Tissue prep for TIRF

[Adele Tufford]

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Hello,

I need to image fixed cryosections of brain tissue on an Olympus TIRF
microscope.
My guess is that I can't use conventional specimen slides (ie Fisher Superfrost
+) to collect tissue. Has anyone had any experience collecting tissue on
Coverslips, and preparing them for TIRF.

Thank you!

Adele Tufford, McGill University

[Gary G. Li-2]

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Hi Adele Tufford,
The coverslip is part of the TIRF imaging apparatus, which produces
total internal reflection at the coverslip-specimen boundary.  The
evanescent wave “leaking” into the specimen, within 100nm immediately
above the coverslip, excites the fluorophone to produce fluorescent
emission.  Therefore, you have to make sure that your tissue specimen
attaches to the coverslip properly.  It does not matter what slide you
use.
Cheers
Gary G Li, PhD

On Sun, Nov 18, 2012 at 2:25 PM, Adele Tufford
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> I need to image fixed cryosections of brain tissue on an Olympus TIRF
> microscope.
> My guess is that I can't use conventional specimen slides (ie Fisher Superfrost
> +) to collect tissue. Has anyone had any experience collecting tissue on
> Coverslips, and preparing them for TIRF.
>
> Thank you!
>
> Adele Tufford, McGill University

[Gary G. Li-2]

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Hi Adele Tufford,
Further to my answer to your last posting, due to microtome damage to
cells on the surfaces of the tissue section, I am not sure how much
useful info you can get by using TIRF.   I would recommend confocal
laser scanning microscopy or widefield fluorescent microscopy with
deconvolution for your application.
Regards
Gary G Li, PhD


On Sun, Nov 18, 2012 at 4:20 PM, Gary G. Li <[hidden email]> wrote:

> Hi Adele Tufford,
> The coverslip is part of the TIRF imaging apparatus, which produces
> total internal reflection at the coverslip-specimen boundary.  The
> evanescent wave “leaking” into the specimen, within 100nm immediately
> above the coverslip, excites the fluorophone to produce fluorescent
> emission.  Therefore, you have to make sure that your tissue specimen
> attaches to the coverslip properly.  It does not matter what slide you
> use.
> Cheers
> Gary G Li, PhD
>
> On Sun, Nov 18, 2012 at 2:25 PM, Adele Tufford
> <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello,
>>
>> I need to image fixed cryosections of brain tissue on an Olympus TIRF
>> microscope.
>> My guess is that I can't use conventional specimen slides (ie Fisher Superfrost
>> +) to collect tissue. Has anyone had any experience collecting tissue on
>> Coverslips, and preparing them for TIRF.
>>
>> Thank you!
>>
>> Adele Tufford, McGill University

[John Oreopoulos]

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I might be stating the obvious, but for TIRF to work well, your sample mounting medium must also be aqueous in order for the TIR effect to occur (strict TIRF requires a refractive index mismatch at the sample/coverslip interface). That's also assuming you really are interested in seeing what happens in the regions closest to the coverslip. If want to simply illuminate your sample (all depths) with laser light in a widefield/epifluorescence condition using a TIRF illuminator, it will work fine for that too.

Now, having said that I recently observed that the TIR effect can occur (disappearance of out-of-focus light) with a fixed monolayer sample of cells (fluorescently labeled membranes) mounted in mowiol - but just barely. I'm not sure what the refractive index of mowiol is, but it must have been such that the critical angle corresponded to a a back-aperture radial position just inside the inner edge of the objective (the NA was 1.47 in that case).

Cheers,
John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2012-11-18, at 5:56 PM, Gary G. Li wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Adele Tufford,
> Further to my answer to your last posting, due to microtome damage to
> cells on the surfaces of the tissue section, I am not sure how much
> useful info you can get by using TIRF.   I would recommend confocal
> laser scanning microscopy or widefield fluorescent microscopy with
> deconvolution for your application.
> Regards
> Gary G Li, PhD
>
>
> On Sun, Nov 18, 2012 at 4:20 PM, Gary G. Li <[hidden email]> wrote:
>> Hi Adele Tufford,
>> The coverslip is part of the TIRF imaging apparatus, which produces
>> total internal reflection at the coverslip-specimen boundary.  The
>> evanescent wave “leaking” into the specimen, within 100nm immediately
>> above the coverslip, excites the fluorophone to produce fluorescent
>> emission.  Therefore, you have to make sure that your tissue specimen
>> attaches to the coverslip properly.  It does not matter what slide you
>> use.
>> Cheers
>> Gary G Li, PhD
>>
>> On Sun, Nov 18, 2012 at 2:25 PM, Adele Tufford
>> <[hidden email]> wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hello,
>>>
>>> I need to image fixed cryosections of brain tissue on an Olympus TIRF
>>> microscope.
>>> My guess is that I can't use conventional specimen slides (ie Fisher Superfrost
>>> +) to collect tissue. Has anyone had any experience collecting tissue on
>>> Coverslips, and preparing them for TIRF.
>>>
>>> Thank you!
>>>
>>> Adele Tufford, McGill University