20240627Thur - summary of sections below on recent reagents for (hopefully) improve your immunofluorescence microscopy

  * 20240701M see nice linkedin thread on topic - both PipeBio's URL and the comments, https://www.linkedin.com/posts/pipebio_recombinant-antibodies-are-less-than-5-of-ugcPost-7213534716845563905-fwuJ 

Product names are not meant as endorsements. Acronyms ("VHH") are explained in "the literature" (peer-reviewed, preprints, product pages, press releases). 

Assumption: standard primary antibody from rabbit or mouse. One hour of confocal microscope time ($30/hr) using standard secondary antibody.

Brighter options imply proportionally greater signal (and signal to noise ration) and/or decrease in acquisition time (lower cost per specimen field of view). Not all possibilities are permuted in the table. I used Fab below (fragment antigen binding; VH_CH1 + VL-CL; historically made by specific protease digestion of IgG ad then purify the Fab's from the Fc fragment crystallizable domain), but scFv (single chain fragment variable, VH-linker-VL or sometimes VL-linker-VH) is single chain equivalent, so simpler for recombinant protein production.

Not in table below: DNA-PAINT (single molecule localization Ab-oligo + fluorophore-oligo, antibody-PAINT (especially Fast Kd Ab-PAINT, scFv-PAINT, nanobody-PAINT), hybridization chain reaction (HCR, detecting either a DNA sequence, RNA sequence, Ab-oligo, protein-protein interaction etc). I am a big fan of many of these. 

Also not in the table: the mounting medium you use matters. Elsewhere in this web site I discuss D2O instead of H2O (Maillard 2020 Chem Sci; also Alexa Fluor 610-X AF610-X, brightest of 42 fluorophores they tested; potentially deuterated glycerol, though expensive compared to D2O) to bosst intensity (photons out) of many read and near infrared dyes; some users still use VectaShield with DAPI: (i) VectraShield quenches many CyDyes (i.e. Cy3, Cy5, also some Alexa Fluor trademark named dyes are CyDyes) maybe not a good idea, (ii) often with DAPI in the VectraShield, which is just a way to badly decrease contrast [you can apply DAPI with the secondary antibodies; if using direct label antibodies, can apply with them ... or switch to a NIR DNA dye, perhaps DRAQ5 or To-Pro-3, apply with antibodies); some users use ProlongGold - a product around longer than some of the users have been alive: consider Prolong Diamond or Prolong Glass ... and read the instructions: need to cure at least 24 hours in open space to vent volatile compound(s) that keep them liquid. 

Ideal future in my (George McNamara) opinion: $1 per slide very bright modern fluorophore(s) all recombinant direct label Fab or scFv or primary nanobodies with custom C-terminal tail, simple and efficient labeling of BV421 (Brightness = 1500 vs Alexa fluor 488 or EGFP Brightness ~60; Brightness = Extinction coefficient * Quantum Yield / 1000, for BV421 2.5M * 0.6 / 1000). Using Fab (~50 kDa)  instead of intact IgG (~$155 kDa) eliminates binding to FcR and FcRn receptors (good to avoid); decreases number of lysine (for NHS ester) or cysteines (for maleimide); for 1 ug protein, monomeric Fab has ~1.5x more antigen binding sites than IgG (two antigen binding sites per IgG); custom C-terminal tail: (i) antigen binding site is near N-terminal end so best to avoid, (ii) tail can be whatever length and sequence is desired (optimal for brightness vs total cost [and for companies, profit ... caveat intellectual property protection and rights). 

20240605W ThermoFisher Superclonal recombinant secondary antibodies ... available with Alexa Fluor PLUS fluorophores

* GM note(s): recombinant antibodies "should" perform better than "out of rabbit", "out of goat", out of donkey" etc, plus no more dead animals haunting you. Of course, ThermoFisher (and other rec Ab vendors) need to deliver conistently high quality manufacturing - and follow through with QA/QC. I do think they would do better by offering scFv-custom-optimized-tail, instead of just Fab and intact IgG.

from marketing info:

https://www.thermofisher.com/us/en/home/life-science/antibodies/secondary-antibodies/superclonal-secondary-antibodies.html

What are Superclonal recombinant secondary antibodies?

Thermo Fisher Scientific Invitrogen Superclonal secondary antibodies represent a recombinant antibody technology designed to provide precise and accurate detection of mouse, rabbit and goat primary antibodies in a variety of applications.

Our proprietary screening and production process yields specific mixtures of recombinant goat or rabbit secondary antibodies that bind with the epitope-precision of monoclonal antibodies, while also achieving the multi-epitope coverage (e.g., H+L) and sensitivity of polyclonal antibodies. Superclonal secondary antibodies are in vitro manufactured using synthetic genes after the first immunization. Each Superclonal secondary antibody is formulated and optimized to help achieve excellent results in ELISA, cell and tissue imaging (ICC/IF and IHC) and flow cytometry applications.

***

Superclonal plus ... 11 products  (as of 20240604U)

https://www.thermofisher.com/antibody/secondary/query/Superclonal%20plus

two examples (one IgG, one Fab):

Goat anti-Human IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor™ Plus 488

Goat anti-Human IgG Fab, Superclonal™ Recombinant Secondary Antibody, Alexa Fluor™ Plus 488

20240511A: 

Daniel Beacham, ThermoFisher - Molecular Probes (Eugene, OR) presented a seminar on 20250508W on fluorescent probes at the JHU SOM campus (Smith Bldg Atrium, Wilmer Eye Institute - attended by researchers from all over campus). Among the highlights:

* Dan is a coinventor of the Bacmam2 product line, Baculovirus derived 30 kb cargo transient transduction reagent, uses VSV-G to bind most mammalian cells (receptor is human, mouse etc LDL-R). After internalization, routed to endosomes/lysosomes, then escape to cytoplasm and cargo genes expressed from appopriate promoter(s) [the remaining bucolovirus vector genes are not expressed in mammalian cells - Bacmam2 is generated in insect cell lines]. The one major cell lineage Bacmam does not work in are hematopoietic cells, ex. hematpoietic stem cells (HSC), T-cells, B-cells, monocytes, neutrophils. Maybe TF-MPI will come up with a "Bacmam3" that does. Dan mentioned "2" binds ok, but do not escape from endosomes.

* Dan said Alexa Fluor Plus products vary in their 'tweaks' (my term) compared to 'regular' antibodies. Can be any (one?) of:

• * tweak to fluorophore (possible methods include, not limited to, add sulfonylation to make more soluble; single isomer AF488).
• * optimized linker (which is what I was informed when Plus came out - maybe marketing dept simplified story?).
• * optimized conjugation site(s) -- that is choices of what amino acids on the antibody heavy and/or light chains (possibly by using non-canonical amino acids and 'click' chemistry; possibly adjusting reactive moieties, pH, etc, to get the fluorophores only onto specific amino acids ... possibly C-termini???).

* Dan alsoe said the key release criteria of Plus products is at least 1.5-fold better signal to background ratio compared to 'regular' secondary antibodies.

• GM note: This implies to me that this makes "titering" each of the Plus antibodies -- and each primary antibody -- for each use may be crucial to see the full benefit of "Plus" prducts. As a further suggestion (by me), titering may need to be different for antigens on (or in) cell types with low expression vs medium or high expression. EGFR is expressed at low level on 'normal kidney' cells (i.e 5,000 EGFR per cell), much higher on some tumor cells (ex. 100,000 EGFR per cell) and very high on the classsic A431 lung cancer cell line (1,000,000 EGFR per cell, perhaps more on some cells -- for example a cell just before division would have ~2,000,000 molecules per cell, then divide to two cells each with 1,000,000 per cell [on average]). the 20-40 range of EGFR on these cells (we'll assume same surface area of each) might benefit from different doses of antibodies.

* I mentioned to Dan (post seminar) my (mis?)understanding of TF-MPI marketing "Plus" as 3x brighter than regular -- he disavowed that Marketing would ever put up such numbers, and remphasized the Signal-to-Background (SNB) 1.5x of more release criteria for each Plus product (he also mentioned not knowing prices). If 'only' 1.5x better, then if 1.3x fold more expensive, Plus would be a marginal win (if user takes the time to optimize titers of each reagent) -- but I suggest (hope) that in many cases "Plus" will be both brighter and higher SNB. 

20240307H:

Two fun facts (or at least colleagues repoorts):

To-Pro-3 DNA counterstain was so bright on Tim Feinstein's Leica SP8 that it destroyed a HyD detector. No additional details - so could have been "crazy concentration", "crazy laser power", "bad settings" (aka bad user), "user not following training", any or all of the above. The Leica HyD edetectors have a safety interlock - Tim told me zap was too fast.

Alexa Fluor 568 Phalloidin worked as a LIVE cells F-actin label for Dowlette-Mary. Normally users fix and permeabilize cells, so any Fluorophore-Phalloidin works. I encourage users to test AF568-Phalloidin on their (your) live cells and let me know if it lights the live cells up ... and that I should update this section with postive and/or negative results.

20240207W

--> see also (above) 20240605W "ThermoFisher Superclonal recombinant secondary antibodies ... available with Alexa Fluor PLUS fluorophores".

"Plan Plus" (see also Plan T below in this box for even brighter)

Alexa Fluor Plus secondary antibodies 3x brighter 1.3x more expensive

Famous quote: "Time is money".

ThermoFisher (Molecular Probes) introduced circa 2023 Alxa Fluor Plus secondary antibodies.

https://www.thermofisher.com/us/en/home/life-science/antibodies/secondary-antibodies/fluorescent-secondary-antibodies/alexa-fluor-plus-secondary-antibodies.html#table

Donkey anti-mouse is $355 for 1 mg (1 mL)

https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Highly-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A32766

If you use 1:100 dilution, then 10 ug (10 uL) is $3.55 per coverglass, imaging dish, etc.

the "standard" prouduct is 1.3 fold less, so $2.55.

the primary antibody would likely be used at the same amount, say $1 per specimen.

GM update #1 20240322F: ThermoFisher SuperBoost tyramide signal amplification is about $5.33 per coverglass, so with primary Ab at $1/coverglass would be $6.33 each coverglass (or 35 mm imaging dish), 10x to 100x higher intensity implying 1/10 to 1/100 imaging time.

Confocal microscope time is $27/hr (work hours, our Leica SP8 or Olympus FV3000RS).

User time making specimens and imaging: $0 from the standpoint of the P.I. = already accounted for.

So 1 hour confocal time:

SuperBoost:        $5.33 + $1 + 27 = $33.33.

AF### Plus:        $3.55 + $1 + 27 = $31.55.

AF### Regular:  $2.55 + $1 + $27 = $30.55.

Or, since Plus is 3x brighter, could reduce confocal time to 1/3 hour ($9) and Plus slide same brightness as regular for 1 hour:

Plus at 20 minutes: $3.55 + $1 + $9 = $12.55.

SuperBoost at 10 minutes (i.e. 1/6 imaging time): $5.33 + $1 + $4.50 = $10.83.

My recommendation is choose the same acquisition time, get "Plus" 3x brighter data, or SuperBoost 10x to 100x brighter.

GM update #2 20240322F: AAT Bioquest now offers "Styramide" (Styryl-phenol aka styryl-tyramide - see freepatentsonline or google patents for patents), which they claim is superior to tyramide - see box below for their marketing info.

MAYBE Styramide will be superior to ThermoFisher Alexa Fluor ### Superboost TSA.

GM update #3: (minimal info here); a Japanese group has published several papers using "methyl-Luminol" as an alternative to tyramide for HRP. See pubmed or ask GM for more info.

**

I suepect many experiments could use lower concentration of secondary antibody to get "same or almost as bright" as the common 1:100 dilution = same more money, similar brightness. There is also potential to optimize incubation time of the primary and/or secondary antibodies, such as dilute further, incubate 24 hours (aking sure the specimens do not dry out), see (direct label flow cytometry experiments:

Whyte, C. E., Tumes, D. J., Liston, A., & Burton, O. T. (2022). Do more with less: Improving high parameter cytometry through overnight staining. Current Protocols, 2, e589. doi: 10.1002/cpz1.589

***

"Plan T" for 10x to 100x brighter:

Big boost in signal: tyramide signal amplification (TSA) has been available since around 1990. Used optimally, can increase signal approximately 100x, while background is still close to zero. This could reduce imaging time by a lot (33 fold compared to "Plus" above) or increase signal for same scan time, or some combination. HRP is "easy to kill" the enzyme (Biocare Medical "PeroxAbolish" is one of the coolest name products for this), then multiplex,

Many companies now offer TSA reagents, see for examples (two of many):

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/immunofluorescence/tyramide-signal-amplification-tsa.html

https://www.tocris.com/product-type/tyramide-signaling-amplification-tsa 

McTip 20240131 Centrifuge down antibodies

GM note1: use cold (stored 4 C) microfuge rotor - if use room temperature rotor, you will cook your antibodies - similar to frying egg whites. You could use ultrafiltration (i.e. 40nm Amicron filte) though some risk of your antibodies sticking to the filter (lose of titer).

GM note 2: Also, if you use BD/BioLegends Brilliant Violets (BV421, etc), Brilliant Ultraviolets (BUV395 etc) you should use BD's Brilliant buffer to avoid aggregation of the Brilliants; quantum dots may or not play well in Brilliant Buffer and vice versa. I also note the authors used several QDot antibodies - quantum dot antibodies originally (first generation thermoFisher/Molecular Probes, acquired QDot Corp circa 2005) were highly prone to aggregation - may or not have been corrected in current (2024) versions. QDots have "blinking issues" which may be good for PALm/STORM/SMLM super-resolution microscopy and/or bad for standard "brighter is better" fluorescence microscopy.

Fun fact: Authors used many Brilliants (BUV, BV, BB) and several quantum dots. Brilliants technology won Nobel Prize in Chemistry in 2000 "conductive polymers" (see Sirigen history page - acquired by BD); Quantum dots won Nobel Prize in Chemistry in 2023 (more for light sources for televisions and computer screens than for fluorescence). 

bioRxiv preprint doi: https://doi.org/10.1101/2023.12.14.571745

50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells
Andrew J. Konecny, Peter Mage, Aaron J. Tyznik, Martin Prlic, Florian Mair

Antibody mixes were centrifuged for 10,000 x g for 5 minutes immediately prior to use to remove antibody aggregates. We have observed a decrease in aggregation of Qdot 605 :: CD2, BUV615 :: CD141, Qdot 625 :: NKp46 (CD335), Pacific Blue :: CD1c, NovaFluor Blue 555 :: CD8a, NovaFluor Blue 585 CD4, and RB744 :: CD127. An example for such aggregates before and after centrifugation is shown in Online Figure 18.

Apple Mac users networking SMB://networkname is equivalent to Windows PC \\networkname

(the above line item is from McTips 2018 index).

for example, if a server is \\TikiGoddess on a Windows PC, then SMB://TikiGoddess on a Mac.We do not currently have a PC or server called TikiGoddess - to see Tiki_Goddess, see http://confocal.jhu.edu/gallery   .

Note: You may need to be logged in as a local user, not any JHU SOM Domaion login (ex: WIN domain). Local login cannot see the Domain assets for security. You can easily log in/out or "switch users" between a local and a Domain login.

Please do not give out the names of our file server or any image core workstations to anyone.

**

You can create a network "share" on YOUR PC. Your office/lab PC is owned by JHU, and managed by your PI, so you should get permission before creating a "shared" folder -- and if you do not fully understand networking and computer security you should probably NOT crete any shartes.

For example, C:\TikiGoddess\Share could be made visible on the campus network (may be restricted to being on -- or not on -- the Domain). Then you could transfer files from our acquisition PC to your PC "share", and as soon as you get back to your lab, move the contents from your "Share" folder to a secure (not visible not network) folder. 

We recommend you upload from our acquisition PC(s) to your JHU OneDrive - this is simpler (though usually slower) than network share drives. MyJHU --> Cloud --> JH OneDrive (can be made a favorite in MyJHU), upload your new data (i.e. save to a folder with today's date, so your new data is segregated from previous data), then sign out of your OneDrive, wait for browser page to update, then sign out of MyJHU, wait for browser page to update, then close the web broswer.

Reminders:

* No USB drives on any of our image core PCs.

* no checking your email (JHU or personal) on any of our PCs. Besides your privacy, if you get an email with a computer virus it could infect our PCs and JHU network (which could lead to you being fired for cause). 

* No surfing the "Dark Net" on any of our PCs - and please use MyJHU just to access OneDrive (not email, not other content ... not your daily covid-19 "click"). 

McTips 2018 PDF (as of 201810002) go to: McTips 2018 

https://works.bepress.com/gmcnamara/84

The McTips 2018 includes some of my thoughts with respect to Fast Photon Counting (FPC) to make fluorescence confocal microscopy both faster and more quantitative than is now practiced by most biomedical researchers (i.e. twiddle the HV gain and offset values until someone proves their boss' hypothesis ... especially when they are using 'Santa Crap' antibodies and don't bother with controls).

https://www.fpbase.org - Fluorescent Proteins Database - including Spectral Viewer and FRET Ro Calculator

Spectral Viewer https://www.fpbase.org/spectra/ 

FRET Calculator https://www.fpbase.org/fret/ 

FRET equation from above:

QY = Quantum Yield, EC = Extinction Coefficient, J(λ) = Overlap Integral, R0 = Förster Radius, �� = refractive index, κ2 = orientation factor
Wu & Brand (1994). Resonance Energy Transfer: Methods and Applications. Analytical Biochem. 218 

 

Note: GM also has a FRET calculator Excel file inside PubSpectra ZIP file.

PubSpectra web page          https://works.bepress.com/gmcnamara/9

PubSpectra download link    https://works.bepress.com/gmcnamara/9/download

20190528U: stay tuned late 2019 for Nathan Shaner's improved GFP and YFP. The GFP is 2x brighter than his mNeonGreen, so 6x brighter than EGFP. Time has come to retire EGFP!!!

ThermoFisher Prolong Glass  (without DAPI)     is now the best choice, if imaging fixed specimens with oil immersion objective lens. 

==> Or- from Marker Gene Technologies (see MGT web site for more options for each product):           * 

           * Opti-Bryt (fixed cells) https://www.markergene.com/opti-bryt-trade-perm-antifade-mount.html

           * Opti-Klear (live cells) https://www.markergene.com/opti-klear-live-cell-imaging-buffer-5x.html

Prolong Glass info states needs to cure for 30+ hours.

My advice:

* grow cells in imaging dishes (mattek or WPI-Inc ... or ibidi imaging quality coverglass chambers)

** at no time should cells be allowed to "air dry" = keep submerged.

* fix (i.e. formaldehyde), permeabilize if needed.

* immunofluorescence (i.e. http://www.nano-tag.com 2ndary nanobodies with each mouse mAb) ... can include DAPI and/or other counterstains here (example: fluorescent phalloidin).

* wash extensively (but quickly).

* "drip on" some Prolong Glass with imaging dish tilted, so that it forces aqueous media away ... pipet out the "run off", drop more (but not too much $) Prolong Glass ... goal is ~100% Prolong Glass, ~0% aqueous.

* allow to "cure" 30+ hours, in the dark, at room temperature, no lid, large volume of air (i.e. not small sealed box) to let volatiles escape.​ ... Probably simplest to go closer to 48 hours (and would be nice to be consistent in experiments).

20180803Fri ... connecting to our file server from Windows (win 7).

* ask George for the name of our file server - and please do not give out the name or IP address of our server.

* you are welcome to set up your own 'share drive to transfer your files (and can we please have 42 Terabytes of space on yours?).

Some Windows PC's are able to see our file server. Some are not. Today we -- "we" being 99% Jim Potter and 1% GM -- were able to trouble shoot the network acess issue. 

0. assumptions:

      (i) windows PC (win7 or ideally win10)

      (ii) plugged into the JHU SOM network (Ethernet cable).

      (iii) you have administrator privileges on the PC (does not need to be 'Administrator' login name).

1. Use JHARS to connect to JHU network (if you have not already done so) https://jhars.nts.jhu.edu/

  1a. after setting  up (or confirming) JHARS, probably useful to power off the PC, wait a few seconds, then power up and log in.

2. enable all the items in the Local Area Connection Properties dialog box (it is probably ok to enable more, but at minimum you need IPv6 and IPv4 and probably more).

3. using CMD prompt -> IPconfig / all (2nd screen shot below) ... see that DHCP Server 10p15.76.226

Windows Start menu ... cmd ... ipconfig / all

    ==> DHCP Server 10.15.176.226

    ==> Subnet mask 255.255.255.0     (if this is not correct, you may not be able to see JHU network at all!).

January 15, 2019 (20190115U) new book and eBook:

Basic Confocal Microscopy second edition
https://link.springer.com/book/10.1007%2F978-3-319-97454-5
W. Gray (Jay) Jerome, Robert L. Price 2018

Chapter 1 includes:
Our Ten Commandments of confocal imaging are as follows.
1. The Perfect Microscope and the Perfect Microscopist Do Not Exist
2. Confocal Microscopy Is More Than a Confocal Microscope
3. During Specimen Processing the Integrity of the Specimen Must Be Maintained as Much as Possible
4. Photons Are Your Friends and Signal-to-Noise Ratio (SNR) Is King (GM note: and Queen and President and Premier ... and Dean, milliDean, microDean, nanoDean)
5. Quantification of Fluorescence in a Confocal Micrograph Is a Challenge and at Best Is Only Semiquantitative 
6. Scientific Digital Imaging and Normal Digital Imaging (Family Photography) Are Not the Same
7. Your Image Is Your Data: Garbage in Will Result in Garbage Out
8. The Resolution and Bit Depth Present in a Digital Image Are a One-Way Street
9. The JPEG (Joint Photographic Experts Group) Image File Format Is EVIL but Useful
10. Storage Media Is Essentially Free and Infinite
Notes:
* JHU staff can download eBook PDF for 'free' (that is, JHU has a subscription to publisher's content ... which comes out of NIH and other grants indirect overhead).
* Springer ofters MyCopy softcover edition $24.99 see "Buy" button at top right of https://link.springer.com/book/10.1007%2F978-3-319-97454-5

GM comments:

#1. reminds me of my high school's National Honor Society slogan (which I'm paraphrasing here): "Those of you who think you're perfect, amuse those of us who are". (our NHS T-shirt had the original slogan and Mr. Wampole's face ... Mr. Wampolewas the advanced math teacher in addition to neing NHS chapter advisor).

#5. fluorescence intensity is 'at best only semiquantitative' ... this is sad & true

  * (I blame it on 30+ years of researchers not requiring 'good intensity quantitation' of manufacturers AND manufacturers not making quantitation easy and reasonably priced).

  * I suggest ACCM's Leica SP8 confocal microscope HyD detectors in photon counting mode enables users to come close to quantitation. There are still issues of laser performance (most lasers fluctuate in power), Z-drift and XY-drift (not a big deal for sngle focus plane, single field measurements), and specimen refractive index induced issues (if any mismatch in R.I., then Z affects intensity -- see Staudt and Hell "TDE" paper and Olympus silicone oil graph).

  * Fast FLIM and - simpler and less expensive to get going and less data deluge - "fast photon counting" (FPC) can be implemented on any PMT or Hybrid or APD based point scanning confocal microscope. Re: Becker&Hickl fast FLIM or ISS FastFLIM" (and either would be less expensive to add to our FV3000RS confocal microscope than buying a new fully loaded Leica SP8 Falcon Fast FLIM ... bonus: Wolfgang Becker correctly disses Leica's featuring 'fast lifetime contrast' (FALCON) over fast TCSPC data).

Grey and Price 2018  Table 1.1:

Reference: 

Pawley J (2000) The 39 steps: A cautionary tale of quantitative 3-D fluorescence microscopy. BioTechniques 28:884–888

https://www.future-science.com/doi/abs/10.2144/00285bt01 

Leica Microsystems - THUNDER Imager Tour at JHU SOM 3/3019

https://www.leica-microsystems.com/jhu-tour/ 

Leica - LIGHTNING and THUNDER

Leica THUNDER Tour - See Through the Haze with Computational Clearing
Johns Hopkins University, School of Medicine, Ross Fluorescence Imaging Center
* Summary:

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MetaMorph Key 3 (#34334)

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q Andor Revolution X1 Spinning Disk confocal microscope -...

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