. FISHscope

FISHscope - use iLab (currently limiting to select users)
Location: Ross Imaging Core (Ross Bldg 9th floor)

FISHscope

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., Peter Brunt (AVR Optics, Erich Zeiss (Lumencor) for working with us and our other vendors, for getting this instrument specified and delivered. 

 

20200831: A few throught about "future directions" (our focus now is on using what we have!).

  • more cameras would enable simultaneous acquisition of additional fluorophores (ex: Brilliants tandems, Phitonex tandems), re: Hazen Babock's published 4cam (2018), has made 8cam (personal commun.), Babcock HP 2018 Multiplane and Spectrally-Resolved Single Molecule Localization Microscopy with Industrial Grade CMOS cameras. Scientific Reports 8: 1726. https://www.nature.com/articles/s41598-018-19981-z ... 4 (or 8) inexpensive CMOS camewras.
  • Bigger field of view and/or back illuminated camera(s) ... i.e. 5.5 megapixel (8 megapixel) "bi" camera(s). Current front  illuminated sCMOS cameras are 82% (modest width spectral peak) quantum efficeincy; back illuminated are 95% QE over a much wider range, and better QE in UV-blue and Red-NIR spectral range. That is, "total area under the curve" greatly favors "BI" over "FI". The only problem: some vendors charge a huge premium for their BI over FI.
  • New PC: PCIe gen4 E-ATX motherboard, big power supply(s) [and line conditioner and UPS to protect), 2 NVidia RTX 3090 GPUs (20+ Teraflops each!!!! this ection written 1 day before NVidia launch date for Ampere architecture GPUs, PCIe gen 4 etc ... goa: "instant gratification" spatial deconvolution, that is deconvolve faster than acquisition time ... ideally "joint spatial deconvolution and spectral unmixing" [JSDSUN] ... re adam Hoppe 2008 Biophys J reported 10x improvement in SNR, but was slow at the time), NVMe gen4 SSDs (on motherboard and ASUS Hyper M.2 PCIe gen 4 NVMe array. Dual HD 4K monitors would be nice, though our current acquisition software (Olympus cellSens 3.0, spring 2020) is not really dual monitor friendly - ideally, most of the time: one monitor for acquisition controls, one monitor for images (i.e. one big picture, and thumbnails for each channel, image histogram, LUT controls, etc (the Leica LAS software for our Leica SP8 confocal microscope is closer to dual monitor friendly, but Leica has "missed the landing" on optimizing GUI controls). 
  • As usual, would also be great for next software release to have "print money" and "direct deposit (to my bank account)" buttons (for my login ... though the touch panel screen could have the buttons, not sure where any printed money would appear). 

20200828F: We won Lumencor's Earthday 2020's grand prize: a $10,000 SOLA u-nIR 350-760nm white light fluorescence light engine. Eligibility rules included using a Lumencor lamp. Ours is Lumencor SPECTRA III-360 8 band light engine(detailed on this web page). Winning slide of 5 color fluorescence (2 color FISH, antibody, MS2, DAPI) was made by Lauren Blake, graduate student in Prof. Bin Wu's lab. Acquired (by GM) on FISHscope. 

20200729W: I realized we have 555nm not 510nm LED in our SPECTRA-III-360 (360nm replaced usual 510nm). I added "Performance Measurements" table below (aka Lumencor Certificate of Conformance), may take some time before I replace all mentions of 510nm with 555nm. I discovered this because of testing with "RT" since 510nm would not be very useful with our Penta cube. 

20200616Tue: new project: R10/T90 beamsplitter filter cube and "R03/T97" glass filter cube (re: Sawano ... Miyawaki 2002 Biophys J) to enable any combination of our Lumencor SPECTRA III-360 light engine and Sutter emission filter wheel with Semrock emission filters.Our preliminary results (6/2020) are each of these work, with somewhat longer exposure time on our Hamamatsu ORCA-FLASH4.0LT sCMOS camera (all controlled by Olympus cellSens). Advantage: not limited to "Penta" cube wavelengths, so we can use any of our Lumencor light engine's 8 exctiation bands. That is: "RT" provides a lot more flexibility than usual dichroics or multichroic beamsplitters. 

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. 

https://calendar.google.com/calendar?cid=c291bHVucDE3M3JyZTIwOWFrOTk1dTc1bG9AZ3JvdXAuY2FsZW5kYXIuZ29vZ2xlLmNvbQ

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. Condenser 0.55 NA (brightfield).
    • 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, Lumencor facilitated).
  • Sutter ten position 32 mm filters emission wheel inside deck 2, Lambda 10-3 controller
  • AVR Optics / Semrock filters: 9 (one empty position ... plan 7/2020 is to buy 10th emission filter to balance our wheel):
    • 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).
    • Semrock four (7/2020 soon to be five) additional emission filters to essentially "fill the gaps" between the Penta emission filters.
  • Thorlabs "RT" cubes (added 06/2020) R10/T90 beamsplitter (Thorlabs BSN10R) and "R03/T97" glass (Thorlabs BCP42R, "compensator"). Enables excitation with ANY of our 8 excitation "bands" from our Lumencor light engine with ANY of our 9 emission filters. No need to change filter cubes (so less risk of image shifts than when using conventional DAPI, Green, Cy3, Cy5, Cy7, etc, filter cubes ... also faster). The "RT" beamsplitter is also less expensive than a standard dichroic or out Penta multichroic. More on "RT" near bottom (Torlabs product details and graphs).
  • 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 [maybe not many given capabilities of our 6/2020 RT cubes) , 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. 

ix83

 


 

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.

How many plex do you want to image?

Note: Olympus cellSens (as of 6/2020) can list 40 acquisition settings in Process Manager.

McNamara 20200624W FISHscope 8 excitation 9 emission bands – table (note; we are in process of ordering filter #10 to balance the wheel and add yet another eission band option).

Lumencor light engine (368 .. 747 nm) --> RT beamspliiter cube --> specimen --> Semrock filters in Sutter filter wheel 392 .. 809 nm) --> Hamamatsu sCMOS camera

 

* GM note; "510" (left column) should be "555" (will fix table), also now have 572/15 emission filter installed (replaces "Empty"). 
 

GM notes: reflection is useful for

(i) find the coverglass-mounting medium surface,

(ii) flatness of the coverglass [or lack thereof .. and whether the specimen should be re-positions to make as flat as possible],

(iii) interference reflection contrast microscopy (IRM, aka RICM … optional dual or triple wavelength quantitative IRM, qIRM). In principle, for fluorescence, could acquire every emission band longer than excitation (which are labeled as ‘reflection’ here): 9 + 8 + 7 + 7 + 6 + 5 + 3 + 1 = 50 images. If each image is 1 second exposure time (data transfer is ~10 ms each, so 500 ms total; emission filter wheel moves could be minimized by stepping only between excitation wavelengths, so 8 moves in ~50 ms each, so ~400 ms; more conservative in case of short wavelength induced photobleaching is to excite longer à shorter excitations, so ~50 moves * 50 ms = 2500 ms) could acquire all in 50.5 seconds, so under 1 minute (in practice, some may be optimal at longer exposure). Note: our Lumencor light engine has “plenty of power” in each excitation band, and our “RT cubes” work at every wavelength (reflection/transmission, R10/T90, R03/R97). Excellent IRM (and qIRM) review: Barr Bunnell 2009 Interference reflection microscopy. Curr Protoc Cell Biol, Unit 4.23. PMID: 20013754.

Below: table 20200623Tue , with four new em filters (392/23, 559/34) and RT10/90 and "RT03/97" beamsplitter cubes (see above for more info on these cubes). Also tabulated are Brilliant Ultraviolets (BUVs) and Brilliant Violets

FISHscope Excitation Emission Table

(BVs). Two BV's available only from BioLegend (BV570 and BV750), which licensed BV's from Sirigen before BD Biosciences acquired Sirigen.

FISHscope excitation and emission table

Below: "Go FISH" Prof. Bin Wu currently likes: Cy3, Alexa Fluor 594, Cy5, Cy7 for single molecule RNA FISH probe sets (alternatives: Atto 594 for AF594, AF750 for Cy7, not shown). Also shown BUV395 and BV421. All accessible using either "RT"cube and one or more emission filters (and enabled by having a nice camera: Hamamatsu ORCA-FLASH4.0LT).

GoFISH

Below: BUVs and BVs with respect to FISHscope (640/40 em and 732/68 on order 6/2020).

All Emission filters, from https://searchlight.semrock.com - Penta cube shown below.

 

Lumencor SPECTRA III-360 Spectra

Power Measurements (Lumencor "Certificate of Conformance") with "color", wavelength (and LED emission filter; last two are laser lines) and power measured at factory.

Lumencor SPECTRA III-360 light engine on FISHscope

***

News 20200624W: Phitonex 19plex Nova's ... can be used with Brilliants BV, BUV, SBs (SuperBrights) ... Phitonex is targeting flow cytometry market, looks promising for fluorescence microscopy. 

Phiton DNA origami ... fluorescent dye(s) on or in the DNA origami ... built from oligonucleotides (see Phitonex web site and white papers for academic references). The Phiton 150 kDa "cruciform structure" is 20nm linear, but folded into a 3D "cup like structure". Upshot is similar in molecular weight (150 kDa) and dimensions to an antibody. Normal use would be direct label on a primary antibody (and probably defined 1:1 or 1:2 antibody:phitons). Would be possible to conjugate to Fab (~50 kDa) or nanobody (~12 kDa). Note: we do not have any financial interest in Phitonex, just intrigued by potential for multiplexing. See also Ron Vale's 2020 Nature Methods "FluoroCube" (6 nm cube, published 6 identical dyes on 6 of the 8 corners, one corner open for conjugation ... I don't understand why they did not put dye on 7 of the 8 corners).

Phitonex 19plex launch

Phitonex phiton

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A key accessory to enable additional uses is the LCI 24 well plate optogenetic device, funded with the P30 supplement + Mark Donowitz matching funds:

optogenetics

 

RT beamsplitters

20200713Mon - more information on our RT Beamsplitters project.

Goal: eliminate need for dichroic (2-color) or multichroic beamsplitters in our microscope. Our initial filter cube was a "Penta" cube (ex+dm+em each "penta") + 5 matched emission filters in wheel from Semrock (U.S. distributor AVR Optics). Works fine for the "penta" (5plex) designed for: DAPI / FITC (green) / Cy3 / Cy5 (Alexa Fluor 647) / Cy7. Can be used for other fluorophores, such as some Brilliant Violets (395nm excitation, various emission), but not all.

Concept - use a piece of glass -- from Sawano ... Miyawaki 2002 Biophysical Journal (see  Sawano ... Miyawaki 2002 Biophys J    open access ... figure 1 modified by GM)

Doable: because we have a powerful lamp (lumencor SPECTRA III-360) and emission filter wheel in the microscope (so safet to use eyepiece or camera) and Hamamatsu ORCA-FLASH4.0LT sCMOS camera.

Implementations:

1. R10/T90 = reflection 10% and transmission 90%. Thorlabs BSN10R beamsplitter.

2. R03/T97 = Reflection ~3% and transmission ~97%  Thorlabs BCP42R compensator (maybe more like 2% reflectance).I find ~4x longer exposure for this compared to R10/T90.

Each in an Olympus IX83 filter cube (these two from Thorlabs, TLV-U-FF filter cube) -- only the beasmplitter, no exciter, no emission filter in cubes, since our microscope has Lumencor lamp (8 single exciter 'bands') and 10 emission filters in Sutter emission wheel (deck 2 of IX83 microscope).

Thorlabs web site has prices for these items and most other products.

Thorlabs beamsplitter specifications.

 

BSN10R

BCP42R compensator

BCP42R reflectance (0 to 10% depending on polarization state and wavelength) and transmittance (90 to ~98%). We care about;

  

BCP42R ... UNpolarized reflectance (excitation light).

7/2020 initial results:

For our Eosin tissue section slide, I obtained equivalent intensities with 555nm excitation and 598/25 emission filter in wheel for:

* Penta cube ...     150ms

* R10T90 ...        1000ms

* R03T97 ...        4000ms

using same Lumencor 555nm intensity level (25%). I could have adjusted lamp level (increase for RT10T90, increase more for R03T97), but did not since 4 second exposure not a big deal time size nor dark current noise on our camera.

 

 

 

 

 


Reserve Equipment