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>The degree of axial confinement is independent of the power; I guess you
mean you have to overfill the objective BFP pretty hard to get good
sectioning, wasting power?

What I was thinking was that there was a loss of spatial confinement
associated with TF and so you would need more average power to get the
same peak power, but looking online, I think I was confusing the
widefield and linescan equations.

Mike

On Fri, Jan 9, 2015 at 5:48 PM, Andrew York
<[log in to unmask]> wrote:
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>
>>
>> Its hard for me to say since I don't have any experience with temporal
>> focusing, but I wonder if part of the problems you observed were related to
>> that technique?
>
>
> Temporal focus is just fast passive scanning, like the lavision technique
> but with shoulder-to-shoulder spots. The typical diffraction-grating
> implementation is equivalent to splitting your beam (or line focus) into a
> bunch of lines (or spots) and delaying each one a different amount.
>
>
>>   From what I've read, getting tight axial confinement is
>> fairly difficult often requiring relatively high power or a low rep rate
>> laser for a given level of 2p excitation.
>
>
>  The degree of axial confinement is independent of the power; I guess you
> mean you have to overfill the objective BFP pretty hard to get good
> sectioning, wasting power? Line scanning temporal focusing should give
> sectioning equivalent  to point-scanning 2p, and that's what we measured (
> http://www.nature.com/nmeth/journal/v8/n4/full/nmeth.1571.html ,
> http://www.ncbi.nlm.nih.gov/pubmed/21317909 Fig 1c, Sup Fig 1, and Sup Note
> 1). It's lots of 2p power compared to point scanning because the scanning
> is so much faster, but the required power nicely matches the output power
> of the Coherent Chameleon.
>
>
>>   Furthermore, if you're doing
>> parallel detection, you tend to have less efficient light collection,
>
>
> I think I might misunderstand what you mean here; I don't think this is
> true, unless the sample scatters a lot. The C. elegans embryo shell
> scatters, but not much. I don't think this explains my experience.
>
> Intuitively at least (and I could be wrong), I would
>> expect that it would be very difficult to read the same level of
>> contrast/power that a point scanning system achieves.
>>
>
> Check out Sup Note 1 from that link above; it seemed to me we had plenty of
> power, mostly because of how ridiculously powerful the Chameleon is. We
> could turn up the power high enough to bleach quantum dots.
>
> An off-list friend directed me to this paper:
> http://www.ncbi.nlm.nih.gov/pubmed/15884061
> If I'm reading it right, the paper suggests that 2p illumination bleaches
> rhodamine about 18x faster than 1p, for the same output signal rates. If
> this similarly true for GFP, it lines up with my suspicions, and explains
> my experience. If generally true, it would also be pretty important for
> everyone else using 2p illumination! Anyone know of similar work on GFP?
>
> -Andy