nerve optical imaging

[Ko, Alex]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I was recently asked this question:  is there an optical modality suitable for nerve imaging ? either using endogenous signal or with contrasting agent.

Any comments are appreciated.

Thanks.

Alex  

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of S Ram
Sent: October-14-16 1:12 PM
To: [hidden email]
Subject: Re: Tyramide superboost kits quantitative?

 

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Mark and George,

Thanks for the feedback and comments! 

 

Sripad

 

 

On Fri, Oct 14, 2016 at 5:40 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Mark,

thanks for the find! the kidney GBM laminae rarae's ~60 nm periodicity could make this a nice nanoscopic calibration standard.

I also realized -- from their mention of 2 minute incubation times to limit the DAB + H2O2 reaction -- that my "Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea below could be greatly simplified: detect with tyramide color 1 briefly, then color 2, then color 3, etc.

George

 

On 10/14/2016 3:07 AM, Mark Cannell wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi

 

This contains some references that may be relevant to the question of how far radicals move?

 

doi: 10.1177/31.7.6304184J Histochem Cytochem July 1983 vol. 31 no. 7 945-951

HTH Mark

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi George,

As always super informative. Is it just me or do others also think that its high time someone invents a device download all that info that's sitting in your head...?

 

Very interesting comment about the diffusion radius of the activated TSA radical being less than the diffraction limit. I think that is a very important parameter. Just wondering if you know a reference for that? 

 

Perkin Elmer recently showed some correlative fluorescence and EM data which suggests similar range of specificity for their TSA staining. 

 

 

Thanks!

 

Sripad Ram

 

 

 

 

 

On Thu, Oct 13, 2016 at 11:24 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Claire,

Depending on temperature, constant reaction time may be bad. Minimal issue is always run on a warm plate (that is perfectly uniform temperature ... there are slide thermometers for this), incubators or ovens.

I suspect the TSA reaction runs to completion usually, with one of these running out:

* active HRP enzyme molecules (self destructs with some probability for every H2O2 catalytic cycle)

* tyrosine "docking sites" ... both in cells/tissues, the antibody molecules, and the HRPs (an advantage for poly HRP)

* fluorescent tyramide (least likely?)

* H2O2 (also unlikely?)

 

I believe PerkinElmer's Opal kits for multiplexing deliberately strips the detection Ab-polyHRP, before the next cycle, but have not been able to get data from PerkinElmer on this (and did not need TSA while I worked in a research lab here in Houston).

Many IHC reagent companies sell HRP polymers conjugated to streptavidin or secondary antibodies. For example
STREPTAVIDIN POLY-HRP80 CONJUGATE (plus stabilizer diluent)
https://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html

Yes, you can replace components in the kit with other reagents ... and conversely, use components in the kit for other purposes. An interesting "head to head" is fluorescent tyramide vs DAB IHC, or whatever favorite HRP substrate detection reagent your friendly neighborhood pathologists like.

I'm a big fan of TSA ever  since Phil Moen of NEN (acquired by PerkinElmer) spoke about and demo'd it at a Cold Spring Harbor fluorescence in situ/immuno courses. Phil mentioned the diffusion radius of the activated tyramide radical is less than the diffraction limit of confocal microscopy (i.e. 100 nm radius, 200 nm diameter).

One potential limitation on quantitation: zero reactable tyrosines implies zero signal ... as I mentioned above, both the Ab and HRPs have tyrosines (some even on the surface if I recall correctly).

Big benefit: when done right, can dilute the primary antibody 100 fold (maybe more ... try it), saving money. In fact, failing to do so will result in massive background.

Automation: in principle, automating TSA should improve reproducibility (assuming constant temperature for the reaction, and either constant enzyme reaction time or "run to completion"). However, not paying attention to the cost of the reagents (or stability or lack thereof) could result in one well, $100 in reagents --> one slide).

One of my U Miami customers published a super-duper brightness CD4 and CD8 2 color T-cells (also CD4 and FoxP3) paper ... they killed off the HRP molecules (and before that, endogenous peroxidases) with a terrific product (both great name and works well): PeroxAbolish   http://biocare.net/product/peroxabolish/

https://www.ncbi.nlm.nih.gov/pubmed/21929847

Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub 2011 Sep 

16.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue

using laser scanning cytometry.

Takahashi H(1), Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R, Barker

S, Burke GW, Tzakis AG, Ichii H.

Author information: 

(1)Miami Transplant Institute, University of Miami Leonard M. Miller School of

Medicine, Miami, FL, USA.

There is abundant evidence that immune cells infiltrating into a transplanted

organ play a critical role for destructive inflammatory or regulatory immune

reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune

cells remains challenging due to a lack of objective methodology. Laser scanning 

cytometry (LSC) is an imaging-based methodology that performs quantitative

measurements on fluorescently and/ or chromatically stained tissue or cellular

specimens at a single-cell level. In this study, we have developed a novel

objective method for analysis of immune cells, including Foxp3(+) T regulatory

cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy

sections using iCys® Research Imaging Cytometer. The development of multiple

immunofluorescent staining was established using FFPE human tonsil sample. The

CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using 

iCys and compared with the results from conventional flow cytometry analysis

(FCM). Our multiple immunofluorescent staining techniques allow obtaining clear

staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with

those obtained by FCM. This method was also applicable for liver, small

intestine, kidney, pancreas, and heart transplant biopsy sections and provide an 

objective quantification of Tregs within the grafts.

DOI: 10.3727/096368911X586747 

PMCID: PMC3777543

PMID: 21929847  [PubMed - indexed for MEDLINE]

and my 2010 book chapter (Yuste 2010 CSHL Press chapter 15 - Imaging in Neurosciences) has a protocol (I would use Mol Probes/ThermoFisher or PerkinElmers ... can probably crosslink the Ab-HRPpolymer onto the cells/tissue instead of their strip).

I also want to acknowledge Molecular Probes - specifically Mike Janes and his team - for visiting Miami (several years ago) for a workshop featuring TSA and Bacmam2.0. Hopefully ThermoFisher still lets the Mol Probes folks out of the lab to do similar workshops.

Speculation (spectral-ation - I currently do not work in a lab, so not going to test this anytime soon -- maybe someone from Mol Probes can try it):

Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea (this will be tedious to do by hand! Payoff: count everything):
=>I suggest using 35 mm imaging dish, so that wash steps can be very large volume (2 mL), and expensive reagent steps just the imaging area (7, 10, 14, 20, 28 mm diameter as appropriate for your expt).
1. incubate primary antibody at ~1/10th of saturation.
2. wash (extensively).
3. incubate secondary antibody-polyHRP.
4. wash extensively.
5. detect with Tyramide color #1.
6. kill HRP, i.e. with PeroxAbolish (http://biocare.net/product/peroxabolish/) AND I suggest gently crosslinking the 2ndAb-HRP-polymer into the specimen (see also Expansion Microscopy approaches).
7. OK, may want to image at this point, ideally with a way to refind the same place later.
*** Repeat 1-6 with different color, optionally also #7.
*. Image everything. See Valm et al https://www.ncbi.nlm.nih.gov/pubmed/27391327 for one example of high multiplexing (not tyramides) using all laser lines and detectors on a spectral confocal microscope.

I see in SuperBoost PDF only 7 colors (7 Alexa's)

https://tools.thermofisher.com/content/sfs/manuals/tyramide_superboost_kits_man.pdf

to get to 10 (ore more) could use biotin. A 1998 paper had do-it-yourself tyramide hapten synthesis
http://jhc.sagepub.com/content/46/6/771.full

Sure, could also do this single color, either add the signals, or photobleach. This could be useful on the MilliporeSigma/EMD Millipore/CellASIC ONIX microperfusion platform (since ONIX only has a few reservoirs and they seem to lack interest in making plates and manifolds for use with 2 or more controllers).

Another option for quantitation: wait for Garry P. Nolan (Stanford Univ.) to publish his immuofluorescence alternative to MIBI-ToF, or collaborate with him to use his MIBI-ToF or buy a Fluidigm imaging CyTOF.

enjoy,

George

 

On 10/13/2016 11:52 AM, Claire Brown, Dr. wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I wonder if anyone knows if the new ThermoFisher Tyramide SuperBoost kits are quantitative?

 

https://www.thermofisher.com/order/catalog/product/B40915

 

 

I would assume that keeping the reaction conditions identical from sample to sample would be really important in stopping the HRP reaction after a fixed time but with such amplification is there any way relative signals could be quantified?

 

I would love to hear what people think.

 

Sincerely,

 

Claire

 

 

 

-- 

George McNamara, PhD

Houston, TX 77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

https://works.bepress.com/gmcnamara/75/

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

Mark  B. Cannell Ph.D. FRSNZ FISHR Professor of Cardiac Cell Biology School of Physiology &  Pharmacology Faculty of Biomedical Sciences University of Bristol Bristol BS8 1TD UK

[hidden email]

-- 

George McNamara, PhD

Houston, TX 77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

https://works.bepress.com/gmcnamara/75/

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

 

[Steffen Dietzel]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

We found that peripheral nerve fibers in the cremaster muscle are strong inducers of Third Harmonic Generation. Another group (I believe working on spinal cord) showed that the myelin sheaths are the cause. Not sure whether this is something which might work in your case.

For details see our paper at

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028237

and references therein.

Steffen

Am 14.10.2016 um 20:53 schrieb Ko, Alex:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I was recently asked this question:  is there an optical modality suitable for nerve imaging ? either using endogenous signal or with contrasting agent.

Any comments are appreciated.

Thanks.

Alex  

[Craig Brideau]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Alex, it varies depending on the exact tissue, but generally white matter is difficult to image through. Optical clearing methods can certainly help improve imaging depth, but this delipidates the sample so if you are interested in myelin it's not very helpful. If you are interested in the axons and associated ion channels, etc, then a good clearing method coupled with two-photon is probably your best bet. If you are directly interested in myelin, CARS is a good technique, or two-photon imaging with a far-red dye at longer wavelengths (>1000nm laser).

Craig

On Fri, Oct 14, 2016 at 12:53 PM, Ko, Alex <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I was recently asked this question:  is there an optical modality suitable for nerve imaging ? either using endogenous signal or with contrasting agent.

Any comments are appreciated.

Thanks.

Alex  

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of S Ram
Sent: October-14-16 1:12 PM
To: [hidden email]
Subject: Re: Tyramide superboost kits quantitative?

 

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Mark and George,

Thanks for the feedback and comments! 

 

Sripad

 

 

On Fri, Oct 14, 2016 at 5:40 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Mark,

thanks for the find! the kidney GBM laminae rarae's ~60 nm periodicity could make this a nice nanoscopic calibration standard.

I also realized -- from their mention of 2 minute incubation times to limit the DAB + H2O2 reaction -- that my "Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea below could be greatly simplified: detect with tyramide color 1 briefly, then color 2, then color 3, etc.

George

 

On 10/14/2016 3:07 AM, Mark Cannell wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi

 

This contains some references that may be relevant to the question of how far radicals move?

 

doi: 10.1177/31.7.6304184J Histochem Cytochem July 1983 vol. 31 no. 7 945-951

HTH Mark

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi George,

As always super informative. Is it just me or do others also think that its high time someone invents a device download all that info that's sitting in your head...?

 

Very interesting comment about the diffusion radius of the activated TSA radical being less than the diffraction limit. I think that is a very important parameter. Just wondering if you know a reference for that? 

 

Perkin Elmer recently showed some correlative fluorescence and EM data which suggests similar range of specificity for their TSA staining. 

 

 

Thanks!

 

Sripad Ram

 

 

 

 

 

On Thu, Oct 13, 2016 at 11:24 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Claire,

Depending on temperature, constant reaction time may be bad. Minimal issue is always run on a warm plate (that is perfectly uniform temperature ... there are slide thermometers for this), incubators or ovens.

I suspect the TSA reaction runs to completion usually, with one of these running out:

* active HRP enzyme molecules (self destructs with some probability for every H2O2 catalytic cycle)

* tyrosine "docking sites" ... both in cells/tissues, the antibody molecules, and the HRPs (an advantage for poly HRP)

* fluorescent tyramide (least likely?)

* H2O2 (also unlikely?)

 

I believe PerkinElmer's Opal kits for multiplexing deliberately strips the detection Ab-polyHRP, before the next cycle, but have not been able to get data from PerkinElmer on this (and did not need TSA while I worked in a research lab here in Houston).

Many IHC reagent companies sell HRP polymers conjugated to streptavidin or secondary antibodies. For example
STREPTAVIDIN POLY-HRP80 CONJUGATE (plus stabilizer diluent)
https://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html

Yes, you can replace components in the kit with other reagents ... and conversely, use components in the kit for other purposes. An interesting "head to head" is fluorescent tyramide vs DAB IHC, or whatever favorite HRP substrate detection reagent your friendly neighborhood pathologists like.

I'm a big fan of TSA ever  since Phil Moen of NEN (acquired by PerkinElmer) spoke about and demo'd it at a Cold Spring Harbor fluorescence in situ/immuno courses. Phil mentioned the diffusion radius of the activated tyramide radical is less than the diffraction limit of confocal microscopy (i.e. 100 nm radius, 200 nm diameter).

One potential limitation on quantitation: zero reactable tyrosines implies zero signal ... as I mentioned above, both the Ab and HRPs have tyrosines (some even on the surface if I recall correctly).

Big benefit: when done right, can dilute the primary antibody 100 fold (maybe more ... try it), saving money. In fact, failing to do so will result in massive background.

Automation: in principle, automating TSA should improve reproducibility (assuming constant temperature for the reaction, and either constant enzyme reaction time or "run to completion"). However, not paying attention to the cost of the reagents (or stability or lack thereof) could result in one well, $100 in reagents --> one slide).

One of my U Miami customers published a super-duper brightness CD4 and CD8 2 color T-cells (also CD4 and FoxP3) paper ... they killed off the HRP molecules (and before that, endogenous peroxidases) with a terrific product (both great name and works well): PeroxAbolish   http://biocare.net/product/peroxabolish/

https://www.ncbi.nlm.nih.gov/pubmed/21929847

Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub 2011 Sep 

16.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue

using laser scanning cytometry.

Takahashi H(1), Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R, Barker

S, Burke GW, Tzakis AG, Ichii H.

Author information: 

(1)Miami Transplant Institute, University of Miami Leonard M. Miller School of

Medicine, Miami, FL, USA.

There is abundant evidence that immune cells infiltrating into a transplanted

organ play a critical role for destructive inflammatory or regulatory immune

reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune

cells remains challenging due to a lack of objective methodology. Laser scanning 

cytometry (LSC) is an imaging-based methodology that performs quantitative

measurements on fluorescently and/ or chromatically stained tissue or cellular

specimens at a single-cell level. In this study, we have developed a novel

objective method for analysis of immune cells, including Foxp3(+) T regulatory

cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy

sections using iCys® Research Imaging Cytometer. The development of multiple

immunofluorescent staining was established using FFPE human tonsil sample. The

CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using 

iCys and compared with the results from conventional flow cytometry analysis

(FCM). Our multiple immunofluorescent staining techniques allow obtaining clear

staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with

those obtained by FCM. This method was also applicable for liver, small

intestine, kidney, pancreas, and heart transplant biopsy sections and provide an 

objective quantification of Tregs within the grafts.

DOI: 10.3727/096368911X586747 

PMCID: PMC3777543

PMID: 21929847  [PubMed - indexed for MEDLINE]

and my 2010 book chapter (Yuste 2010 CSHL Press chapter 15 - Imaging in Neurosciences) has a protocol (I would use Mol Probes/ThermoFisher or PerkinElmers ... can probably crosslink the Ab-HRPpolymer onto the cells/tissue instead of their strip).

I also want to acknowledge Molecular Probes - specifically Mike Janes and his team - for visiting Miami (several years ago) for a workshop featuring TSA and Bacmam2.0. Hopefully ThermoFisher still lets the Mol Probes folks out of the lab to do similar workshops.

Speculation (spectral-ation - I currently do not work in a lab, so not going to test this anytime soon -- maybe someone from Mol Probes can try it):

Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea (this will be tedious to do by hand! Payoff: count everything):
=>I suggest using 35 mm imaging dish, so that wash steps can be very large volume (2 mL), and expensive reagent steps just the imaging area (7, 10, 14, 20, 28 mm diameter as appropriate for your expt).
1. incubate primary antibody at ~1/10th of saturation.
2. wash (extensively).
3. incubate secondary antibody-polyHRP.
4. wash extensively.
5. detect with Tyramide color #1.
6. kill HRP, i.e. with PeroxAbolish (http://biocare.net/product/peroxabolish/) AND I suggest gently crosslinking the 2ndAb-HRP-polymer into the specimen (see also Expansion Microscopy approaches).
7. OK, may want to image at this point, ideally with a way to refind the same place later.
*** Repeat 1-6 with different color, optionally also #7.
*. Image everything. See Valm et al https://www.ncbi.nlm.nih.gov/pubmed/27391327 for one example of high multiplexing (not tyramides) using all laser lines and detectors on a spectral confocal microscope.

I see in SuperBoost PDF only 7 colors (7 Alexa's)

https://tools.thermofisher.com/content/sfs/manuals/tyramide_superboost_kits_man.pdf

to get to 10 (ore more) could use biotin. A 1998 paper had do-it-yourself tyramide hapten synthesis
http://jhc.sagepub.com/content/46/6/771.full

Sure, could also do this single color, either add the signals, or photobleach. This could be useful on the MilliporeSigma/EMD Millipore/CellASIC ONIX microperfusion platform (since ONIX only has a few reservoirs and they seem to lack interest in making plates and manifolds for use with 2 or more controllers).

Another option for quantitation: wait for Garry P. Nolan (Stanford Univ.) to publish his immuofluorescence alternative to MIBI-ToF, or collaborate with him to use his MIBI-ToF or buy a Fluidigm imaging CyTOF.

enjoy,

George

 

On 10/13/2016 11:52 AM, Claire Brown, Dr. wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I wonder if anyone knows if the new ThermoFisher Tyramide SuperBoost kits are quantitative?

 

https://www.thermofisher.com/order/catalog/product/B40915

 

 

I would assume that keeping the reaction conditions identical from sample to sample would be really important in stopping the HRP reaction after a fixed time but with such amplification is there any way relative signals could be quantified?

 

I would love to hear what people think.

 

Sincerely,

 

Claire

 

 

 

-- 

George McNamara, PhD

Houston, TX 77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

https://works.bepress.com/gmcnamara/75/

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

Mark  B. Cannell Ph.D. FRSNZ FISHR Professor of Cardiac Cell Biology School of Physiology &  Pharmacology Faculty of Biomedical Sciences University of Bristol Bristol BS8 1TD UK

[hidden email]

-- 

George McNamara, PhD

Houston, TX 77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

https://works.bepress.com/gmcnamara/75/

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650