FISHscope - use iLab (restricted to select users)
Location: Ross Imaging Core (Ross Bldg 9th floor)
Main application: single molecule RNA fluorescence in situ hybridization (smFISH).
Our thanks to Prof. Mark Donowitz for supporting our NIH NIDDK P30 grant (P30 DK089502) supplement application and matching funds, and to NIH -- U.S. taxpayers !!! -- for providing the supplement $. Our thanks to John Gibas, Olympus Corp., for working with us and our other vendors, for getting this instrument specified and delivered.
20191111Mon" The JHU SOM Johns Hopkins Transcriptomics and Deep Sequencing Core Facility (Miller Research Bldg, room 351 and 360), http://www.microarray.jhmi.edu , is a good core to go to "upstream' of our FISHing capabilities. Use transcriptomics to identify the key RNAs, and then "Go FISH" with us.
20191107Thur: all is working. Lumencor SPECTRA III-360* installed, run through Olympus cellSens, "one click" for the up to five fluorescence channels (can be single plane or Z-series).
20191104Mon: Ross Imaging Center On iLab since November 4, 2019 ... restricted use (GM, Ana De La Cruz, maybe another Wu lab member(s), a very limited # G.I. Center members, future a few others), started 20190802Fri (August 2, 2019 after 'first light' 20190719F). We anticipate expanding use in the future (early 2020?), with priority being Ana De LaCruz experiments validating and using smFISH probe sets for G.I. experiments.
location: Ross Imaging Core (Ross Bldg 9th floor).
- Acknowledgement: All users are REQUIRED to include proper acknolwedgement of our funding sources. We suggest this text: The Ross Fluorescence Imaging Core FISHscope was funded by NIH NIDDK P30 DK089502 grant supplement to Prof. Mark Donowitz and Prof. Bin Wu, with JHU matching funding provided by Prof. Donowitz.
smFISH: one dot = one RNA molecule - below: POLR2A mRNA (green), EGFR mRNA (red), DAPI DNA counterstain (blue). See below for more details and full field of view.
- Olympus IX83 inverted microscope ... Olympus UPlanSApo 60x/1.35NA oil immersion objective lens (108 nm pixel size at camera). Also: Olympus 20x/0.75 NA lenses.
- We note that our main objective lens, the 60x oil immersion lens, and our GPU Deconvolution module license (see next bullet) was purchased with our NIH shared instrumentation grant 1S10OD025244-01 (Prof. Brian O'Rourke and Prof. Mark Donowitz) for our Olympus FV3000RS confocal microscope. For logistic reasons, we suggest just acknolwedge our P30 supplement funding in your manuscripts.
- Olympus Cellsens Dimensions with GPU deconvolution (deconvolution license transferred from our Olympus FV3000RS confocal IX83 microscope NIH S10 grant purchase).
- Lumencor SPECTRA III-360* lamp, 8 excitation 'bands', 360 to 747 nm (initially using a demo SPECTRA X provided by John Gibas, Olympus) (Erich Zeiss, Iain Johnson, et al).
- Sutter ten position 32 mm filters emission wheel inside deck 2, Lambda 10-3 controller
- Semrock Penta cube (ex + em + em filters in cube) LED-DA-FI-Cy3-Cy5-Cy7 + five emission filters in Sutter wheel. Semrock filters purchased through AVR Optics (Peter Brunt especially helpful).
- Hamamatsu FLASH4.0LT sCMOS camera (purchased through BBMicro / Hunt Optics) (Butch Granada, Hamamatsu, especially helpful).
- Our thanks to Kevin Murphy PhD, JHU Cardiology, and John Gibas, for leading getting our PC to Windows 10, Cellsens, all drivers, and improved performance.
- Our thanks to Ron at Olympus TAC (technical support) for microscope and Cellsens Dimensions assistance ("one click" Penta acquisitions).
- future ... we anticipate adding more fluorescence filter cube(s) and emission filters in the future, to enable more applications (Brilliants [BUV, BV, BB, BYG], SuperBrights, S.ChuQDots, more ... i.e. BUV_longpass, BV_longpass, BB_longpass complete cubes --> full set of "adjacent emission bands" in Sutter emission filter wheel ... think "21plex" immunocytochemistry (I.C., aka "eye see") / immunofluorescence + smFISH ... Go "IC & FISH" ... pronounced "go ice fish(ing)"). For now focus is smFISH.
- No plans for environemental controls (for now): the FISHscope does not have a 37 C incubator, and we have no plans to purchase one. That said, in the future, we can contemplate - some day - moving the Tokai Hit incubator from our Andor X1 spinning disk confocal microscope, designated "Legacy status" August 2019, over to FISHscope.
smFISH: one dot = one RNA molecule - below: POLR2A mRNA (green), EGFR mRNA (red), DAPI DNA counterstain (blue).
Olympus UPlanSApo 60x/1.35 NA oil immersion lens, IX83 microscope, Lumencor SPECTRA X lamp (demo unit from Olympus), Semrock Penta cube (ex, dm, em filters in cube), single emission filters in Sutter 10-3 wheel (IX83 lower deck), Hamamatsu sCMOS camera (2048x2048 pixels), Olympus Cellsens constrained iterative deconvolution (NVidia GPU card accelaration), contrast adjustments and color combine with MetaMorph Imaging System. 108nm pixel size, ~200x200 um field of view. Note: pixel scale 1/4 and saved as JPEG quality 9 (max is quaality 12) due to limitations of our web site host.
Image credit: Ana De La Cruz, JHU SOM.
McNamara 20200214Fri FISHscope Lumencor Spectra III-360 - AVR Semrock Penta cube - Sutter em wheel - table.
DAPI / FITC (GFP, AF488) / TR = TRITC / Cy5 (AF647) / Cy7 (AF750, IRDye800)
Emission filters, from https://searchlight.semrock.com
A key accessory to enable additional uses is the LCI 24 well plate optogenetic device, funded with the P30 supplement + Mark Donowitz matching funds: