HILO ?

[lechristophe]

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Dear microscopists,

There was a recent publication of a method of "highly inclined and
laminated optical sheet (HILO) microscopy" in a Nature Methods article
by Tokunaga et al.
(http://www.nature.com/nmeth/journal/vaop/ncurrent/abs/nmeth1171.html).
I cannot understand how exactly this method works : it consists in
being at the limit between epifluorecence illumination and TIRF
illumination using an laser objective-type TIRF setup, but there is
also an angle that is added between the incident beam and the
azimuthal direction.The authors claim a SNR eight times better than
regular epifluorescence when doing Z-stacks images of cells in
culture.

Could someone explain more precisely what is this method, how it
works, and if it is really new ? Near-TIRF imaging has already been
used, for exemple by Nakada et al (JCB 2003,
http://www.jcb.org/cgi/content/full/162/6/1045) that called it
"critical angle fluorescent microscopy" (CAFM). I've tried it and it
is surprising how good the images are, like a TIRF with more
penetration depth (you dont loose vesicles along neurites in neurons
in culture) or epifluorescence with less blur. What is the rational
explanation behind this ?

Thank you for your advices,


Christophe Leterrier

Postdoc
INSERM UMR641 Neurobiology of ionic channels
IFR Jean Roche - Mediterranee University
Marseille, France

[John Oreopoulos]

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You mentioned the use of "critical angle fluorescence microscopy" by  
Nakada in 2003, and I can point out another example of this where it  
was called "Low-Angle Oblique (LAO) illumination". See for example:

"Imaging single molecules in living cells for systems biology"
by Yasushi Sako
in Molecular Systems Biology 2:56, 2006 (doi:10.1038/msb4100100)

Look at the figure in that paper and you'll see it's nearly identical  
to the one in this new paper.
In my opinion, it's all the same thing, namely, TIRF microscopy  
performed near or at the critical angle. I guess you could think of it  
as a darkfield version of fluorescence microscopy when performed at  
sub-critical angles. But there's nothing really surprising about the  
improved SNR either. Dan Axelrod explains this near critical angle  
fluorescence intensity effect in some of his earlier papers on TIRF  
microscopy as well.
The only novelty I can see in this new paper is that they examined the  
effect in CAF/LAO/HILO microscopy of changing the diameter of the  
illuminating beam, and it is interesting to see how this can be used  
as a form of optical sectioning.

John Oreopoulos, BSc,
PhD Candidate
University of Toronto
Institute For Biomaterials and Biomedical Engineering
Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

Quoting Christophe Leterrier <[hidden email]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear microscopists,
>
> There was a recent publication of a method of "highly inclined and
> laminated optical sheet (HILO) microscopy" in a Nature Methods article
> by Tokunaga et al.
> (http://www.nature.com/nmeth/journal/vaop/ncurrent/abs/nmeth1171.html).
> I cannot understand how exactly this method works : it consists in
> being at the limit between epifluorecence illumination and TIRF
> illumination using an laser objective-type TIRF setup, but there is
> also an angle that is added between the incident beam and the
> azimuthal direction.The authors claim a SNR eight times better than
> regular epifluorescence when doing Z-stacks images of cells in
> culture.
>
> Could someone explain more precisely what is this method, how it
> works, and if it is really new ? Near-TIRF imaging has already been
> used, for exemple by Nakada et al (JCB 2003,
> http://www.jcb.org/cgi/content/full/162/6/1045) that called it
> "critical angle fluorescent microscopy" (CAFM). I've tried it and it
> is surprising how good the images are, like a TIRF with more
> penetration depth (you dont loose vesicles along neurites in neurons
> in culture) or epifluorescence with less blur. What is the rational
> explanation behind this ?
>
> Thank you for your advices,
>
>
> Christophe Leterrier
>
> Postdoc
> INSERM UMR641 Neurobiology of ionic channels
> IFR Jean Roche - Mediterranee University
> Marseille, France
>