FISHscope Quick Tips

FISHscope Quick Tips - started 20220712U - startup / use / finish steps probably will be edited (and then further edited) in future.

 

Filter sets

20230125W * Semrock LED-DA/FY/TR/Cy5/Cy7 ... DAPI, Fluorescein (Alexa Fluor 488), TRITC (not Cy3, not Alexa Fluor 610), Cy5 (Alexa Fluor 647), Cy7 (Alexa Fluor 750 ... often superior to IRDye800) * Semrock AF700 ... new (12/2022) Alexa Fluor 700 filter cube * LED-mCherry ... Alexa Fluor 610 (AF610) (and mCherry, Texas Red, Alexa Fluor 594, etc) ... move from FV3000RS (since Redundant with TRITC cube "orange" fluorescence for looking by eye) * R10T90 (R10/T90) and R03/T97) reflection/transmission beamsplitters in cubes ... flexible choice of Lumencor SPECTRA III-360 lamp channels and emission filters in Sutter wheel.

LEDs and laser lines
368
395
440
475
555
575
635
747

Brightfield multichannel imaging including RGB

20230703M FISHscope has a monochrome camera (Hamamatus ORCA-FLASH4.0LT), can acquire brightfield images with any, some or all of the 10 emission filters (Sutter 10-3 weheel) and colorize them (cellSens LUTs = lookup tables ... or colorize in Adobe Photoshop, fiji ImageJ, MetaMorph, etc). GM reconfigured the cellSens hardware tryy (the underlying settings for Process Manager) to add brightfield settings for EACH of the TEN emission filter wheels, 395-809 nm. Filter        Range 392/23    381 - 403 447/60    417 - 477 515/30    500 - 530 559/34    542 - 576 572/15    565 - 579 598/25    586 - 610 640/40    620 - 660 685/40    665 - 705 732/68    698 - 766 809/81    769 - 849 * ranges: low values are rounded up, high values are rounded down. For example 392/23 is 381 - 403 instead of 380.5 - 403.5.

 

 

Stage Tiling (and auto-stitching) with cellSens MIA

20230703M FISHscope quick tip 02 - simple tile scan using MIA - George McNamara 20230630F instructions: read all the instructions. slide: clean slide, flat, coverglass side down. You may need to specify an Overview Area (some or most of slide).Do this with low power objective lens (large working distancefor safety). 1. Stage Navigator - Position list (2nd icon from right): Delete any pre-existing positions. 2. Process Manager - turn on XY-Positions/MIA icon (3rd of 5 icons above Channels list). 3. Stage Navigator - Create MIA Scanning Area with Stage (5th icon from left) DROP DOWN LIST: Define MIA Scanning Area with Stage   use Rectange with Stage Position this is top left and bottom right corners)    a. activate the item using the drop down list (2nd itemin menu) ... image goes LIvE.    b. Move to top left of your MIA area ... Press "Next" to confirm position.    c. Move to bottom right of your MIA scanning area ... Press "Finish" to confirm the position. 4. turn off live. 5. double check that Z-stack, other unwanted settings inProcess Manager are NOT selected (or are selected with approporiateranges). 6. Process Manager - Start acquisition. Note: if acquiring multiple channels, you may be able to acquire faster by acquring one channel at a time, saving, then acquiring next channel, saving, etc (ex. red, then green, then blue). Note: most histology dyes have "little or no absorbance" in the near infrared, so you may find 809_81m emission filter of interest. Expectations: have LITTLE OR NO Expectations on the quality of the stitching = do not expect seamless tiles. 10x lens pixel size 648nm. 20x lens pixel size 324nm. 60x lens pixel size 108nm.

 

FISHscope main page is http://confocal.jhu.edu/current-equipment/fishscope 

Keep the PC on - just login, logout.

Get training from George - get retraining from George if you do not remember what to do - better to get retraining than to break our microscope (you might have to buy us a new one out of your own pocket, FISHscope was ~$95,000). Note; dynamic range of camera is much greater than your eyes, so use "Adjust Contrast" as needed to see your data (more info duringtraining). 

Start:

• Login to PC (George will make you a login on PC - we generally do not use "WIN" domain).
• Turn on the microscope hardware, simplest to go in order: 1,2,3,4,5,6,7 (#4 is camera, switch faces to the left). If key (#7) was clockwise, need to move back to counterclockwise before #5).
• Lumencor lamp (#5, do #6, then turn #7 counterclockwise) takes ~30 seconds to start, wait for LEDs on front panel to come on (two rows of text) before next step.
• Lumencor lamp software GUI steps:
  • Start Google Chrome web browser - first web page is Lumencor control panel ... set all to 25% (standard setting - you can change if useful ... modest power good to reduce photobleaching)
  • Lumencor cotnrol panel - toggle one channel on, then off (light may or not come out of objective lens depending on microscope settings).
  • Go to top right of web page ... click the "chevron" to get to the setup page ... click in the etext field, select the "SET ... 999999" text, click OK, exit the setup page (not the Lumencor main page).
• start Olympus cellSens.
• Use Process Manager.
• imaging assumes you have been trained by George and remember what to do. 

Use:

• Olympus cellSens ... Process Manager ...  
• Every acquisition gets saved to a folder on C: ... YOU should Save As to G: to your folder (I usually make a new folder per day, optionally per slide, etc). Simplest for you to make new folder in Windows File Explorer, then assign to "Quck Access" menu. Best to save as VSI format (small file in main folder, data in subfolder of matchign name). 
• Please use your own PC to export to TIFF and/or OIR ... doing so on FISHscope wastes time. You can open VSI with Fiji ImageJ and the free Olympus viewer software ("FV-31" - installer is on FISHscope and our file server, can be uploaded to your JH OneDrive).

Finish:

• Upload your data to your JHU OneDrive ... my JHU --> Cloud --> OneDrive (click heart icon to put at favorites at top of myJHU).
• You may want to transfer as much data as possible during your session, so just have minal files to do at end of session.
• When done uploading, Sign out of OneDrive, wait for browser page to close, sign out of myJHU, then CLOSE the web browser (you may want to save other URLs and/or print web pages to PDF or copy/paste to Paint and save, etc).
• Lower objective lens to ZERO.
• Remove your specimen.
• If using 60x/1.35NA oil immersion lens, clean with LENS paper ... swipe one time, flip over, swipe one time, discard. Repeat if needed (usually two swipes is enough).
• Switch to 10x objective lens. 
• Turn off the microscope hardware (unless next user is waiting): simplest to do in reverse orderr 7, 6, 5, 4, 3, 2, 1. (reminder: camera switch is on left, facign keyboard).

Reminder: No USB devices on our microscope PCs. Use your JH OneDrive or for external users, your personal/company OneDrive (do NOT use other online storage sites, per JHU I.t. policy for employees, also applies to visitors). JHU users normally log in using the local PC login, not WIN domain, so lack access to your "H:" drive - this is not a big deal because your JH OneDrive has 5TB capacity (and you can contact I.T. for more space). You can discuss with your P.I. and I.T. about making space on your PC or your lab file server andshare it in a way that lets you access from our FISHscope PC. We have a file server - it is for us, not you - please do not leave data for more than one week on our file server -- you and your P.I. are responsible for your data at all times, and NIH and other funders could censure you and your P.I. for failure to archive your data properly. Not our responsbility.