Posted by
John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/why-does-high-NA-excitation-illumination-give-better-resolution-in-fluorescence-microscopy-tp7581519p7581528.html
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In the context of widefield epifluorescence, evidence of "accidental TIRF" is presented in this article:
Burghardt, T.P., Evanescent field shapes excitation profile under axial epi-illumination. Journal of Biomedical Optics, 2012. 17(6).
I would think that the effect described in this paper could be extended to confocal imaging as well. Quotes from the paper:
"Generally, TIRF or epi- illumination excitations pertain to evanescent or propagating field microscopies that are appropriate for different applications. I show here that the TIRF objective under common axial epi-illumination conditions produces an evanescent field that favorably remodels the excitation volume for samples near the coverslip."
"Curve fitting of the axial emission profile (Fig. 10) showed that for the fluorescent sphere at the lower coverslip, exciting light axial polarization from the evanescent field contributes substantially to the observed fluorescence. This is additional evidence for the significance of the evanescent field under axial epi-illumination excitation. It shows that evanescent excitation contributes to observed fluorescence whenever a TIRF objective is used and suggests that the sample material nearest the coverslip disproportionally contributes to the observed fluorescence signal."
John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca
On 2014-02-05, at 6:42 AM, Guy Cox wrote:
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> I think SA would negate any resolution improvement. But what many people have suggested is that these people are getting 'accidental TIRF' - in other words, TIRF-enhanced fluorescence of structures very close to the coverslip. I don't know of any serious studies investigating this.
>
> Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Steffen Dietzel
> Sent: Wednesday, 5 February 2014 4:56 AM
> To:
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> Subject: Re: why does high NA excitation illumination give better resolution in fluorescence microscopy?
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> Am 03.02.2014 18:39, schrieb Reto Fiolka:
>
>> I have seen many papers where people employed high NA oil objectives for
>> confocal imaging in biological samples. Besides not being indexed matched, they
>> will not provide more resolution, as any illumination above the critical angle is
>> lost in the near field (critical angle for n=1.33 is reached at NA 1.33).
>
> Well, NA 1.33 does sound like better resolution than the NA 1.2 of the
> best Water-Coverslip-Objectives, does it not?
>
> And just for the fun of it, if cells grow directly on the coverslip, n
> is somewhat larger than 1.33 since you stay inside the cell for the
> important parts of the image. (not that I would expect this to make a
> big difference though)
>
> Steffen
>
>
> --
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> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
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