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Suggestion 1:

Custom SuperBoost tyramide (plus poly-HRP, H2O2 etc) using Alexa Fluor 
750 or Alexa Fluor 790.

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

Suggestion 2:

Biotin-XX tyramide https://www.thermofisher.com/order/catalog/product/B40951

with Alexa Fluor 790 (or 750) conjugate 
https://www.thermofisher.com/order/catalog/product/S11378?SID=srch-srp-S11378

Suggestion 3:

tyramide etc from some other company. For example, table 2 Styramide(s)

https://www.aatbio.com/catalog/power-styramide-signal-amplification-psa

has iFluor 750 Styramide, and if click on "all" option for items per 
ppage (below right of table), also iFluor 790 Styramide, with links going to

https://www.aatbio.com/products/ifluor-750-styramide-superior-replacement-for-alexa-fluor-750-tyramide?unit=45065

https://www.aatbio.com/products/ifluor-790-styramide-superior-replacement-for-alexa-fluor-790-tyramide?unit=45070

On a good day, on the right specimen, tyramide signal amplification 
might be 100x brighter than typical unlabeled primary antibody + 
fluorescent secondary antibody. See Andrew Belmont's  paper(s), PubMed 
32609799, MMB chp might also be helpful 35867249,  for activated 
tyramide usable diffusion distances (and reagents to tune distance).

For iterative multiplexing (ThermoFisher, AATBio above, Akoya, Tocris, 
etc, have lots of fluorophore-tyramides),

* a nice way (and great product name) to inactivate HRP between rounds 
is Biocare's PeroxAbolish.

* Fitzgerald Industries streptavidin poly-HRP 
https://shop.bio-connect.nl/chemicals/streptavidin-poly-hrp80-conjugate/65r-s105phrp_100ug/sfid/4747310 
is an alternative to the tyramide vendors kit HRP conjugates.

* I note that in (theoretical) a "tyrosine desert", activated 
tyramide-fluorophores might not have any tyrosines to react with. More 
likely, previous round(s) of TSA could have reacted with all available 
tyramides.

***

pco.edge 5.5 ... QE peak 60% https://www.excelitas.com/product/pcoedge-55-usb-scmos-camera

Next camera could be a back-illuminated sCMOS if you want to routinely 
have good NIR sensitivity.

I do note our FISHscope 
http://confocal.jhu.edu/current-equipment/fishscope  Hamamatsu 
ORCA-FLASH4.0v2 (front illuminated sCMOS, ~80% peak QE in the visible) 
works well with RNAscope NIR (Cy7?) and Alexa Fluor 750 antibodies.

Maybe not cost feasible (though might be able to decant the D2O to save 
some money?), Maillard 2020 Chem Sci reported the (the OH) in H2O 
quenches many red and NIR fluorophores, and that D2O does not quench, 
see Fig 2g, for example Cy7 in H2O vs D2O

https://pubmed.ncbi.nlm.nih.gov/34163898/#&gid=article-figures&pid=fig-2-uid-1

I hypothesize that deuterated (just need the OD's) glycerol would work 
as well, and higher refractive index than D2O (for viscosity reasons, 
something like 80% "D" glycerol : 20% D2O, careful to not stir in O2, 
another quencher, may be more practical).

I also note that AF610X (X is just a linker, all their expts are in 
solution) is more than 2x brighter than Alexa Fluor 594 or Alexa Fluor 
568. Yet ThermoFisher has never featured Alexa Fluor 610(X) on 
antibodies, tyramides, etc.

***

happy 2023 and beyond,

George


On 10/26/2023 12:33 PM, Ariel, Pablo wrote:
> *****
> To join or leave the confocal microscopy listserv or to change your email address, go to:
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> *****
>
> I am looking for information about near IR fluorophores, to be used under the following conditions:
>
>    *   785 nm excitation
>    *   Compatibility with iDISCO+ and related clearing methods, which use methanol, dichloromethane and dibenzyl ether (for final imaging and clearing)
>    *   Fluorophores conjugated to primary or secondary antibodies.
>
> We have had adequate results using secondary antibodies conjugated with AlexaFluor 790, and better results with AlexaFluor800 plus. I am particularly interested in any comparisons of these Alexa dyes with other options like DyLight800, IRDye 800CW, CF770, Cy7.5, etc. I have not found any in the literature, but am hoping someone here may know of a relevant reference or have some hand's on experience.
>
> Unfortunately, even AlexaFluor800 Plus signals are typically significantly dimmer than with AlexaFluor 647. Part of this is the lower quantum efficiency in the camera (pco.edge 5.5) in the near IR, but we see a larger drop in brightness than what we would expect from just doing the arithmetic based on filters, lasers and camera QE (roughly 5-10X lower instead of 2X lower). So if people have done AF647 and compared with a near IR dye, that might also be very informative.
>
> I am hoping that if there are brighter fluorophore options we can better exploit this region of the spectrum that has much less autofluorescence and scattering.
>
> Thanks in advance for any suggestions!
>
> Pablo
>
>
> Pablo Ariel, Ph.D.
> Associate Professor
> Director of the Microscopy Services Laboratory
> Brinkhous-Bullitt Bldg B04
> Department of Pathology and Laboratory Medicine
> University of North Carolina at Chapel Hill
> http://www.med.unc.edu/microscopy
> Tel: 919-966-2413