McTips
Tips & Procedures (web page) http://confocal.jhu.edu/mctips/tipsprocedures (please contact GM directly for PDF).
** 20240123U note: I am currently 20% time at Ross Image Center, so new McTips will be rare, and new 2024 page not worth organizing. I put an antibody solutions centrifugation (cold minifuge rotor) tip near the top of this page (just past the web links).
20240809F Leica SP8 HyD detectors linear range up to 60 photons per second (each) - see http://confocal.jhu.edu/mctips/leica-sp8-hyds-linear-range-to-60-photons-per-microsecond
2021 PC Tips for microscope and analysis workstations http://confocal.jhu.edu/mctips/pc_tips_2021
2022 PC Tips for microscope and analysis workstations http://confocal.jhu.edu/mctips/pc_tips_2022
2022_part2 PC Tips (started 20220705U) http://confocal.jhu.edu/mctips/pc_tips_2022_part2
Confocal Sweetest Spots (web page) http://confocal.jhu.edu/mctips/confocalsweetestspot
Online Image Data Repositories - blog (20210427W web page) image-data-repositories
MPMicro "Multi-Probe Microscopy" (1500 pages - please do not print out) https://works.bepress.com/gmcnamara/2/
Multiplex fluorescence microscopy http://confocal.jhu.edu/mctips/multiplex
Alexa Fluor 610-X performance calculations wrt FISHscope http://confocal.jhu.edu/mctips/af610-x (gm note: SulfoRhodamine 101 = SR101, Brightness=125 aboutthe same as AF610)
StreamBio UK LinkBright CPNs labeling kits available (1/2003) from Sigma-Aldrich (hopefully low shipping and total costs) http://confocal.jhu.edu/mctips/streambiouk-linkbright-cpn-kits
McTips 2023 http://confocal.jhu.edu/mctips/mctips_2023 (no PDF)
McTips 2020 PDF go to: McTips 2020 download at https://works.bepress.com/gmcnamara/90
McTips 2019 PDF go to: McTips 2019 download at https://works.bepress.com/gmcnamara/85
McTips 2018 PDF go to: McTips 2018 download at https://works.bepress.com/gmcnamara/84
McTips 2017 PDF go to: McTips 2017 download at https://works.bepress.com/gmcnamara/81/
McTips 2017 Direct download is https://works.bepress.com/gmcnamara/81/download
Factoids (started 01/2022) http://confocal.jhu.edu/mctips/factoids
FISHscope Quick Tips (startup, close out session) http://confocal.jhu.edu/mctips/fishscope-quick-tips -- FISHscope main page is http://confocal.jhu.edu/current-equipment/fishscope
* Quick Leica SP8 confocal microscope tip (see more info on our SP8 page from current equipment):
I recommend (we have Leica SP8 Klondike linear scanner - most SP8's are the same sub-model):
HyD detectors, Photon counting mode ... not max linear counting rate is 6 Mcps (million counts per second) = 6 counts per microsecond or 0.6 counts per 100 nanoseconds.
At very high counts the safety interlock should trigger, resulting in all values ZERO until the user stops scanning (i.e. 1000x1000 x 100 planes Z-series could be lots of zeros).
600 Hz line scan rate (enables full range of zoom
10 Line accumulation as a starting point, more or less depending on user needs. Max is 16 line accumulation.
1 Frame accumulation as starting value, more if needed.
Modest laser power, usually 0.5% or 1% for 405nm laser, 1 or 2 percent for any of 488, 552, 638nm lasers, depending on what fluorophores are used (ex. DAPI, Alexa Fluor Plus 488, Alexa Fluor Plus 555, Alexa Fluor Plus 647).
Sequential scan tracks, usually ... can combine blue (DAPI) and NIR (AF647Plus) in moany experiments (3plex plus DAPI), such as: (i) AF555Plus, (ii) AF488Plus, (iii) DAPU + AF647Plus.
Always have detection bandpass AT LEAST 10nm away from any laser line - even if the laser line is not used in that wscan track. If no NIR, turn off the 638nm laser.
Ca++ ion experiments of FV3000RS
20240801H - pasted from an email to a new user whose lab has a lot of experience on the FV3000RS confocal microscope.
Ca++ imaging expt advice – from long experience: start with cells with a simple positive control – and this might take more than one imaging dish (helps if your lab mates tell you the folder/file of one of their recent successful experiments, so we can load their FV3000RS settings). Classic control is Ionomycin (A23187) in Ca++ containing medium. For cell the right receptor, ATP, Carbachol (Ach analog), or some other “high positive control” ligand is ok (many GPCR’s signal predominantly through IP3 second messenger to activate Ca++ ion responses). Some researchers go the extra step of: (1) start timelapse (say 1 minute at 30 fps), (2) ATP or similar physiological ligand and let Ca++ signal subside (1-5 minutes), then (3) ionomycin+Ca++ ions.
In the early days of Ca++ imaging, researchers also included an EDTA (or EGTA?) chelator step, to measure “F0” (lowest fluorescence). This has mostly gone out of fashion when using “intensitometric” Ca++ indivators (GCaMP# fluorescent protein biosensor, Fluo-3, Fluo-4, etc), vs early research ratiometric indicators such as Fura-2 (340/380nm excitation ratio, ~510nm emission, widefield microscopes since nearly all confocal microscopes lack those UV wavelength, and 340nm is phototoxic).
FYI - Fluo-3AM, Fluo-4AM, or (in the old days) Fura-2AM esters (or similar indicators loaded with "AM": easy to overload the cells. This can cause the Ca++ indicator to become a Ca++ chelator, that suppresses Ca++ ion response; The AM ester gets cleaved to result in the ion sensitive indicator + formaldehyde, which means high concentration of the AM ester could result in "fixation" of your cells. Rare(?) but can happen: some cell types (or cells from unusual organisms than typical human HeLa experiments) migt not have sufficient cytoplasmic "non specific esterases" to cleave the AM ester - this would result in the added reagent simply equilibrating in the cytoplasm, resulting in signals that are not Ca++ responsive. The reagent can also end up in non-cytosolic compartment(s), such as lysosomes, other endosomes, possibly ER - or extruded from the cytoplasm (usually through an MDR 'drug resistance' transporter), resulting in usually unwanted or no signal.
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Brilliant and SuperBright Blocking Buffers for BV421 etc
20240730U
Brilliant and SuperBright Blocking Buffers for BV421 etc
* I am a big fan of BV421 and related "polymer fluorophores". Won Nobel Prize in Chemistry in 2000 (electron conducting polymers).
BV421 is much brighter than standard small dye fluorophores, E.c. 2.500,00 M-1cm-1, QY 0.6, Brightness = Ec*QY/1000 = 1500 vs Fluorescein (pH 8.0) 81 and Alexa Fluor 488 similar B.
* one mystery is why two or more Brilliants tend tio bind each other and/or "non-specifically" bind cells, calibration beads, etc (and why single product is not a problem).
https://www.colibri-cytometry.com/post/blocking-brilliant-dye-interactions
introduction -- see web page for many more details, lots of flow cytometry scatterplots:
The issue
Interactions between Brilliant dyes on antibody conjugates. Creates compensation-like artefacts where cells staining with one Brilliant-conjugated antibody are artificially positive for others.
The reagent
The appropriately named Brilliant Stain buffer.
Available from BD and ThermoFisher:
https://www.thermofisher.com/order/catalog/product/00-4409-42
https://www.thermofisher.com/order/catalog/product/SB-4401-42?SID=srch-srp-SB-4401-42
https://www.bdbiosciences.com/en-gb/products/reagents/flow-cytometry-reagents/research-reagents/buffers-and-supporting-reagents-ruo/brilliant-stain-buffer.566349
Also available in a more concentrated format:
Brilliant Stain Buffer Plus (bdbiosciences.com)
What is it?
Probably monomers of the base SIRIGEN dye, according to Florian Mair. The spectrum of the BS buffer resembles that of Super Bright 436, as we'll see later.
Alternatives
None, just the variants above. Using less antibody limits the artefacts.
What it’s supposed to do
Reduce dye-mediated interactions between Brilliant Violet, Brilliant Ultraviolet, Brilliant Blue and Super Bright conjugated antibodies.
Brilliant Stain Buffer also helps reduce background in human whole blood assays because it contains polyethylene glycol (PEG). A lot of fluorophores contain PEG, so the presence of PEG in the buffer swamps interactions between serum antibodies and the fluorophores. Why do people have anti-PEG antibodies? Well, there's PEG in vaccines, too, and a lot of people got vaccinated recently. Read more here.
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Alexa Fluor Plus secondary antibodies 3x brighter 1.3x more expensive
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).
Some options for immunofluorescence microscopy detection
Option |
Brightness |
Reagent Price (est)
[1st or 1st + detection reagents)
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Comment |
Nano-Tag or Chromotek VHH nanobody 2ndary Ab
ex: Alpaca anti-rabbit AF488 VHH
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1/3x |
$1 + $1 |
As formatted from Pleiner et al 2018 JCB (PubMed 29263082; Ewers comment 29444803) (licensed to both Nano-Tag.com and Chromotek (now ptglab.com see Nanobody page) dimmer; but enables multiplexing of multiple mouse monoclonal antibodies etc.
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Hypothesis: nanobodies with optimized tail (academic lab with protein synthesis and labeling capability; ThermoFisher etc if they get their act together) |
whatever the maker wants (ex. 1x, 3x, 5x ...) |
$1 + $1
(or multiplier for the detection reagent)
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GM hypothesis/suggestion: Pleiner 2018 suppl table has the sequences of all the VHH used --> instead of "default C-terminal tail" in Pleiner (20+ years of nanobodies) a "custom tail' with optimized conjugation sites for NHS ester (lysine), maleimide (cysteine), non-canonical amino acid(s) (ncAA; click chemistry) could enable optimized brightness, photochemistry (no cross quenching of the fluorophores; optional FRET to diversify colors; FLIM etc).
Further refinement: BV421-streptavidin to mAb-biotin, etc.
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Direct labeled Fab - classic dye, ex. Alexa Fluor 488-anti-CD4
ex: mouse (recombinant) Fab fragment monoclonal antibody (~50 kDa) with degree of labeling (DoL) 1-2 Alexa Fluor 488 fluorophores.
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1/9x (?) |
$0.33 |
Direct labeled antibody (IgG) - classic dye, ex. Alexa Fluor 488-anti-CD4
GM note: this could be much brighter as Fab-(custom tail) as hypothesized above for 2ndary VHH nanobody. Could further optimize with BV421 (one or multiple) or BV-tandem (multiplex) on custom tail.
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Direct labeled antibody (IgG) - classic dye, ex. Alexa Fluor 488-anti-CD4
ex: mouse monoclonal antibody (~155 kDa) with degree of labeling (DoL) 4 Alexa Fluor 488 fluorophores.
|
1/3x |
$1 |
Common in flow cytometry; rare in fluorescence microscopy |
Direct labeled antibody - modern fluorophore, ex. BV421-anti-CD4) |
3x |
$1 |
Common in flow cytometry; rare in fluorescence microscopy. BV### also available (ex. BV570) |
Standard 2ndary antibody (ex. goat-anti-mouse; donkey-anti-rabbit)
|
1x |
$1 + $1 |
Standard of care since ~1965 (the fluorophores, microscopes, detectors have improved a lot over ~60 years) |
Recombinant 2ndary antibody (ex. goat-anti-mouse; donkey-anti-rabbit)
|
1x |
$1 + $1 |
==> no more sacrificing small and/or large furry animals for your experiments; more consistent reagents.
Pricing (usually) identical to standard from dead animals 2ndary antibodies - likely more profitable since no animal feed, more consistent products.
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"Plus" fluorophore 2ndary Ab |
3x |
$1 + $1.30 |
See below for thermoFisher Alexa Fluor Plus secondary antibodies - Plus recombinant secondary antibodies also available. |
"Plus" Recombinant 2ndary antibody (ex. goat-anti-mouse; donkey-anti-rabbit) |
3x |
$1 + $1.30 |
* Plus = High brightness.
* Recombinant = high consistency, no more dead furry animals.
https://www.thermofisher.com/antibody/secondary/query/Superclonal%20plus
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Tyramide signal amplification (TSA) * ThermoFisher Superboost; other vendors (AATbio STyramide; Akoya Biosciences ...) |
10x-x100x |
$1 + $6
potentially
$0.20 + $6
if re-titer primary antibody (and test qualit of all other reagents)
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TSA is more complicated (HRP polymer conjugated 2ndary antibody; H2O2 + fluorophore-tyramide) than 'standard' immunofluorescence methods, but might brighter - and when done optimally, essentally zero background.
IHC labs: those labs that routinely use HRP with DAB (and H2O2) COULD substitute the DAB solution with a fluorescent tyramide, for example, Alexa Fluor 488-tyramide (ThermoFisher / Molecular Probes). Best to be mindful of cost of reagents and that fluorescent tyramide is much more sensitive that DAB IHC. You will very likely need to re-titrate the concentrations (amount of volume of each reagent) onto the specimen of ALL your reagents, including the primary antibody (andHRP-polymer-2ndAb, etc). Any background from any reagent will increase background, which you may mis-interpret. At some point, you might even want to deliberately adjust the amount of primary antibody to NOT saturate binding to the antigen (ex. 50% of saturation), or more elaborately, make a series of specimens, dilution series of primary antibody, so understand the "binding curve". I am assuming/hoping your imaging pipeline is quantitative (a digital camera can be tested with a series of exposure times usinga bright, photostable specimen, such as 10 ms exposure ... series in 10 ms steps to 2000 ms [2 seconds]). tip: there are many places in a typical research fluorescence microscope where linearity can be lost (trivial example: photobleaching).
You can "quench" (aka kill) HRP enzyme activity with PeroxAbolish or other reagents / methods (heat for example), then do multiple rounds. See Berrry, ... Szalay, Taube 2021 Science, PubMed 34112666 (7plex Akoya TSA reagents and slide scanner). Tyramide signal amplification has been around since ~1990 (so long time off patent).
* Tip from 1999: Brian Van Tine(at the time a MD/PhD student at UAB) at at CHLS in situ hybridization & immuocytochemistry course did 6plex tyramide signal amplification of PBMCs in solution (ex. CD3, CD4, CD8, CD20, CD64 plus one more),all very bright. I do not have the protocol details - probably spun cells down in a microfuge (cold rotor, balanced tubes) for wash steps. Very bright, very specific. I do not see references off hand but someone (1990s?) published tyramide flow cytometry.
See (2023 cmmentary) https://pubmed.ncbi.nlm.nih.gov/37199234/
1997 research paper https://pubmed.ncbi.nlm.nih.gov/9071319/
TSA in flow (with some quantitation vs standard luorescent antibody labeling:
Clutter MR ... Nolan GP 2010 Tyramide Signal Amplification for Analysis of Kinase Activity by Intracellular Flow Cytometry. Cytometry A 77A: 10201031. Clutter references of prior paublications using TSA of cells in supsension:
23. Kaplan D, Smith D. Enzymatic amplification staining for flow cytometric analysis of cell surface molecules. Cytometry 2000;40:81–85.
24. Kaplan D, Meyerson H, Lewandowska K. High resolution immunophenotypic analysis of chronic lymphocytic leukemic cells by enzymatic amplification staining. Am J Clin Pathol 2001;116:429–436.
25. Kaplan D, Smith D, Meyerson H, Pecora N, Lewandowska K. CD5 expression by B lymphocytes and its regulation upon Epstein-Barr virus transformation. Proc Natl Acad Sci USA 2001;98:13850–13853. 26. Karkmann U, Radbruch A, Holzel V, Scheffold A. Enzymatic signal amplification for sensitive detection of intracellular antigens by flow cytometry. J Immunol Methods 1999;230:113–120.
27. Kaplan D, Meyerson H, Husel W, Lewandowska K, MacLennan G. D cyclins in lymphocytes. Cytometry A 2005;63A:1–9.
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Conceptual |
? |
?$? |
Ideally we would like an immunostaining and immuno-imaging method that produced quantitative data over the full dynamic range over the relevant biology. This is non-trivial.
Imaging side: most modern scientific cameras for widefield and spinning disk cofnocal microscopy (sCMOS) are listed a 16-bit dynamic range, implying 0 .. 65535 "ADU" (analog to digital units") within one exposure. However, usually the camera offset is ~100, and at the high end, Poisson distribution noise is such that one would not want to come too close to saturation, so the dynamic range maybe ~100 .. ~59,000 (within ~10% of the pixel max), ~100 is ~7 bits (0..128), so realistically 16 - 7 - 1 = ~8-bit (albeit in wonderfully small steps). The best camera (and essentially most expensive) camera GM knows on the market in mid-2024 is the Hamamatsu ORCA-Quest2, with photon resolving range of 0 .. 200 (it also has a more sCMOS like ~100 - ~65535 readout mode).
F-actin in the border of a 'typical' human cell (i.e. flat HeLa, U2OS, megakaryocyte) can be "lit up" with fluorescent Phalloidin (exclusively light up F-actin), and one could also express monomeric AausFP1 GFP-actin (G-actin monomers). Good luck seeing both G-actin monomers (in live cells they may diffuse the expected size of a 44 kDa monomer) and sub-plasma membrane F-actin bundles.
Another examples are EGFR (erbB1) and Her-2 (ErbB2), which might be expressed at ~5000 molecule per cell (and mostly plasma membrane) of a normal human cell, but 500,000 to over 1,000,000 molecules for roughly the same size cancer cell (famously breast cancer, potentially glioblastoma and others). So adjacent normal and cancer cells might be 5000 and 500,000 molecule per cell, or for nearby plasma membrane pixels, say 1 and 1000 (if the plasma membrane was 1000 pixels or voxels).
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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
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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.
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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.
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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.
SuperBoost is only 4 HRP per Antibody
https://www.thermofisher.com/order/catalog/product/B40943
Benefits of SuperBoost kits
Enhancement of signal using Alexa Fluor tyramides: SuperBoost kits utilize Alexa Fluor tyramides, which react with HRP to ultimately deposit bright and photostable Alexa Fluor dye on surrounding proteins and other similar molecules. SuperBoost kits are the only kits that combine the brightness of Alexa Fluor dyes with the enhancement of tyramide signal amplification to produce a superior signal.
Poly-HRP enhancement: Unlike TSA, SuperBoost kits employ poly-HRP-conjugated secondary antibodies. In such systems, several HRP enzymes are conjugated with short polymers, enhancing the signal by several fold over regular HRP systems. The poly-HRP is structured in such a way that the antibodies penetrate cells or tissue as efficiently as regular HRP-conjugated secondary antibodies. The molar enzyme/antibody protein ratio has an average value of '4'.
Reaction stop solution: Like any enzyme-based labeling system, it is possible to overdevelop the signal. SuperBoost kits include an HRP stop solution to halt the HRP reaction. HRP stop solution can be used to obtain maximum signal, without increase of background signal. Images produced with optimized HRP reaction times are as sharp as images produced with standard ICC/IHC/ISH methods, but with 10-200 times more sensitivity.
Reduction of background: SuperBoost kits include blockers for the elimination or reduction of endogenous peroxidase and fluorescent background signals. These blockers help ensure that only specific signals are enhanced while keeping non-specific/background signals in check.
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GM: statement above that is is "possible to overdevelop the signal" - I suggest more likely the number of surface exposed tyrosines (phenylalanines, tryptophans, histidines are 'somewhat reactive' as well) will be limiting in TSA (or Styramide, see below) immunofluorescence experiments.
SuperBoost 4:1 molar ratio:
HRP is 44 kDa
IgG is approximately 155 kDa (isoforms vary in MW: IgG1 vs IgG2a etc).
F(ab')2 is approximately 110 kDa (without Fc)
So: 4 HRP (~176 kDa) + 1 IgG (~155 kDa) is ~331 kDa (I suggest ~333 as an easier number to remember).
I also note that Fitzgerald Industries sells HRP20, HRP40 and HRP80 conjugates (ex. to streptavidin, see also their product list for secondary antibodies) ... I believe number of HRP per polymer is 5x more than the number in the name, that is: HRP20-Streptavidin has 100 HRP:SA, HRP40 has 200 HRP:SA, HRP80 has 400 HRP:SA. So, if access is not a problem (thin specimen or antigen/biotin on a coverglass or ELISA SBS plate surface) then HRP80 would be a whole lot of HRP molecules -- and bring in a whole lot of surface tyrosines to "dock" fluorescent tyramide or fluorescent Styramide (Styr below in AATbio section).
* as of April 2023, F.I. is now part of Biosynth,
https://www.biosynth.com/news/biosynth-agree-acquisition-of-fitzgerald-industries-international-from-trinity-biotech
example product pages (see website)
https://www.biosynth.com/p/65R-S103PHRP/streptavidin-poly-hrp20-conjugate
https://www.biosynth.com/p/65R-S112/streptavidin-poly-hrp40-conjugate
Weird (if not DY-tyramide) and/or interesting: HRP40 with DY647 (DyLight 647)
https://www.biosynth.com/p/65R-S128/streptavidin-poly-hrp40-conjugate-dy647
https://www.biosynth.com/p/65R-S105PHRP/streptavidin-poly-hrp80-conjugate
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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.
AATbio Styramide (styryl-phenol aka styryl-tyramide)
https://www.aatbio.com/catalog/power-styramide-signal-amplification-psa
Similar to tyramide signal amplification (TSA), PSA™ imaging uses the analyte-dependent reporter enzyme, horseradish peroxidase (HRP), to catalyze the covalent deposition and binding of labeled-Styramide™ substrates onto a target protein or nucleic acid sequence in situ. In the presence of hydrogen peroxide (H2O2), HRP converts labeled Styramide™ substrates into highly-reactive, short-lived Styramide™ radicals that rapidly bind to tyrosine residues on and proximal to the enzyme site. Styramide™ radicals have much higher reactivity than tyramide radicals, making imaging with PSA™ significantly faster, more robust, and sensitive than conventional TSA labeling. Since the added labeled-Styramide™ are deposited close to the HRP-target site, there is a minimal diffusion-related loss of resolution. PSA™ imaging technology can be readily added to any application that allows for integrating HRP into its protocol. Such applications include IHC, ICC, IF, in situ hybridization, and ELISA.
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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.
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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
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"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
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McTip 20240131 Centrifuge down antibodies
McTip 20240131 Centrifuge down antibodies (minifuge, 4 C rotor)
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).
20240806Tue update: now published in Cytometry A - centrifuge details (though no mention of whether the minifuge rotor should be kept refrigerated):
https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.24841
OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells
Andrew J. Konecny, Peter L. Mage, Aaron J. Tyznik, Martin Prlic, Florian Mair
Cytometry A Volume105, Issue6 June 2024 Pages 430-436 (online April 2024).
Colibri Cytometry Blog - provided Cytometry A reference - has good stuff
Acknowledgement for the Koncny 2024 Cytometry A publication:
Colibri Cytometry Blog - provided Cytometry A reference - has good stuff
https://www.colibri-cytometry.com/blog
Nice summary on Blocking reagents at
https://www.colibri-cytometry.com/post/blocking-a-cure-for-all-your-problems-or-a-cause-of-all-your-problems
Centrifuge tip in:
in https://www.colibri-cytometry.com/post/blocking-cellblox
"As noted in OMIP-102, some of the non-specific binding is due to aggregate formation and can be mitigated by centrifuging the product at high RCF prior to use. With any product that forms aggregates and requires a hard spin before use, you will reach a point where there is unusable product (most aggregates) left in the vial. Knowing when you have reached that point is a gamble. Personally, I dislike using any reagent that has the potential to cause a large, variable amount of non-specific staining simply by using it up, but this is a post about CellBlox, not NovaFluors."
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* RCF ... https://en.wikipedia.org/wiki/RCF ... Relative centrifugal force, the acceleration in a centrifuge normalized to Earth's gravity.
* NovaFluors are now a ThermoFisher product line - they acquired Phitonex, which (who) had developed them. Colibri Cytomtry (same blog) nice summary:
The issue
NovaFluors are DNA origami structures (Phitons) that allow for specific placement of fluorophore FRET partners via oligo hybridization. These are very large molecules, much bigger than antibodies. The naked DNA can be bound by cells via receptors designed to recognize and internalize foreign DNA. If you're working with immune cells, as most of us are, this is a problem. DNA also binds to dead cells, perhaps via histones, although it may just be because these are large molecules with many sites for potential interaction. The DNA-based fluorophores also interact with each other, like Brilliant dyes do, but much more so.
**
ThermoFisher NovaFluor web page (with products in table):
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry/novafluor-dyes.html
GM notes:
* ThermoFisher does not provide any schematic (figure) showing Phiton structure (or a simplification of structure), see https://www.youtube.com/watch?v=_3IMEfR-ZsM at 8:00 (m:sec) mark for a picture. To me, looks similar to a tRNA, which is much smaller than a 155 kDa antibody.
* I like the Colibri Cytometry blocking blogs, but the major goal of blocking reagents is to eliminate binding of the Fc domain of an antibody to Fc Receptors on and in cells being analyzed. These are mostly FcRgamma receptors (bind Fc domain of IgG's - CD marker names are CD16, CD32, CD64). Potentially also FcRn (Fc Receptor neonatal; pH sensitive in order to recycle or transcytose IgG or albumin [biologists come up with a lot of dumb undescriptive names, though the more accurate FcAndAlbuminReceptorNeonatal would be kind of awkward). There are also receptors for FC's of IgA, IgE and IgM. Back to but: the Fc of IgG can be removed from antibodies with appropriate protease (papain or pepsin) or by modern recombinant DNA technology to make expression constructs that (i) do not have Fc domain, (ii) have a better tail, (iii) optional reformat as scFv[tail] to be monomeric. There are now several companies selling recombinant antibodies, or recombinant Fab or scFv (Miltenyi Biotec, ThermoFisher, etc), though typically do not mention whether they have installed an optimized tail and fully optimized fluorophore(s). See "recombinant secondary PLUS" antibodies on this page (just search for PLUS -- now many mention, read them all) for more.
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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.
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Network Address Tip
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.
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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").
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* Scroll down for some more tips.
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).
George's Quick Tip on Optimizing Confocal Microscope Image Acquisition
20190603Mon (ok, not that quick ... please be patient)
Applies to: Olympus FV3000RS (analog PMTs), Leica SP8 (photon ounting HyDs), and other point scanning confocal microscopes (that is, whatever instrument you are using, whether at JHU or elsewhere).
Goals - sequential!!!:
1. acquire optimally, then explore your data (initially at the confocal PC, then at your office PC) visualization and quantitation.
2. compare acquired image data to your experiment requirements -- what hypothesis(es) are you testing? how close to resolution limit(s) do you need to be to acquire the data to critically test your hypothesis(es) - then figure out what settings to use in the future.
Quick Summary for 1.4 NA objective lens:
- Use pixel size 60 nm, Z-step size 180 nm.
- Use fast pixel dwell time, ex. 2 microseconds per pixel on FV3000RS ... small pixel dwell time minimizes photobleaching and phototoxicity.
- Use "a few" line averaging, try 3 line avg on FV3000RS, so total pixel dwell time (t-PDT) = 6 microseconds (2 us/pixel * 3 line average). More averaging if needed.
- FV3000RS: low laser power, HV 500 mV, offset = 0 (can subtract later), always keep "Gain" at 1.0 ... Leica SP8 is simple: use HyD photon counting mode.
- Unidrectional scanning -- because (i) less prone to jaggedness artefacts, and (ii) every pixel gets identical "dark time", since flyback is dark. That is, in bidirectional scanning, the end pixels get re-illuminated immediately, whereas middle has delay. For more on jaggedness, and a fix, see Papiez et al 2019.
- I generally operate FV3000RS in galvo mode, 2 microsecond pixel dwell time ("PDT") ... resonant scanner mode is faster (typically average more to get usable "total Pixel Dweel Time") ... in fact, RS mode likely to benefit the most from my proposal below.
Proposal (written here 20190603Mon): Acquire onto MULTIPLE DETECTORS, by setting the detectors to ADJACENT wavelength bands, equal area under the emission spectrum curve (on FV3000RS assumes dichroic beamsplitters enable optimal split).
FV3000RS: 4 internal GaAsP detectors (but also dichroic beamplitters that may not be optimal for your fluorophore ... consider switching to fluorophores that are ... and/or help us buy optimal). Example:
Standard scan: 2 microsecond pixel dwell time. one detector, 500-560 nm, 8 line average.
VS
Proposed scan 2 microsecond pixel dwell time. FOUR detectors, 500-515, 515-530, 530-545, 545-560 nm, 2 line average. Note: FV3000RS (currently., 6/2019) does not have optimized dichroics to take advantage of this hypothetical setting. It would be interesting for Olympus to (i) install 50/50 beamsplitters, and (ii) add to FluoView software the image math to combine (add, 12-bit 0 ... 4095 each --> 14-bit 0 ... 16380) the signal of all four detectors, (iii) optimize C.I. deconvolution to take aqdvantage of extended dynamic range. Bonus: "discard outlier" option when operating PMT's in high HV mode (13-bit if always throw out most extreme value ... Max Krummel has punlished similar idea). I also note ISS.com may have electronics (and software) to enable "fast photon counting" with current FV3000RS PMTs, and also pulsed laser to enable "fast FLIM". going from current analog (0 ... 4095) to photon counts (photon counts!) would be a "game changer" for our FV3000RS in terms of simplfying quantitation (the deconvolution module would also need to be optimized for photon counts).
Leica SP8: 2 HyD photon countng hybrid detectors. Can split the emission spectrum (area under the curve) 50:50, add together ... enables image acquisition in half the time (or get 2x more photons for 'full time). Reminder: scan as fast as possible for whatever zoom you ae using. 600 Hz enables full field of view (0.75x zoom by Leica convention). Example: 600 Hz with 10 Line Accumulation is superior to 60 Hz no accumulation, because less photobleaching and less phototoxicity for 600 Hz. SP8's fastest speed is 1800 Hz (requires 7.5x zoom or greater). Note: SP8 has spacefor two more internal HyD detectors AND Leica introduced (Spring 2018) third generation "SMD HyDs" (cooled, so less thermal noise, resulting in fewer dark counts, and faster counting by about 2x than our 2nd gen HyDs, so effectively bigger dynamic range per pixel dwell time), see below for all four seeing one fluorophore together ... would be great if you could provide us the $$$ to get a full set of four SMD HyD's (trade in our current two) + chiller + a (yes, even more $$ for) pulsed laser to enable the FALCON (Leica Fluorescence lifetime contrast ... generically "Fast FLIM") + PC and Leica LAS X software upgrade.
- if we can get our Leica SP8 upgraded to four SMD HyD's (per above) we would be able to go 8x faster than now (single 2nd gen HyD), by the ~2x faster photon counting rate of SMD HyD * using all four SMD HyDs to observe a single fluorophore.
- SMD HyD's count at 80 MHz, that is 1 photon is detected in each 12.5 nanosecond reptitition.
Related References:
Vinegoni ... Weissleder 2016 Nature Communications https://www.nature.com/articles/ncomms11077
whose high dynamic range by multiple detectors featured 50/50 beamsplitters and neutral density filters (ND filters kill photons: how dumb!), did have supplemental figure on 'asymmetric' beamsplitters of 90%/10%, so PMT1 = 90%, PMT2 = 10%, PMT3 = 1% (ignoring tiny losses on each optical surface). One unknown to me is how damaging to PMT1 is zapping it with a lot of light in bright area ... ex: at the limit, entire field of view might be bright, so saturate PMT1 the entire time (our Leica SP8 HyD's have a safety cutoff, so prolonged saturatin would simply cutoff the experiment until the user stops scanning).
Pinkard ... Krummel 2016 PloS One https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150430
Nice approach ... rank filter 50% = median filter, so rank filter 75% could be used to "pick" second highest value of the FV3000RS's internal PMTs ... I suggest "discard outlier" (usually highest intensity) and ACCUMULATE the other three PMTs values (i.e. 3500, 1000, 1000, 1000 --> discard 3500, accumulate 1000+1000+1000 = 3000, data is 16-bit anyway), would work nicely. If doing averaging, PC electronics now fast enough (and RAM cheap enough) that Olympus could keep each value in RAM, wait until that pixel is done acquiring, then rank filter (Max K) or 'discard outlier(s)' and accumulate. Optionally always rescale as if all values were good, i.e. for four data points, discard one, accumulate, multiply by 4/3 ... for 12 data points, if discard two, accumulate ten, multiply by 12/10.
Papiez et al 2019 IEEETBE https://www.ncbi.nlm.nih.gov/pubmed/31034401 and https://ieeexplore.ieee.org/document/8701671
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Back story:
Assumes:
- you have optimally labeled fluorescence specimens, including negative controls (labeling details is outside the scope of this quick tip).
- You are interested in seeing what you have on your specimen, and have time to explore the resolution limits of the specimen and optics.
- You have 'good' understanding of your microscope and/or can work with an expert (ex. me), and have the time and interest (and money) in getting your confocal imaging right.
- Operating at high NA (yes, also assumes you know what NA is!).
- operation at confocal pinhole 1 A.U. (yes, assumes you know what A.U. is). I mention briefly 0.5 A.U. below -- the penalty of 0.5 A.U. is pinhole is 1/4 area, so ~1/4 the number of photons gets through (most confocals can set pinhole to ~0.31, implying 10% area and number of photons compared to 1.0 A.U.).
- your primary interest is in a green fluorophore, emission 520 nm, ex. Alexa Fluor 488, EGFP, mNeonGreen, "mXX" (Nathan Shaner's 5/2019 alias for his new 6x brighter than EGFP green fluorescent protein ... also has new bright YFP). I note that shorter wavelength enables proportionally better resolution, ex. BV421 (em center wavelength 430 nm) enables 1.2 fold better resolution than emission 520 nm (520/430 = 1.2, that is, 520 nm is 1.2 fold worse, so 430 nm is 1.2 fold better).
- Using fluorescence ... I note that reflection from appropriate nanoparticle(s) could have advantages (I would like to see FL-Nanogold tested on our confocals! See www.nanoprobes.com and check for distributors).
- Access to spatial deconvolution algorithm, with GPU accelaration (Olympus: Cellsens "C.I." decovnvolution; Leica: HyVolution = Leica HyD detectors -> Hu
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