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

"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 imagign dish), 10x to 100x higher intensity implying 1/10 to 1/100 imagign 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: alternative to 'optical clearing' (which requires fixation and chemical clearing) for your fluorescent specimens.
* Demonstrations March 12-15, 2019. Two instruments:
   1. "Model organisms": Leica M205, 5x 0.5NA objective lens ... large field of view.
   2. "Live cells": Leica DMi8 inverted microscope, environmental controls (37 C, 5%CO2) available, full set of objective lenses.
* Seminar: THUNDER Imagers – Decode 3D Biology in Real-time.
RSVP please.
Computational Clearing – available exclusively on Leica Microsystems THUNDER Imagers – offers groundbreaking ease of use, throughput, speed and sensitivity for 3D tissue, live cell and model organism imaging. Unparalleled image quality from stereo, upright and inverted live cell microscopes, with no special sample preparation needed.

THUNDER Imager - how it works (pdf) ... two imaging systems platforms.

https://www.leica-microsystems.com/science-lab/thunder-technology-note/ 

LIGHTNING info (web link below - pdf download at bottom of that page) ... 'adaptive deconvolution' (GPU enabled).

https://www.leica-microsystems.com/science-lab/how-to-extract-image-information-by-adaptive-deconvolution

20190712Fri - JHU Data Repository (Repositories)

==> JHU now (2019) uses Microsoft OneDrive for single user backup and sharing, ex. a graduate student or postdoc can share specific OneDrive folders with JHU colleagues and P.I. (not sharable outside JHU) Each JHU employee or student gets 5 Terabytes (5 Tb) OneDrive storage for free. Additional space should be arrangable through I.T. 

***

For published data:

Craedl - Collaborative Research Administration Environment & Data Library 

https://craedl.org/docs/

Craedl is hosted by MARCC Maryland Advanced Research Computing Center https://www.marcc.jhu.edu  

***

See also:

JHU Sheridan Libraries 

https://guides.library.jhu.edu/dataservices/data/analyze

* Data Services   https://archive.data.jhu.edu/

20210510M Excitation power at the specimen for widefield (Lumencor SPECTRA III) for Olympus IX83 and confocal (Olympus FV3000RS)

Note: numbers are approximate to simplify the math. Also going to ignore usual S.I. units (Joules, etc)

Widefield:  http://confocal.jhu.edu/current-equipment/fishscope

confocal:   http://confocal.jhu.edu/current-equipment/fv3000 

Our Lumencor lamp has ~500 mW per channel (8 channels, 368-747nm). Entire eyepiece field of view (and objective lens field of view) is illuminated usually [a square aperture COULD be used to block the area outside the camera field, is not), imaging area is ~50%, so "power to the camera field of view is ~240 mW per channel for field of view for 1 second (and normally only one channel on, since monochrome camera ... Olympus cellSens "Process Manager" lists up to 20 acquisition settings, lamp channel --> filter cube --> Sutter 10 position filter wheel). .Maximum exposure time on ORCA FLASH4.0LT is 10 seconds. Typically acquire between 100 ms and 1000 ms (1 sec). Will use 1 second here. Pixel size for the 60x/1.4NA objective lens --> camera is 108nm, round to 100nm.

Our Olympus confocal has ~20 mW per laser lines (7 laser lines, 405-730nm). Fastest pixel dwell time: 2 microseconds, if 5 line average, then 10 microsecond "total pixel dwell time". For ~4 million pixels (2048x2048), laser(s) on for 40 seconds (actual scan time approximately double sincewe usually use uni-directional scanning). While I typically acquire at 50nm pixel size for 60x/1.4NA (with GPU deconvolution), I use 100 nm here to match the camera rounded pixel size above. The confocal spot size is NA and wavelength dependent (and is "Airy" or roughly Gaussian), will simplify to in focus pixel is getting ~50% of the excitation photons, so 10 mW per channel per pixel..

100 nm pixel size * 2000x2000 pixels = 200x200 um image size

Widefield: every pixel 1 second exposure (simultaneously). 240 mW / 4,000,000 pixels = 0.00006 mW = 0.06 uW = 60 nW (1 second). 

Confocal: 10 mW for 10 usec (focused spot, 'typical' confocal pixel dwell time). In practice, probably <=50% of the power reaches the specimen (light loss in coupling to the scanhead, in the scanhead, through objective lens back aperture). so would be ~5 mW for 10 usec ... but typically use 0.1% laser power so ~5 uW for 10 usec.

Real-life excitation power is much lower than the "max power" above.

Lumencor on FISHscope: we typically use 10% of maximum, so 500 mW --> 50 mW, etc. ... so with 10% power, 6 nW / pixel for 1 second.

Olympus FV3000RS confocal: we typically operate the confocal with the "10% Neutral Density" option selected, limiting power control range to 0.01 - 10% power (zero is also available) AND "typical" power setting range is 0.02% to 1.00%, and 0.10% is common, hence the ~5 uW for 10 usec.

Less than maximum power: We do this to minimize photobleaching (users can contribute to this by using optimal fluorophores and op[timcal mounting medium).

***

background info on fluorescence imaging:

Widefield camera: ~80% peak quantum efficiency.

Olympus confocal GaAsP detectors: ~40% peak quantum efficiency (of face plate, and will ignore a lot of details that may lower the effective QE, since the poibnt of a PMT is to multiply the photon signal, and at typical HV "gain", one photon --> 100,000 electrons, Analog-to-Digital Converter produces 'nice' signal to the software).

Widefield: in plus ALL the out of focus light ... Z-series followed by GPU deconvolution can produce "confocal like" results.

  Widefield XY resolution: dxy = 0.61 * Lambda / NA = 0.61 * 500nm / 1.4 = 214 nm

   Widelield plus deconvoluton: ~10% improvement, so 214 - 21 = 193 nm.

   GM interprets Nyquist sampling theorem for 2D as needing ~3 to ~3.5 pixels across resolution, so 214 / ~3 is ~70nm pixels. On FISHscope, we deliberately chose to purchase a 60x/1.4NA objective lens so more light would reach a pixel (108x108nm of specimen = ~10,000 nm^2) than if we purchased a 100x/1.4NA lens (~65x65nm, 4,225nm^2, which is 42% as many photons for same exposure time, implying need ~2.5x longer exposure time ... and assuming all the excitation power of the lamp reaches the specimen).

Confocal, at standard of care 1.0 Airy unit pinhole: optical section, mostly blocking the out of focus light, so only "in focus plus lamost in focus" light  detected. For same field of view as our FLASH4.0LT, acquisition time takes a lot longer (nominally 40 seconds, see above). Each focus plane is "clean" (in and nearly in focus light, no out of focus light), that is, every photon is a "good" photon. 

  Confocal XY resolution (1.0 Airy unit): dxy = 0.51 * Lambda / NA = 0.51 * 500 / 1.4 = 182 nm.. (if pixel size is ~50nm XY).

    Confocal XY resolution (1.0 Airy unit) plus deconvolution: ~10% improvement, so 182 - 18 = 164nm. (if pixel size is ~50nm XY).

Confocal can be used at SMALLER pinhole size (Zeiss has a nice appnote with the details), one example here: 0.5 airy unit

  Confocal XY resolution (0.5 Airy unit): dxy = (0.51 * Lambda / NA) / 1.11 = 0(.51 * 500 / 1.4 ) / 1.1 = 165 nm.. (if pixel size is ~50nm XY, maybe useful to use 40nm).

  Confocal XY resolution (0.5 Airy unit) plus deconvolution): 165 - 16 = 149nm (if appropriate pixel size). 

Leica appnote claims "not muich loss of fluorescence emission" at oderately small pinhole size (vs 1.0 airy unit). Best to do the test yourself, using out Leica confocal and HyD detector(s) in photon counting mode (converting PMT --> ADC values to photon counts is not trivial, and most publications that report confocal PMT data do so in "Arbitrary Units" -- quite literal, since no one "proves" that 1000 digitiizer counts is double that of 500 ... on Leicawith HyD in photon counting mode, the counts are the photon counts)..

Nyquist sampling microscopy data - good calculator at https://svi.nl/NyquistCalculator

Better resolution: use shorter wavelength fluorophores, such as BUV395 or BV421 or SB436 or SuperNova v428 (assuming microscope has proper excitation wavelength, filters, good detector QE in blue, good transmission through objective lens), and no background fluorescence (from specimen or optics). 

Conclusion: both widefield and confocal are useful. I strongly encourage decovnolving each.Good to great data if you use each correctly. 

*   Widefield: usually faster acquisition, limited flexibility in acquisition field of view (wrt illumnination field). 

*   Confocal: massively flexible in acquisition settings (more on Leica SP8 than FV3000RS).

There is a saying in the restaurant business (and most other retail businesses) 3 keys to success: "Location, location, location". 

Fluorescence microscopy success, assuming good widefield and/or confocal microscope available (and the user knows what they are doing, or gets help from the image core manager); has 3 keys to success: "sample preparation, sample preparation, sample preparation".

Last thoughts:

* Bring a nicely labeled specimen (i.e. 4 color fluorescence microscope slide-sample-coverglass) with you and image at the START of every session (can be single in focus plane, modest field of view if confocal, so not take long). Helps make sure microscope (and user) are operatign correctly. Sometimes a lase may be "dim", or oil on the 20x dry lens, or 'just some weird shit' (though more likely previous user did something wrong).

* Every experiment (every replicate) needs an appropriate negative control or controls. If using "old school" primary + secondary antibodies, usual key cotrol is "no primary, ALL secodnary" antibodies. Can be "quick look by eye, then small field of view acquisition" (of the brightest relevant area you see by eye, and focus with the instrument). The hope is to document that the negative control worked -- but if the cotnrol is 'surprisingly bright' better to find out at the start of the session, and troubleshoot (and think!), than to assume life is good. I note that users of labeled primary antibodies (ex: Brilliant Violet 421 anti-CD8 monoclonal antibody from the flow cytometry world) need to think through their cotnrols. Ideally: cell linke(s) with the gene of interested transfected/transduced in or knocked out. For many cells types and tissues, controls can be other cells, such as for T-cells, most blood, lympgh node, or spllen T-cells are CD3+ and either CD4+ or CD8+ (assduming you block FcReceptors and are aware that a few t-cells are CD4+ CD8+, some are CD4- CD8-, some macrophages are CD4+, some dendritic cells are CD8alpha positive, and some cells trogocytose (eat and/or nibble on) other cells; also G.I. (gut) T-cells are ~50% alpha-beta TCR (either CD4+ or CD8+) and the other half are gamma-delta TCR (may be CD4- CD8-, but could be positive ... "cells do not read Cell, Immunity, Nature, Science or ELife").

* "Old antibodies die, please through them away". I now offer the chicken egg rule: if youi put a (raw) chicken egg into a refrigerator the same time you put a new antibody in a

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