limited field of view in TIRF

[mmodel]

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Dear Microscopists

We had TIRF installed on an Olympus IX81, and the side effect of this was a greatly reduced size of illuminated field under normal epifluorescence (TIRF also illuminates only a small portion of the field). Is this how it is supposed to be? Is there a way to get around this limitation? Thank you

Mike Model

[John Oreopoulos]

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Hi Mike,

What camera are you using - sCMOS, EMCCD, CCD? I haven't used the latest generation Olympus TIRF modules, but my recollection of the older version of the same instrument is that there was a field diaphragm which could be removed and allowed the entire eyepiece field of view to be seen. I was never quite sure why the field diaphragm existed other than perhaps for spot photobleaching purposes. Back then I was using a CCD camera and my whole camera was illuminated, albeit with a Gaussian laser beam profile that rolled-off to the edges of the camera. You could still collect useful data in this manner, however.

If you're using an sCMOS camera which has a much larger field of view, perhaps the Gaussian nature of TIRF illumination beam is playing a role here. Again, I have no experience with the current module, but that's one way I could imagine the problem you describe occurring.

Cheers,

John Oreopoulos


On 2015-01-28, at 8:57 AM, MODEL, MICHAEL 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.
> *****
>
> Dear Microscopists
>
> We had TIRF installed on an Olympus IX81, and the side effect of this was a greatly reduced size of illuminated field under normal epifluorescence (TIRF also illuminates only a small portion of the field). Is this how it is supposed to be? Is there a way to get around this limitation? Thank you
>
> Mike Model

[Michael Model]

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Hi John,
Thanks for the response. I am talking about direct viewing through eyepieces

On Wed, Jan 28, 2015 at 9:31 AM, John Oreopoulos <
[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 Mike,
>
> What camera are you using - sCMOS, EMCCD, CCD? I haven't used the latest
> generation Olympus TIRF modules, but my recollection of the older version
> of the same instrument is that there was a field diaphragm which could be
> removed and allowed the entire eyepiece field of view to be seen. I was
> never quite sure why the field diaphragm existed other than perhaps for
> spot photobleaching purposes. Back then I was using a CCD camera and my
> whole camera was illuminated, albeit with a Gaussian laser beam profile
> that rolled-off to the edges of the camera. You could still collect useful
> data in this manner, however.
>
> If you're using an sCMOS camera which has a much larger field of view,
> perhaps the Gaussian nature of TIRF illumination beam is playing a role
> here. Again, I have no experience with the current module, but that's one
> way I could imagine the problem you describe occurring.
>
> Cheers,
>
> John Oreopoulos
>
>
> On 2015-01-28, at 8:57 AM, MODEL, MICHAEL 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.
> > *****
> >
> > Dear Microscopists
> >
> > We had TIRF installed on an Olympus IX81, and the side effect of this
> was a greatly reduced size of illuminated field under normal
> epifluorescence (TIRF also illuminates only a small portion of the field).
> Is this how it is supposed to be? Is there a way to get around this
> limitation? Thank you
> >
> > Mike Model
>