisition software Mantra Snap 1 seat Image analysis software inForm Tissue Finder 2 seats 1 3 System Requirements Mantra System Power Requirements C input power 00 230 VAC 1 0A 50 60Hz 1 uses 2x 2 0A 250V P N CLS 139063 Mantra Fluorescence Illuminator AC inputpower 100 240 VAC 2 0 50 60Hz 3 Mantra Snap Mantra Fluorescence Illu
45. cus of your slide on the Mantra microscope stage Mantra Front Control Panel contains switches for changing the Mantra s epi fluorescence filters and objectives a button to change modes from brightfield BF to fluorescence FL and a large knob surrounded by active indicators to adjust the illumination level Mantra Snap Shutter Buttons located on a hand controller to manually snap pictures and open or close the Fluorescence Illuminator shutter Mantra Interface Panel located on the left side of the Mantra instrument as you sit in front of it contains the power input connector and fuse USB ports for the computer optional footswitch not yet enabled and optional microphone not yet enabled and an RS232 port for control of the microscope objective and epi fluorescence filter turrets as well as the shutter Mantra Computer comes with the Mantra instrument and has the Mantra Snap and inForm software installed and activated at the factory Also included are a wireless keyboard and mouse Mantra Widescreen Monitor ultra widescreen monitor enables side by side viewing of both the Mantra Snap and inForm program windows Welcome to Mantra 1 2 System Specifications maging Modes Brightfield and fluorescence multispectral and color imaging Microscope Customized research grade Olympus microscope with e 8 position motorized epi fluorescence filter turret e 7 position motorized objective nosepiece e Active management of intensity per obje
46. d to take your reference images if this is the first time you have used the system You might also be prompted to slide the Beam Block shown in Figure 10 out of the way if it is covering the brightfield illuminator Figure 10 The Beam Block the plastic cover shown in the red box slides over the brightfield illuminator when you are using the Mantra system to take fluorescence image The Mantra Snap program will prompt you to slide it into or out of place when necessary Imaging With Mantra 21 Taking References You can skip ahead past this step if reference images have already been taken Follow the directions in the Mantra Snap program to take your reference images The Mantra system will select different objectives adjust exposures by itself and prompt you to take actions such as to change the setting of the trinocular optical path switch or move the slide to a new location using the stage controls When the references have been taken click Done and you will be taken back to your initial settings your previously selected objective and illumination setting NOTE Reference images help produce optimal images no matter what objective is used They compensate for small optical imperfections that are a normal part of any optical system You will need to have a clean microscope slide available in order to follow the directions for taking your reference images You wil need to take a set of reference images the fi
47. e Select Stains button When the Select Stains window opens click the checkboxes representing the stains that are present on your sample slide and then click OK W form vasi An e Project Me id Vem Teo Lemoe Hep Figure 22 Back to the Manual Analysis tab in inForm where you can now click on the Select Stains button to identify the stains for which you previously saved the spectra New information and options will appear below the Select Stains COE button including a list of the Spectra that will be used for Unmixing spectrally separating the two stains from another in your image Click 420 460 500 5 550 620 660 700 Prepare Image to oem pany unmix the image o pee 3 Source C Users mantra Desktop samie_2 im Notes Figure 23 You can now select the spectra of the two stains that are presentin your sample slide Once the image has been unmixed you can use the Pathology Views button to show just the hematoxylin or just the eosin You can also show the image as it would appear in fluorescence oa Mantra Snap E E pr a pa Eosin a nw Hematoxylin aa E E Figure 24 The Pathology Views button left allows you to select and view the individual stains in this case eosin middle and hematoxylin right Now that you have successfully chosen stains and unmixed an image you can return to the Mantra Snap program and continue to take images As long as the inForm progr
48. e inForm User s Sarie Foret Sih Sa ta A Manual Building Libraries for more details _ ns Ye ee ae pan we fat a y E S nN Zr E b f nf RR z Review Merge Batch Analysis Manual Analysis Budd Libraries s gt ae Be R Figure 46 Once you have selected a stain or fluorophores in the Choose Which Types of Stains and Fluors You Lab Uses window of the Stain Store Manager the stain or fluorophore will appear in the Stain drop down menu in the inForm Build Libraries tab Frequently Asked Questions and Troubleshooting a 5 Appendix A Mantra Hardware Setup This appendix covers both the Mantra Hardware Setup program as well as adjusting the Mantra microscopy hardware to suit your particular individual operating preferences e g eyepiece viewing and ergonomic adjustments Under normal circumstances you should not need to perform additional setup or maintenance procedures If you have any questions do not hesitate to contact PerkinEIlmer s Technical Support 14 team o Mantra Hardware Setup Program 50 o Mantra Microscopy Hardware Adjustmentl 58 5 1 Mantra Hardware Setup Program When your Mantra system is first set up by the PerkinElmer technician who visited your laboratory the Mantra Hardware Setup program is run allowing the technician to configure your hardware with the proper objectives epi fluorescence filters 2 communications settings as well as to properly align the optical system
49. e number 20 digits in length ready If you wish to capture images using the Mantra instrument power up the Mantra system as described in the previous section and start your computer When Windows has finished booting start the Mantra Snap program by double clicking on the Mantra Snap icon on the td desktop shown below in Figure 5 or go to Mantra 1 0 0 Start gt gt All Programs gt gt PerkinElmer gt gt Mantra gt gt 1 0 0 gt gt Mantra 1 0 0 pe Snap Desktop Icon Understanding the Mantra Snap Work Area The Mantra Snap program is designed to help you capture the images you want with as little effort as possible You can keep your hands on the controls built into the Mantra instrument instead of making multiple clicks on a mouse button and looking back at the computer monitor You can even configure the Mantra Snap software to automatically send your images to the included inForm software for processing while you take more pictures Shown below in Figure 6 are the important interface elements of the Mantra Snap program 16 Mantra Snap D Settings Area i E Focus Indicator Figure 6 Mantra Snap brightfield interface is shown above Note the large Image Window with the Settings Area on the right Different settings can be selected using the Settings Button in the upper right side of the window justbelow the red Close Window button Imaging With Mantra a MenteaSngp DAPI 3 S Figure 7 Man
50. e table below lists the warnings that may appear on the display on the front of the Mantra Fluorescence Illuminator and the recommended actions to address the 2 Message Alarm Reason Quiet Alarm Bulb Fault 1 Software not No acions off unit recognizing hour count Check bulb for damage and from bulb check connections Bulb Fault 2 Bulb failure Yes Switch off unit Hold Alarm Reset button for 5 10 Check bulb for damage and seconds check connections Reset to clear alarm Over Temp Bulb over temperature Yes Check vents at rear of unit Fault Hold Alarm Reset button for 5 10 Make sure that they are not seconds blocked or covered Reset to clear alarm CHANGE Bulb reached 200 hour Yes BULB lifetime limit Hold Alarm Reset button for 5 10 seconds Reset to clear alarm gs Mantra Snap Software EULA The following is an agreement the Agreement between you and Cambridge Research amp Instrumentation Inc 68 Elm St Hopkinton MA 01748 CRI for software known as Mantra Snap and its accompanying documentation collectively the Software By installing and or using the Software you agree to the following terms and conditions If you do not agree to all of the terms and conditions in this Agreement you may not install or use the Software 1 Single Use License The Software is licensed to you and not sold Subject to the terms and conditions of this Agreement CRI hereby grants to you a restricted non exclusive
51. ed This is very important in order for the installation to proceed correctly IMPORTANT Figure 58 You must unplug the MantraUSB connection on the Mantra interface panel before performing an installation or an upgrade of the Mantra software Prior to running the installer program unplug the Mantra USB connection Once installed the Mantra Snap software wil not run without a working Mantra Instrument attached You will need to be logged in as 9 Mantra License Applicat an Administrator on your Windows computer To activate ee eee T your Mantra Snap license derail akg online start up the licensing app at Start gt gt PerkinElmer gt gt Mantra gt gt 1 0 0 gt gt Mantra License SSS ee ee App Figure 59 Mantra Licensing Application Have your 20 character license number ready and follow the instructions You can also reinstall inForm if necessary Please see the section Activating the License of the inForm Manual Appendix B Installing the Mantra Software i at 7 Appendix C Mantra Fluorescence Illuminator Setup and Maintenance The Mantra Fluorescence Illuminator is a 200W module that provides epi fluorescence illumination of slides on the Mantra microscope stage It has 6 levels of illumination 0 10 25 50 75 and 100 using a self aligning temperature controlled 200W metal halide bulb The Mantra Fluorescence Illuminator is normally unpacked and installed by a PerkinE lmer trai
52. eeeeees 10 6 Table Of SYMONS ees E E 11 T Technical SUPPO eiren emesis TEE EOT E AEE EEE E 11 S About MiS Mantakna E E E E A 12 9 CE Testing and CernincaiONesse ieii a R canhndecouahgeccansaiee 12 Part II Imaging With Mantra 13 1 Hardware Setup siecs ecteien cisiees accuse E EE E E E E ganar EEE E 14 2 Installing and Starting the Mantra Snap Software cccececceseeeeeeeneeeeeeeeeeeeeeaaeeeeeeaaeeeeeeaaeeeeees 15 3 Understanding the Mantra Snap Work Area ccceececeneeeee cena ener eeaaeeeeeaaeeeeeeaaeeeeeeaaeeeeeeaaeeeees 16 4 Taking Brightfield Images A Practice Exercise ccccceececenneeeeeeeeeeeeeeaaeeeeeeaaeeeeeeaaeeeeeeaaeeeeees 21 Configuring Mantra for Brightfield IMaQiING ceeceeeeeeeeeneeeeeeaeeeeeeeaaeeeeeeaaeeeeeeaaeeeeeeaaanees 21 Taking a Brighttiold Images fccsushascesexadecestsetueter ta decead saguenne tudes dedavGneceedvdveccdatenecnatatceantaaences 24 Processing a Brightfield Image in INFOIM ccceeeeeeeeeeeeeeeeeeeeeeeeaeeeeeeeeaeeeeeeeaeeeeeeeaeeeeenees 26 5 Taking Fluorescence Images A Practice EXxerciSe cccececesneeeeeeaeeeeeeeaaeeeeeaaeeeeeeaaeeeeeeaaanees 31 Configuring Mantra for Fluorescence Imaging eeeeeeeneeeeeeneeeeeeeaaeeeeeaaeeeeeeaaeeeeeeanenees 31 Taking a Fluorescence IMaQe sensisse eiei e EEEE EE EEEE 38 Processing a Fluorescence Image in iNFOrm cceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaeeeeeeaaeeeeeeaes 41 Par
53. eyepieces using the BF Lamp knob on the right Imaging With Mantra 23 2 4 2 Taking a Brightfield Image Locating Your Sample With your sample on the stage locate a portion of the slide that you wish to image You may use the Stage Control knob shown in Figure 14 to move the stage or handle the slide directly The knob is normally located to the right of the stage next to one of the large focusing knobs You can have the stage control knob as well as the main focusing knob moved to the opposite side Contact PerkinElmer s Service and Support team 111 for details on performing the procedure Figure 14 Use the Stage Control knob located by default on the right side of the Mantra instrument to move the microscope stage along the X and Y axes Destination Folder Select a destination folder for your images by clicking on the Save Snap To Browse button at the bottom of the Mantra Snap Settings area to the right of the live image The default location is D Save Snap To Browse Data MantraSnap C Users mantra Desktop NOTE the Mantra computer comes with two hard disk drives Base Filename sample_ one for the Windows operating system and the Mantra Snap and Swing Number Ti inForm software C drive and one for your data D drive PerkinElmer recommends saving your data to the D drive and making back up copies whenever possible to avoid data loss in case your hard disk drive ever fails Your insti
54. g to the Settings button and choosing Post Processing You can refer to the inForm Users Manual version 2 1 0 or later for additional details on the program s capabilities and features In order to save you from flipping back and forth amongst the two separate user s manuals for this particular exercise the directions in Processing a Fluorescence Image in inForm 41 pertain to the inForm Advanced Image Analysis software program that comes with each Mantra system 2 5 3 Processing a Fluorescence Image in inForm Under normal circumstances you would capture images from single stained slides as well as from some unstained tissue in order to build up a library of spectra precise colors that are really unique multispectral profiles of your stains PerkinElmer calls this a Stain Store of the stains and fluorophores that you would see in the normal course of your research To help get you started PerkinElmer includes some commonly used and characterized fluorescence spectra referred to as synthetic stains because they were not captured and characterized on your particular Mantra system However these synthetic stains will be close enough for instructional purposes that we will use them here and note when you could use single stained samples if and when they are available to you We highly recommend having an unstained slide in order to obtain the spectra of your tissue sample s autofluorescence Imaging With Mantra Loa
55. he full length of the indicators space So simply strive for the longest green bar and the highest number possible When you are ready push the Snap E button on the handgrip located to the left of the Mantra lower front control panel or click on the Snap button at the bottom right of the Mantra Snap program You may observe the slide through the eyepieces or watch the LCD monitor while snapping your images The system will acquire imagery of your imaging bands returning back to the original epi filter when it is done NOTE If you hold down the Alt key on your keyboard while clicking on the Snap button in the Mantra Snap program the button changes from Snap to Snap Color and you wil simply save a color TIFF image instead of a full multispectral IM3 file ao Mantra Snap The Mantra Snap program will warn you if you have inadvertently set the trinocular optical path switch directing all of the light to the eyepieces and none to the camera if you try to snap a picture when in that mode You can take pictures with either some or all of the light directed to the camera You can click on the Full Screen Mode g button and the image will fill the Mantra Snap window hiding the Settings Area This can be very useful if you are only using the hardware controls and wish to have as large an image as possible on the screen Take a few more images to familiarize yourself with the Snap process Next you could set up a Post Process procedure by goin
56. hlight the milliseconds current Imaging Band Set exposures for all of your imaging bands and for the objectives that you want to use Click Done when complete and you will be taken back to the Mantra Snap editor or select another editor from the Settings menu NOTE If you would like to set exposures for bands that are not used for imaging you can do so by selecting the correct 250 filter using the Epi Filter Rocker Switch on the hardware Use the Non imaging band exposure field to autoexpose or manually set your exposure Figure 32 Set exposures for non imaging bands Imaging With Mantra 2 5 2 Taking a Fluorescence Image Locating Your Sample Locate a suitable fluorescence slide and place it onto the stage below the objectives You may set the trinocular optical path switch so that some or all of the light goes to the eyepieces Choose the objective and select an epi fluorescence filter by clicking on the Epi Filter and Objective switches located on the Mantra s lower front control panel Epi Fitter Objective Figure 33 You can use the Epi Filter rocker switch to select the epi fluorescence filter that you wish to use and the Objective rocker switch to select the objective you want to use In fluorescence mode the blue light above the FL fluorescence label is lit The BF Lamp knob for setting the brighttfield illumination level is deactivated when you are in fluorescence mode With your sample
57. is equipped with a 40X objective CFR with 50 or 100 of the light sent to the Mantra camera Focus on a detailed Py T specimen on a slide on the slide stage Then with 50 or 100 of the light sent lt to the eyepieces adjust the diopter settings in the eyepieces to the image as seen through the eyepieces is equally sharp Normally the image on the computer monitor will be as sharp as the image seen through the eyepieces but if it is not and you prefer to fine tune your eyepieces and then make adjustments to the focus while looking at the monitor before taking pictures perform the following routine 1 Set the diopter adjustment rings on both sides to scale 0 2 Engage a high power objective 20X or 40X in the light path look into the right eyepiece with your right eye and rotate the coarse and fine adjustment knobs to bring the specimen into focus Do not use an oil immersion objective 3 Engage a low power objective 10X or so in the light path rotate the right diopter adjustment ring to bring the specimen into focus Do not touch the coarse and fine adjustment knobs during the focusing 4 Looking into the left eyepiece with your left eye rotate the left diopter adjustment ring to bring the specimen in focus The above procedure adjusts the diopter with reference to the right eye but it is also possible to adjust with reference to the left eye In this case read the above procedure by inverting right and
58. itation cubes are located in their own motorized turret 7 between the objectives and the eyepieces IMPORTANT The objectives and the epi fluorescence filter cubes are installed in specific numbered locations in their respective turrets Do not move them into different locations or replace them wth other objectives or epi fluorescence filter cubes unless instructed to do so The default positions associated with each epi fluorescence filter are labeled on the epi fluorescence filter turret cover shown in Figure 3 far right Position 1 BF brightfield no filter cube present Position 2 DAPI Position 3 FITC Position 4 CY3 Position 5 TXRED Texas Red Position 6 CY5 Position 7 CY7 if present optional Position 8 QUAD quad band filter for visualization through the eyepieces optional If you wish to perform fluorescence imaging the Mantra Fluorescence Illuminator 62 shown below in Figure 4 should be connected to the rear of the Mantra instrument by a liquid light guide The a Mantra Snap 2 2 illuminators power cable should be connected to a power outlet Switch the power switch located on the lower left front of the Mantra Fluorescence Illuminator to the ON position The Mantra Fluorescence Illuminator is a 200W module that provides fluorescence epi illumination of slides on the Mantra microscope stage It has 6 levels of illumination 0 os a 10 25 50 75 and 100 usi
59. levels can also result in the fluorescence signals 33 Mantra Snap appearing to be washed out the highlights being clipped and lacking detail Set the trinocular optical path switch so that all or part of the light goes to the camera and lets you see a live streaming image of the slide on the monitor NOTE If this is the first time you have opened Mantra Snap you might not see a clear image because you may need to set much longer exposure times for each fluorophore in your sample in the Exposures editor Figure 35 The Mantra Snap editor allows you to select a protocol see your exposure times in milliseconds and specify a location to save your images You can also specify a base filename and a starting number for capturing sequences of images Destination Folder Select a destination folder for your images by clicking on the Save Snap To Browse button at the bottom of the Mantra Snap Settings area to the right of the live image The default location is D Data MantraSnap NOTE the Mantra computer comes with two hard disk drives one for the Windows operating system and the Mantra Snap and inForm software C drive and one for your data D drive PerkinElmer recommends saving your data to the D drive and Figure 36 Mantra Snap Controls making back up copies whenever possible to avoid data loss in case your hard disk drive ever fails Your institution will almost certainly have a network to allow access t
60. lone or by combination of their Contribution s with the Work to which such Contribution s was submitted If You institute patent litigation against any entity including a cross claim or counterclaim in a lawsuit alleging that the Work or a Contribution incorporated within the Work constitutes direct or contributory patent infringement then any patent licenses granted to You under this License for that Work shall terminate as of the date such litigation is filed 4 Redistribution You may reproduce and distribute copies of the Work or Derivative Works thereof in any medium with or without modifications and in Source or Object form provided that You meet the following conditions a You must give any other recipients of the Work or Derivative Works a copy of this License and b You must cause any modified files to carry prominent notices stating that You changed the files and Software EULA lon c You must retain in the Source form of any Derivative Works that You distribute all copyright patent trademark and attribution notices from the Source form of the Work excluding those notices that do not pertain to any part of the Derivative Works and d If the Work includes a NOTICE text file as part of its distribution then any Derivative Works that You distribute must include a readable copy of the attribution notices contained within such NOTICE file excluding those notices that do not pertain to any part of the Derivative Works
61. matter what objective is used They compensate for small optical imperfections that are a normal part of any optical system You wil need to take a set of reference images the first time you start using the system but not necessarily for every imaging session As the lamp ages or if you notice that there are unwanted non uniformities in the images you can take new reference images by choosing References in the drop down menu Taking new reference images can take 15 20 minutes but it is not an especially challenging task We recommend that your lab decide upon a procedure that specifies when new references should be taken Fluorescence References Unavailable Missing references for the following gt Reference for 4x objective and filter DAPI Reference for 4x objective and filter FITC Reference for 4x objective and filter CY3 Reference for 4x objective and filter Texas Red Reference for 4x objective and filter CYS Reference for 10x objective and filter DAPI J Reference for 10x objective and filter FITC Reference for 10x objective and filter CY3 Reference for 10x objective and filter Texas Red Reference for 10x objective and filter CY5 m Choose this to take the reference images now Choose this to choose a different protocol Change Protocol Choose this to quit loading this protocol This will cause you to exit the Mantra Snap program Figure 28 Taking reference images is made easy by
62. mber 2006 17 This software uses the libjpeg turbo 1 5 13 library http libjpeg turbo virtual gl org This software makes use of the libjpeg turbo library This library has not been modified by CRI and all rights are reserved by the copyright holder This software is provided as is without express or implied warranty and with no claim as to its suitability for any purpose The library is covered by the libjpeg turbo License Additionally libjpeg turbo incorporates parts of libjpeg which is covered by the BSD license The libjpeg turbo license is reproduced below Redistribution and use in source and binary forms with or without modification are permitted provided that the following conditions are met Redistributions of source code must retain the above copyright notice this list of conditions and the following disclaimer Redistributions in binary form must reproduce the above copyright notice this list of conditions and the following disclaimer in the documentation and or other materials provided with the distribution Neither the name of the libjpeg turbo Project nor the names of its contributors may be used to endorse or promote products derived from this software without specific prior written permission THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS AS IS AND ANY EXPRESS OR IMPLIED WARRANTIES INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE
63. minator Fuses C S C CCCC dR 5 OAA BV Mantra System Environmental Requirements berating Environment perating Temperature perating Humidit erating Altitude torage Temperature torage Humidit hipping Temperature 24 hours nominal ollution Degree 1 4 Operator and Equipment Safety It is the responsibility of the purchaser to ensure that all persons who operate the Mantra Quantitative Pathology Imaging System are aware of the following cautionary statements As with any scientific instrument there are important safety considerations which are highlighted throughout this User s Manual General Cautionary Statements READ AND UNDERSTAND THIS USER S MANUAL BEFORE ATTEMPTING TO OPERATE TROUBLESHOOT OR MAINTAIN THE MANTRA SYSTEM READING THIS MANUAL FIRST MAKES IT EASIER AND SAFER TO OPERATE AND MAINTAIN THE SYSTEM The Mantra system contains a motorized objective changer and a motorized epi fluorescence filter changer Always keep your fingers long hair loose clothing dangling jewelry etc away from the objective and filter changers to avoid personal injury and possible system damage Use only as specified by the operating instructions The protections may be impaired if the equipment is used in a manner not specified by the manufacturer Do not operate the system if there has been a malfunction of any component as this could lead to equipment damage or physical injury Contact PerkinElmer for assista
64. n Neither the name of the copyright holder s nor the names of its contributors may be used to endorse or promote products derived from this software without specific prior written permission THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS AS IS AND ANY EXPRESS OR IMPLIED WARRANTIES INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED IN NO EVENT SHALL THE COPYRIGHT HOLDER S BE LIABLE FOR ANY DIRECT INDIRECT INCIDENTAL SPECIAL EXEM PLARY OR CONSEQUENTIAL DAMAGES INCLUDING BUT NOT LIMITED TO PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES LOSS OF USE DATA OR PROFITS OR BUSINESS INTERRUPTION HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY WHETHER IN CONTRACT STRICT LIABILITY OR TORT INCLUDING NEGLIGENCE OR OTHERWISE ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE Should you have any question concerning this Agreement you may contact CRI by writing to CRI 68 Elm St Hopkinton MA 01748 You may also call 1 508 435 9500 Software EULA Index Base filename 24 39 Color image 25 D Ls Disclaimers design change 12 reproduction 12 Py ee EULA 67 Fe Focus indicator 18 Full screen mode button 18 H Hardware epi filter turret 7 eyepieces 7 focus knob 7 front control 7 imaging module 6 interface panel 7 objective turret 7 shutter button 7 snap button 7
65. n 6 CY5 e Position 7 CY7 if present optional e Position 8 QUAD quad band filter for visualization through the eyepieces optional The Brightfield filter is permanently assigned to position one its position cannot be changed Also note that as you assign filters to their turret positions the system prevents you from assigning the same filter to more than one position To change a filter s location first you need to remove it from its current position and then select its new position The Load Factory Filters button automatically populates the Available Epi Filters box with the default factory filters Click the Next or Bands button to continue with system setup Or close the window if you are finished setting up the hardware Appendix A Mantra Hardware Setup 53 Bands You can also setup the bands associated emeas with each epi fluorescence filter The Bands panel mirrors the System Epi Filters panel in that it lists all the installed epi fluorescence filters It also lists all of the Bands created for each of those filters Bands specify how Mantra measures light over the filter s range of wavelengths By adding multiple bands to a filter you can quickly select how you want to use the filter during a specific protocol e g within a narrow wavelength range in high definition 10 nm step size mode etc Figure 49 You can add or delete bands associated with each epi fluorescence filter wi
66. n let you view your spectrally unmixed Mantra images as if they were brightfield IHC images In this case the FITC CD4 is shown in brown overlaid onto a blue hematoxylin like DAPI counterstain There are many other features in inForm and you should refer to the inForm User s Manual for more detail Now that you have successfully chosen fluorophores and unmixed an image you can return to the a Mantra Snap Mantra Snap program and continue to take images As long as the inForm program window is open the new images will be processed just like the first image that you worked on Of course if you switch to a different slide that was prepared with different fluorophores your results will not be consistent with the first slide Imaging With Mantra as 3 Post Processing Your Images By default each time you snap a picture the Mantra Snap program Saves Post Processing your image to the location that you selected for your Destination folder But as demonstrated in the previous sections Mantra Snap can do much Launch inForm Analysis more than that You can tell the Mantra Snap program to automatically launch the included inForm advanced image analysis program and oe E Ee S perform a pre arranged operation such as tissue and cell segmentation and phenotyping on each of your images even while you continue to snap pictures Browse Launch a Custom bat file Browse If this is the first time someone has opened the Post Proces
67. nce Operate the system on a flat stable surface Do not drop the Mantra imaging module illumination module or any other system component Do not expose the imaging module to prolonged heat above 40 C 104 F Do not operate the system in an environment with explosive or flammable gases Do not operate the system in places where it may be splashed with liquid Use only the power supply cord set provided with the Mantra system If the correct cord set for the location was not provided please contact PerkinElmer for a replacement Do not use power supply cords with inadequate ratings Some cables and adapters supplied with the system have proprietary specifications Do not connect components supplied by PerkinElmer using unqualified cables or adapters Doing so could result in damage and voids the Warranty Contact PerkinElmer for a replacement if necessary Welcome to Mantra 9 e Use only a properly grounded power outlet when connecting the system to power e Place equipment and devices in a manner such that the power switches and disconnecting devices are accessible at all times e Follow the recommended maintenance procedures to help ensure optimal performance Caution Installing servicing and moving of the Mantra system should be performed by PerkinElmer authorized and trained personnel only Power must be disconnected from the system before servicing 1 5 Important Safety Information for the Fluorescence Illuminator
68. ned technician Unless specifically asked to perform the unpacking installation or setup please contact Technical Support 17 Setup Procedure Ensure that there is a minimum of 100 mm 4 inches clearance around the Mantra Fluorescence Illuminator at all times Equipment and objects should be placed in m such a manner that the power switch and disconnecting ane C TER devices are accessible at all times Menu imei r D 33 Under Mues Damages BE eN I Figure 60 The Mantra Fluorescence Illuminator should be placed in a location where there is atleast 100 mm around it at all times Installing or Replacing the Bulb Required Equipment e Hex Key supplied with the unit e 200 Watt Metal Halide Bulb P N 132364 WARNINGS e Use only the specified 200W Metal Halide bulb Attempting to install any other bulb may cause damage to the unit e Do not touch the inside of the reflector of the bulb e The bulb is delicate so handle it carefully e Mantra Snap Make sure the Illuminator is turned OFF and disconnect the power supply Wait 30 minutes to allow the bulb to cool after turning the unit off N Lay the Illuminator upside down on a flat padded surface 3 Remove the four hex screws shown below and remove the panel 4 Carefully remove the bulb from the packaging Do not touch the silver surface of the lamp or the PCB on the back of the bulb Figure 61 Mantra Fluorescence Ill
69. ng a self aligning temperature controlled 200W metal halide bulb If you need to replace the bulb see Appendix C Mantra Fluorescence llluminator Setup and Maintenancel 62 IMPORTANT A minimum of 4 inches 100mm clearance 0 fl around the Mantra Fluorescence Illuminator should be maintained at all times Equipment and objects should be placed in such a manner that the power switch and disconnecting devices are accessible at all times Figure 4 Switch on the Mantra Fluorescence Illuminator if you wish to perform fluorescence Although the illuminator will reach 70 output in 1 5 imaging The illuminator should be allowed 30 minutes allow 30 minutes for it to reach operational minutes to reach operational temperature temperature before imaging begins WARNING Do not switch the unit off within 5 minutes of switching it on Not complying wth this warning may result in damage to the bulb After switching the unit off allow the bulb to cool for at least 30 minutes before changing the bulb or switching the unit back on Failure to do so is likely to result in damage to the bulb See Appendix C Mantra Fluorescence Illuminator Setup and Maintenancel 6 for a list of warnings that might be displayed on the illuminator s front panel display and that have corresponding actions You can adjust the Mantra microscope hardware including the slide stage focusing controls and eyepieces by referring to Appendix A Mantra Ha
70. non transferable non assignable non sublicensable and revocable license to use for your internal purposes only the executable code version of the Software and the accompanying documentation in hard copy or electronic format CRI RESERVES ALL RIGHTS NOT EXPRESSLY GRANTED BY THIS AGREEMENT 2 Specific Restrictions You may use the Software only on a single computer You may make only one 1 copy of the Software solely for backup purposes You agree that except as expressly permitted by applicable law neither you nor a third party acting on your behalf will i decompile disassemble or reverse engineer the Software ii modify or create derivative works of the Software iii use the Software in any manner to provide service bureau commercial time sharing or other computer services to third parties iv transmit the Software or provide its functionality in whole or in part over the Internet or other network except as expressly permitted v use the Software for any purpose other than solely for your internal research except as may be otherwise agreed in writing by CRI vi use the Software to provide any analytics or diagnostics or otherwise for the benefit of any third party except as may be otherwise agreed in writing by CRI or vii sell distribute rent lease sublicense or otherwise transfer the Software to a third party 3 Ownership of Software CRI and or its suppliers own all right title and interest including all copyrights tr
71. o larger storage areas for your data The Mantra computer comes wth a high speed Gigabit Ethernet port for this purpose Base Filename Enter a new name in the Base Filename text box and choose a Starting Number New images will be placed into a folder named after the base filename that you have entered Those images will be similarly named with the base filename and a number beginning with the starting number you have entered The default starting number is 1 one The file format suffix is PerkinElmer s IM3 This file format preserves relevant information from the Mantra system including but not limited to the imaging mode magnification exposure times stains that were present on the slide and creation date So if you entered a Base Filename of My IHC Slide and chose a Starting Number of 5 the first image you take will be named My IHC Slide 5 or My IHC Slide 5 im3 if you have chosen to have Windows Imaging With Mantra 2 show file type extensions and placed into a folder named My IHC Slide The next image you take will be named My IHC Slide 6 and placed into the same folder You may choose any starting number but you will be warned if your choice inadvertently results in a file with the same name in the same location as an existing file You will be given a choice to overwrite the existing file or to select a different filename Taking an Image Unlike taking brightfield images using just
72. objectives NOTE The Mantra s Shutter button is disabled when you are in Brightfield mode If you push on the button located below the Snap button you wil hear a clicking sound but the shutter setting for the fluorescence illumination system wil not change Take a few more images to familiarize yourself with the Snap process Next you could set up a Post Process procedurel 4 by going to the Settings button and choosing Post Processing You can refer to the inForm User s Manual version 2 1 0 or later for additional details on the program s capabilities and features In order to save you from flipping back and forth amongst the two separate user s manuals for this particular exercise the directions in Processing a Brightfield Image in inForml 2 pertain to the inForm Advanced Image Analysis software program that comes with each Mantra system 2 4 3 Processing a Brightfield Image in inForm Under normal circumstances you would capture images from single stained slides in order to build up a library of spectra precise colors that are really unique multispectral profiles of your stains PerkinElmer calls this a Stain Store of the stains and fluorophores that you would see in the normal course of your research To help get you started PerkinElmer includes some commonly used and characterized fluorescence spectra referred to as synthetic stains because they were not captured and characterized on your particula
73. omatically post processed an image phenotyping selected cells taken with the Mantra Snap program An image processing algorithm had been created using a previously captured image and can be applied to subsequent images taken with a Mantra microscopy workstation in fluorescence mode Post Processing Your Images 4 Frequently Asked Questions and Troubleshooting Why do I occasionally see portions of my sample turn a solid color such as red green or blue when I am rapidly moving the stage or switching objectives This is completely normal In order to obtain rine Be such high spatial and spectral resolution with its scientific grade CCD imaging sensor the Mantra contains a solid state liquid crystal tunable filter LCTF The LCTF rapidly changes wavelength color within milliseconds during live imaging so that you see a high quality color image on the screen Moving the stage rapidly or switching objectives eor can briefly cause just one color to be displayed while the filter is tuning from one color to another When you snap an image it actually cycles through many different wavelengths of z light within a few seconds taking a picture at each wavelength and then the Mantra Snap Figure 44 Rapidly moving the microscope slide stage can program combines them into a multi layered file cause brief color shading as the specimen moves before containing all the information necessary the solid state tunable imaging filter which enables
74. or while they are powered up or immediately after power down as Mantra Snap l am trying to extract a pure hematoxylin signal from a singly stained slide but after open the image that I took in Mantra in the Build Libraries tab in inForm don t see hematoxylin in the Stain drop down menu How can add hematoxylin to the choices The Stain drop down menu items in the Build stain store meneger E Libraries tab are populated from the Stain Store as described in the inForm User s Manual in Understanding the inForm Work Area Stain Store Management and Build Libraries Family Name Active Stains Active ls Nuclear Counterstain You might not have selected hematoxylin as a stain that your lab uses Go to Tools gt gt Stain Store Manager and click on Choose Which Types of Stains and Fluors You Lab Uses Other User Specified TSA Click on Brightfield under Family Name and then select the Hematoxylin checkbox on the right Figure 45 You can add stains and fluorophores to the i Stain drop down menu in the inForm Build Libraries tab Close the Stain Store Manager and return to the o mom 20 Bult inForm Build Libraries tab Hematoxylin will now appear in the drop down menu s Took License Help Of course you can also measure new stains and i Build Library Settings 7X x fluorophores that do not already exist in the losd rage Browee N Gg inForm Stain Store See th
75. or written communication sent to the Licensor or its representatives including but not limited to communication on electronic mailing lists source code control systems and issue tracking systems that are managed by or on behalf of the Licensor for the purpose of discussing and improving the Work but excluding communication that is conspicuously marked or otherwise designated in writing by the copyright owner as Not a Contribution Contributor shall mean Licensor and any individual or Legal Entity on behalf of whom a Contribution has been received by Licensor and subsequently incorporated within the Work 2 Grant of Copyright License Subject to the terms and conditions of this License each Contributor hereby grants to You a perpetual worldwide non exclusive no charge royalty free irrevocable copyright license to reproduce prepare Derivative Works of publicly display publicly perform sublicense and distribute the Work and such Derivative Works in Source or Object form 3 Grant of Patent License Subject to the terms and conditions of this License each Contributor hereby grants to You a perpetual worldwide non exclusive no charge royalty free irrevocable except as stated in this section patent license to make have made use offer to sell sell import and otherwise transfer the Work where such license applies only to those patent claims licensable by such Contributor that are necessarily infringed by their Contribution s a
76. pier eight position turret and the software wa om See Ss FITC rion Pat 8 requires the presence of 8 positions All oa Spee Excitation Wavelengths 3 a Lele Le Le le Le le 8 positions need not be populated though Tam Use the Available Epi Filters box to create or edit entries for each of the epi fluorescence filters mounted in the filter turret Notice that as you highlight each filter the information for the filter displays in the space at the right Emission Wavelengths Figure 48 Adding or deleting epi fluorescence filters from within the Mantra Hardware Setup program Add Filter To add entries for filters you have purchased separately click the Add button Then enter a short descriptive name Enter the vendor and vendor part number Select the Band Pass type either Single Double Triple or Quad band pass Finally enter the excitation and emission cut on and cut off wavelengths in nanometers Click OK when finished Band Name Whenever you add a new epi filter you will be prompted to enter a band name for the emission cut on cut off range you entered Every epi fluorescence filter in the Mantra filter turret must have at least one band associated with it and this is the filter s default band Enter a name that includes the filter s specific target and intended wavelength range See the next section Setup System Bands for more about bands Edit Filter The software will only let you edit filter names
77. plied warranty In no event will the authors be held liable for any damages arising from the use of this software Permission is granted to anyone to use this software for any purpose including commercial applications and to alter it and redistribute it freely subject to the following restrictions 1 The origin of this software must not be misrepresented you must not claim that you wrote the original software If you use this software in a product an acknowledgment in the product documentation would be appreciated but is not required 2 Altered source versions must be plainly marked as such and must not be misrepresented as being the original software 3 This notice may not be removed or altered from any source distribution 19 This software may use the RS erveCLI2 library https github com S urajGupta Rserve CLI2 Original work Copyright c 2011 Oliver M Haynold and other contributors Modified work Copyright c 2013 Suraj Gupta All rights reserved Mantra Snap Redistribution and use in source and binary forms with or without modification are permitted provided that the following conditions are met Redistributions of source code must retain the above copyright notice this list of conditions and the following disclaimer Redistributions in binary form must reproduce the above copyright notice this list of conditions and the following disclaimer in the documentation and or other materials provided with the distributio
78. r Mantra system However because of the variability of stains and the staining process in brightfield IHC there are no synthetic spectra provided for brightfield microscopy Nevertheless with an H amp E or hematoxylin amp DAB slide that features areas of relatively pure staining of one or both stains you can capture and save representative spectra that will be useful for instructive purposes only 25 Mantra Snap Mantra Snap Brightfield Post Processing Launch inform Analysis Batch Process Using inForm Launch a Custom bat file Save Image Only Figure 17 You can configure Mantra Snap to simply save your images or you can configure it to automatically launch the inForm Advanced Image Analysis Software program perform a batch process using inForm or even launch a custom Windows bat file In the Post Processing settings editor seen in Figure 17 above select Launch inForm Analysis Locate an area on your sample slide presuming that you have a properly stained H amp E slide for purposes of instruction here that features a large area of nearly pure eosin and snap a picture using the Mantra Snap program In a moment the inForm program window will appear as shown in Figure 18 below Imaging With Mantra ATEA Fe if Wen Teb Ume Hep fi ii T ry i i a H N gt ET Figure 18 When you select Launch inForm Analysis in the Mantra Snap program the inForm prog
79. ram window will appear as shown with the image that you snapped loaded into the Image Display Area of the inForm workspace You can see the Mantra Snap program behind and to the left of the inForm program window Click the lower Build Libraries tab in the far left of the inForm program You will be asked to save the current project You should click No and then load the image that you just captured by clicking on the Load Image Browse button and selecting the saved image When the image appears in the inForm work area it will be automatically detected and classified as a brightfield image see the grayed out Sample Format Brightfield Choose Stain Eosin from the drop down menu Most likely you will receive a message that more than one stain was detected in your particular specimen This is normal if the slide contains more than one stain in our example a large amount of eosin and a very small amount of hematoxylin If that is the case dick on the Draw Processing Regions button at the top of the work area and draw one or two regions around areas that feature just the eosin and little if any hematoxylin 8 Build Library Settings Sample Format Brightfield M 3 i Review Merge Batch Analysis Manual Analysis o amp Build Libraries Figure 19 You can choose from a drop down menu to identify the pure stain presentin your sample a Mantra Snap Click Extract ol Foioajiione bah A
80. rdware Setupl 50 for instructions If you have installed a new lens or epi fluorescence filter need to reconfigure your computer interface or want to Koehler align the microscope optics refer to the section that describes the Mantra Hardware Setup Program Installing and Starting the Mantra Snap Software The Mantra Snap software requires an activated software license to run The software was installed and activated on the Mantra computer at the factory If you need to re install or re activate the Mantra Snap software please contact PerkinElmer Service amp Support at US Toll Free 1 800 762 4000 or your local PerkinElmer Custom Care Center or authorized distributor You may download the most recent version of the software by visiting us at www perkinelmer com See Appendix B Installing the Mantra Software 6 for a detailed description of the software installation and activation processes Imaging With Mantra 15 2 3 IMPORTANT The Mantra instrument must be attached to your computer and powered up in order to run the Mantra Snap software and acquire images If the Mantra ever loses power Mantra Snap will shut down Recovering a Lost Seat License Mantra Snap licenses are not recoverable If your Mantra Snap seat license is damaged or inadvertently lost e g your hard drive crashes contact Technical Support 11 for assistance Please have your Mantra system serial number 5 digits in length and or your Mantra Snap licens
81. rent filename Taking an Image Focus the image using one of the focus control knobs on the Mantra microscope body You can see the slide in real time on the monitor The green bar of the Focus Indicator in the lower left of the Mantra Snap program grows longer as the image displayed is sharper and becomes shorter as the image displayed is less sharp The green bar may not extend to the full length of the indicators space So simply strive for the longest green bar and the highest number possible When you are ready push the Snap Ee button on the handgrip located to the left of the Mantra lower Figure 16 The Mantra Snap button to take pictures is front control panel shown in Figure 16 or click on located on the handgrip on the left side of the Mantra the Snap button at the bottom right of the Mantra instrument base In the picture above the Snap button Snap program as seen in Figure 15 You may is located under the user s left thumb The Objective observe the slide through the eyepieces or watch the rocker switch to change objectives is located under the i user s right thumb LCD monitor while snapping your images NOTE If you hold down the Alt key on your keyboard while clicking on the Snap button in the Mantra Snap program the button changes from Snap to Snap Color and you will simply save a color TIFF image instead of a full multispectral IM3 file The Mantra Snap program will warn you if you have inadver
82. rst time you start using the system but not necessarily for every imaging session As the lamp ages or if you notice that there are unwanted non uniformities in the images you can take new reference images by choosing References in the drop down menu Taking new reference images can take 5 10 minutes but it is not an especially challenging task We recommend that your lab decide upon a procedure that specifies when new references should be taken 5 White References Unavailable Missing references for the following Reference for 4x objective Reference for 10x objective Reference for 20x objective Choose this to take white references now Choose this to choose a different protocol Choose this to quit loading this protocol This will cause you to exit the Mantra Snap program Change Protocol E Figure 11 Taking reference images is made easy by following the directions in the Mantra Snap program Good reference images ensure the high quality images from your slides You do not have to take reference images every time you use the Mantra but you certainly must take them the first time you use it 22 Mantra Snap If you have already taken references you can view Roforaeces them using the references editor References are stored for two trinocular positions Camera Only and a ee ae aye ae re Shared Adjust your trinocular to one of those two camera only position positions then click View References to
83. see the reference images for that position View References If you would like to retake your references click Take References Click Done when you are finished or use the menu to select another editor References will be acquired with the trinoc in the camera only and shared positions Take References Figure 12 Brightfield References Editor Adjusting the Lamp Locate a suitable brightfield slide e g H amp E or hematoxylin amp DAB and place it onto the stage below the objectives You may set the trinocular optical path switch so that some or all of the light goes to the eyepieces You will see a live streaming image of the slide on the monitor Use the Objective rocker switch on the left side of the lower front control panel to choose the objective and adjust the illumination level using the knob on the right side of the panel as shown in Figure 13 The level that you set for each objective is automatically remembered by the Mantra system So once you have adjusted the a illumination level for each objective switching from one to another does not require that you re adjust Objective Mode the level by hand lt Figure 13 You can use the Objective rocker switch to select the objective you want In brightfield mode the blue light above the FL fluorescence label remains off and the Epi Filter switch is deactivated You can set the illumination level that is comfortable for you through the
84. sing editing Save Image Only area the Save Image Only option will be selected You can choose Launch inForm Analysis which will start the inForm program if it is not Figure 42 You have three already running after you snap a picture If the program is open and an options to post process your algorithm is loaded inForm will automatically apply the algorithm to each Mantra images as well as the new Mantra image as you take them Figure 43 shows a phenotyping option to simply save them algorithm developed inForm that can be applied to new images are without any post processing captured in Mantra You can choose Batch Process Using inForm which will not bring up the actual inForm user interface but will run a specified algorithm selected using the Browse button on each picture you snap in the background You can also choose Launch a Custom bat file for additional expert control of automated processes within the Windows operating system environment Click Done when you ve completed your selection or use the Settings menu to navigate to another editor For more information on the capabilities of the inForm program see the inForm Users Manual NOTE This automatic transfer of Mantra Snap images to inForm is only available with inForm version 2 1 and later as Mantra Snap m Ge i a ERREBEE Figure 43 The PerkinElmer inForm Advanced Image Analysis software program shown here has aut
85. sisi Figure 20 You can select areas in your slide than contain pure or nearly pure signal in this case eosin if you do not have samples of tissue prepared with only the single stains The inForm program will extract the pure signal or a signal that is very close to a pure signal in this case representing eosin Save the stain in a Group with a name of your own choosing Try to pick a name that differentiates this stain as a test rather than a stain from an image and spectra gathered from a real pure single stained sample You can now repeat the above steps to extract the spectra for the second stain on your slide or if you have a previously saved image with in this case pure hematoxylin you can open the Mantra IM3 file An example of this is shown below Select Stain Hematoxylin from the drop down menu and then click Extract Save the pure spectra from the second stain just as you did with inform 20 Build fle Edit Views Tools License Help Build Library Settings toed trege Comme z Semple fomat Bigio v Z an t Eom an f i E 1 eat Figure 21 For your second stain choose from the drop down j menu If this is from a real single stained slide inForm will be able to easily extract the pure spectra from it Imaging With Mantra 2 the first Then click on the Manual Analysis tab in the upper left side of the inForm work area Click on th
86. software uses the log4net libraries http logging apache org This software makes use of the log4net libraries These libraries have not been modified by CRI and all rights are reserved by the copyright holder This software is provided as is without express or implied warranty and with no claim as to its suitability for any purpose The libraries are covered by the Apache License The Apache License is reproduced below Apache License Version 2 0 January 2004 http Awww apache org licenses TERMS AND CONDITIONS FOR USE REPRODUCTION AND DISTRIBUTION 1 Definitions License shall mean the terms and conditions for use reproduction and distribution as defined by Sections 1 through 9 of this document Licensor shall mean the copyright owner or entity authorized by the copyright owner that is granting the License Mantra Snap Legal Entity shall mean the union of the acting entity and all other entities that control are controlled by or are under common control with that entity For the purposes of this definition control means i the power direct or indirect to cause the direction or management of such entity whether by contract or otherwise or ii ownership of fifty percent 50 or more of the outstanding shares or iii beneficial ownership of such entity You or Your shall mean an individual or Legal Entity exercising permissions granted by this License Source form shall mean the preferred form for
87. state or federal courts in New York County New York State and you hereby consent to the exclusive jurisdiction of those courts This Agreement shall not be governed by the United Nations Convention on Contracts for the International Sale of Goods the application of which is expressly excluded You may not assign or otherwise transfer this Agreement or any of your rights or obligations therein without the prior written consent of CRI You may not use the Software for any unlawful purpose nor export or re export the Software except as authorized by law 10 TIFF module This software uses the LibTIFF 4 0 3 library http www libtiff org misc html The TIFF module used is Copyright 1988 1997 Sam Leffler Copyright 1991 1997 Silicon Graphics Inc Permission to use copy modify distribute and sell this software and its documentation for any purpose is hereby granted without fee provided that i the above copyright notices and this permission notice appear in all copies of the software and related documentation and ii the names of Sam Leffler and Silicon Graphics may not be used in any advertising or publicity relating to the software without the specific prior written permission of Sam Leffler and Silicon Graphics THE SOFTWARE IS PROVIDED AS IS AND WITHOUT WARRANTY OF ANY KIND EXPRESS IMPLIED OR OTHERWISE INCLUDING WITHOUT LIMITATION ANY WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL SAM LEFFLER
88. symbols that identify particularly important information and alert you to the presence of hazards These symbols may appear in this manual and or on the product it describes Warning Read instructions to determine possible hazard Caution Read these operating instructions fully before use and pay particular attention to sections containing this symbol Warning Surface may be Hot Warning UV Output Danger Electrical Shock hazard AC Input PPSD DC Input USB Ports Connects to Computer Connects to Foot Pedal This port is not currently used Do not plug other devices into this port Connects to Microphone This port is not currently used Do not plug other devices into this port rG 1 7 Technical Support lf you experience any difficulty setting up operating or maintaining the Mantra system please contact your PerkinElmer representative Office hours are 8 00 a m to 8 00 p m Eastern Standard Time Monday through Friday Welcome to Mantra 11 e Telephone 800 762 4000 or 1 203 925 4602 e Fax 1 203 944 4904 e Email global techsupport perkinelmer com To contact a local PerkinElmer representative outside the United States go to e Website www perkinelmer com 1 8 About This Manual This manual describes the use and functionality of the PerkinElmer Mantra Quantitative Pathology Imaging System and the Mantra Snap 1 0 0 software Operating instructions functional descriptions troubleshooting ill
89. t End Users The Software qualifies as commercial computer software for purposes of the Federal Acquisition Regulations FAR 52 227 19 and the Department of Defense Supplement to the FAR DFARS 52 227 7013 If the Software is acquired by a civilian government agency it is furnished with only the minimum Restricted Rights provided by FAR 52 227 19 If the Software is acquired by a military agency it is furnished with only the minimum Restricted Rights provided by DFARS 52 227 7013 c 1 i 9 Miscellaneous This Agreement contains the entire agreement of the parties with respect to the subject matter hereof and supersedes any proposal or prior agreement written or oral and any other communications between the parties relating to the subject matter hereof No modification or waiver of any provision of this Agreement shall be effective unless in writing and signed by the parties No delay or failure on the part of any party in exercising any right under this Agreement shall impair any such right or any remedy of such party nor shall it be construed to be a waiver of any continuing breach or default under this Agreement In the event any provision of this Agreement is held to be unenforceable the remaining provisions of this Agreement will remain in full force and effect This Agreement shall be governed by the laws of the State of New York without regard to principles of conflicts of laws Any disputes relating hereto shall be adjudicated only in the
90. t Ill Post Processing Your Images 46 Part IV Frequently Asked Questions and Troubleshooting 48 Part V Appendix A Mantra Hardware Setup 50 1 Mantra Hardware Setup Program cccecceeeeeeeeneeeeeeeeeeeeeeeeeeeeeeeaeeeeeeeaeeeeeeeaaeeeeeeaaeeeeeeeeeeeeeeees 50 2 Mantra Microscopy Hardware AdjUStMent 0 ccceeeeeceneeeee cena eeeeeeaaeeee essai eeeeaaeeeeeeaaeeeeeaaeeeeees 58 Part VI Appendix B Installing the Mantra Software 61 Part Vil Appendix C Mantra Fluorescence Illuminator Setup and Maintenance 62 Part Vill Software EULA 67 Contents 3 Index 76 4 Mantra Snap 1 Welcome to Mantra The topics in this part of the User s Manual provide general information about the Mantra Quantitative Pathology Imaging System It is important to read and understand the General Cautionary Statements before using the system The Hardware Overview provides a description of each of the hardware components that constitute the Mantra system Please review the Fluorescence Illuminator User Information so that you are familiar with the fluorescence illuminator and its safety precautions The Mantra system is an easy to use semi automated quantitative pathology workstation for the development of multiplexed immune cell and expression profiling assays in situ It includes inForm Advanced Image Analysis Software that enables immune cell phenotyping within the tumor microenvironment in FFPE tissue sections The Mantra Snap softw
91. tently set the trinocular optical path switch directing all of the light to the eyepieces and none to the camera if you try to snap a picture when in that mode You can take pictures with either some or all of the light directed to the camera lf you wish you can click on the Full Screen Mode o button and the image will fill the Mantra Snap window hiding the Settings Area This can be very useful if you are only using the hardware controls and wish to have as large an image as possible on the screen As previously described if you push the switch to change your objective as shown in Figure 16 Mantra Snap remembers the illumination level and adjusts it for you So if you have optimized the illumination level for your eyes and or camera while at the 4x setting and then decide to change to the 10x or 20x setting you do not have to re adjust the illumination level when the new objective clicks into place Mantra does that for you Of course you can adjust the illumination level at any time Mantra will not override your choice You can see a representation of the illumination level by observing the blue dots that surround the knob that controls the illumination level on the right side of the lower front control panel as shown in Figure 13 Imaging With Mantra 25 IMPORTANT While Mantra can adjust the illumination level from objective to objective it does not adjust the focus setting So you might need to fine tune the focus when switching
92. the a A A eee eon edges of the field stop are sharp Finally p o ae The Koehler adjustment is now complete use the Objective rocker switch to select your lowest magnification 4X and open the field stop until your field of view is unobstructed Figure 53 Step 2 of the Koehler alignment procedure requires you to center and focus on the edges of the field stop Step 3 Set the condenser numerical Mare Harare Saup ee SS GSS Gs aperture NA to your preferred value highlighted in green in Figure54 If you do amp Q not have a preference a list is provided of ent the NA values for your objectives PerkinElmer recommends first trying the oat aioe highest value in the list 1 Set the adjustable NA control to your preferred value If u do not have an NA preference make a list of the NA lues for your objectives and set the condenser to the highest value in that list Figure 54 Step 3 of the Koehler alignment procedure requires you select an optimal condenser NA setting Appendix A Mantra Hardware Setup Step 4 When complete close the Mantra eemo Hardware Setup program NOTE PerkinElmer strongly recommends w O that you retake your brightfield references See after completing the Koehler adjustment saup compiete Congratulations Your Mantra setup is complete n ve J lt gt Back ext Figure 55 Step 4 When you complete the Koehler alignment procedure you should
93. thin the Mantra Hardware Setup program Clicking on an Epi Filter reveals the bands that have been created for it When you click on a band the details of the band appear in the space on the right Name The name given to the band when it was created Factory band names are not editable you can only edit the names of bands you create Imaging Mode Always Brightfield for any bands created for the brightfield filter and is always Fluorescence for bands created for any of the fluorescence filters Range The starting and ending wavelengths specified when the band was created This range is limited by the emission filter You can edit these values for non factory bands only by clicking the Edit button Step Size The default step size is Normal 20nm A High Definition 10nm step size is also available You can choose either step size when adding or editing a band Note that High Definition bands will increase the file size significantly and may not improve your data quality Peak Wavelength The peak wavelength is the wavelength that you will see live on the monitor and that will be used for focusing Typically the peak emission is approximately 20nm after the filter s emission cut on wavelength so this peak wavelength is used for the live view as well as for focusing So for example if the epi filter s emission cut on wavelength was 633nm or any value between 630 640nm the default band s starting wavelength would be 630nm and its peak
94. to the following The copyright notices in the Software and this entire statement including the above license grant this restriction and the following disclaimer must be included in all copies of the Software in whole or in part and all derivative works of the Software unless such copies or derivative works are solely in the form of machine executable object code generated by a source language processor THE SOFTWARE IS PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE TITLE AND NON INFRINGEMENT IN NO EVENT SHALL THE COPYRIGHT HOLDERS OR ANYONE DISTRIBUTING THE SOFTWARE BE LIABLE FOR ANY DAMAGES OR OTHER LIABILITY WHETHER IN CONTRACT TORT OR OTHERWISE ARISING FROM OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE 13 This software uses the Quan 1 0 0 library https Aists sourceforge net ists istinfo quan matters Software EULA This software includes machine executable object code generated by a source language processor from the Quan libraries covered by the Boost license These libraries have not been modified by CRI and all rights are reserved by the copyright holder This software is provided as is without express or implied warranty and with no claim as to its suitability for any purpose 14 This software uses the CommandLine 1 6 0 0 library http commandline codeplex
95. tons Mantra Trinocular Optical Path Switch Mantra Fluorescence Illuminator EEn z s os Figure 1 The Mantra Quantitative Pathology Imaging System with important components identified al e Mantra Imaging Module contains a cooled scientific grade camera system and PerkinElmer s solid state tunable imaging filter as well as circuitry for USB control of the instrument A green linear LED display on the front panel indicates the current wavelength as well as when the system is initializing Mantra Snap Mantra Eyepieces allow you to see your slide directly Mantra Trinocular and Optical Path Switch diverts the image from your slide to either the eyepieces only the camera only or to both eyepieces and camera shared Mantra Epi Fluorescence Filter Turret a motorized turret containing epi fluorescence filters IMPORTANT Do not move the epi fluorescence filters from their designated and marked positions unless directed to do so by a PerkinElmer technician Mantra Power Switch main power switch for the Mantra instrument Mantra Objectives a motorized turret containing your microscope objectives IMPORTANT Do not move the objectives from their designated and marked positions unless directed to do so by a PerkinElmer technician Mantra Stage Controller moves the microscope stage There are separate knurled knobs to allow movement along both the X and Y axis Mantra Focus Knob allows coarse and fine fo
96. tra Snap fluorescence interface is shown above Note that the work area has a more subdued darker theme than the brightfield interface This darker theme reduces the amount of stray light in your laboratory if you are intentionally working in reduced lighting conditions There are also some additional menu items that are unique to taking pictures in fluorescence mode The Mantra Snap program in brightfield mode has a white background while the Mantra Snap program in fluorescence mode has a dark gray background The darker theme shown in Figure 7 reduces light scatter from the computer monitor when the imaging system is being used in a darkened laboratory area Both Mantra Snap interface themes share the same basic elements settings you would like to change The list of settings for Brightfield and for Fluorescence is slightly different as shown in Figures 8 and 9 A Settings Button allows you to choose in a drop down menu which x ao B Full Screen Mode Button toggles between displaying just the live image or showing the live image and all available tools such as the Settings Area and Focus Indicator C Image Window the large window in the work area where the live image is displayed D Settings Area the vertical area to the right of the Image Window where Mantra Snap settings are displayed and can be edited E Focus Indicator green bar that changes length and a number that Focus jaa 82 increases or decreases based on how
97. tution will almost certainly have a network to allow access to larger storage areas Figure 15 Mantra Snap Controls for your data The Mantra computer comes wth a high speed Gigabit Ethernet port for this purpose Base Filename Enter a new name in the Base Filename text box and choose a Starting Number New images will be placed into a folder named after the base filename that you have entered Those images will be similarly named with the base filename and a number beginning with the starting number you have entered The default starting number is 1 one The file format suffix is PerkinElmer s IM3 This file format preserves relevant information from the Mantra system including but not limited to the imaging mode magnification exposure times stains that were present on the slide and creation date So if you entered a Base Filename of My IHC Slide and chose a Starting Number of 5 the first image ra Mantra Snap you take will be named My IHC Slide 5 or My IHC Slide 5 im3 if you have chosen to have Windows show file type extensions and placed into a folder named My IHC Slide The next image you take will be named My IHC Slide 6 and placed into the same folder You may choose any starting number but you will be warned if your choice inadvertently results in a file with the same name in the same location as an existing file You will be given a choice to overwrite the existing file or to select a diffe
98. uminator bulb housing with the location of the four 4 retaining screws shown Open the top of the carton remove the V shaped cardboard holder push back the cardboard flaps and lift the bulb out of the carton 5 Turn the bulb so that the cables and connector hang down into the opened bulb housing O Plug the brown connector from the bulb into the power socket in the bulb housing Make sure it is pushed firmly into position N Make sure the bulb is oriented so that the Hg label faces upwards Then place the bottom side of the bulb into the groove in the bottom of the lamp housing 8 Lift the spring towards the bulb This pushes the bulb into the correct Daami position Click the spring all the way Figure 62 The 200W metal halide bulb installed in the enclosure into the Lamp Spring Restraints to lock the bulb in place 9 Slightly rotate the lamp in its holder and set it in the middle of its travel Appendix C Mantra Fluorescence Illuminator Setup and Maintenance i 3 10 Plug the data connector into the base 7 of the bulb 11 Re install the bulb housing cover using the four hex screws Data connector Figure 63 The 200W metal halide bulb data connection Liquid Light Guide connection to the Illuminator and the Mantra instrument Before turning the illuminator on confirm that the Liquid Light Guide is properly and firmly attached to the front of the illuminator and the rear of
99. unt of stray light entering the objective improving image quality In fact Mantra will not let you proceed with taking fluorescence images until you have slid the Beam ee Block into place covering the brightfield illuminator Figure 26 The Beam Block the plastic cover shown in the red box slides over the brightfield illuminator when you are using the Mantra system to take fluorescence image The Mantra Snap program will prompt you to slide it into or out of place when necessary 32 Mantra Snap Taking References You can skip ahead past the E steps describing how to take fluorescence reference images if they have already been taken Locate the blue fluorescence reference slide included with your Mantra system Follow the directions to take your reference images The Mantra system will select different objectives select the _ epi fluorescence filters by Figure 27 The Mantra fluorescence reference itself and prompt you to take slide is blue in color and is used for taking your actions such as to change reference images when in fluorescence mode the setting of the trinocular Store itin a safe location If you damage or optical path switch or move misplace it contact PerkinElmer to obtain a replacement slide the slide to a new location on the stage When the fluorescence references have been taken click Done Imaging With Mantra 33 NOTE Reference images help produce optimal images no
100. ustrations and other relevant information are contained in this manual Design Change Disclaimer Due to design changes and product improvements information in this manual is subject to change without notice PerkinElmer reserves the right to change product design at any time without notice to anyone which may subsequently affect the content of this manual PerkinElmer makes every reasonable effort to ensure that this User s Manual is up to date and corresponds with the shipped Mantra Quantitative Pathology System Reproduction Disclaimer This User s Manual is solely for the use of the owner and operator of the PerkinElmer Mantra Quantitative Pathology Imaging System Any reproduction of this publication in part or in whole without the express written consent of PerkinElmer is strictly prohibited Neither may this publication be made available for electronic download without the express written consent of PerkinElmer 1 9 CE Testing and Certification The Mantra Quantitative Pathology System has been tested by an independent CE testing facility and bears the appropriate CE mark Note Changes or modifications to this equipment not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment Remarque Tout changement ou modification apport a cet instrument non express ment approuv par l entit responsable de la conformit peut annuler l autorisation d op rer l appareil
101. utput may drop very rapidly Liquid light guides have an expected lifetime of 4 years The suggested replacement interval is every 3 years These figures are based on a temperature of 73 4 F 23 C and 60 humidity If the system is used at ambient temperatures beyond the recommended range it is likely that bubbles ea Mantra Snap could form inside the liquid These bubbles may be reabsorbed by the liquid by storing the light guide at the recommended temperature for several days Starting the Mantra Fluorescence Illuminator WARNING Make sure that the power switch is in the OFF position when the unit is plugged in Plugging the unit into a power outlet when the swtch is in the ON position can result in damage to the unit Do not power up the fluorescence illuminator without the light guide attached to both the illuminator and the Mantra instrument Only power up the illuminator when it is on a flat stable surface Confirm that the power switch is in the OFF position Plug in the power cable to a power outlet Switch the power switch to the ON position Although the illuminator will reach 70 output in 1 5 minutes allow 30 minutes for it to reach operational temperature Shutting the Illuminator Off WARNING Do not switch the unit off within 5 minutes of switching it on Not complying with this warning may result in damage to the bulb After switching the unit off allow the bulb to cool for at least 30 minutes before

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