Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deep-tissue-imaging-with-spinning-disc-comparison-with-other-confocal-techniques-tp7561610p7562946.html
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Hi Jan,
The reason to use confocal - whether spinning disk or laser scanning -
is to block the out of focus light. In their Figure 1, two-photon did an
excellent job through the entire 200 um range shown (they should have
kept going!).
Confocal laser scanning cut out at ~80 um as the authors mention.
The Yokogawa shows a couple of in focus, or maybe "not quite in focus"
features at 100 um, but everything at and below 120 um looks blurry =
out of focus.
Bottom line: Multiphoton excitation wins again.
George
p.s. the authors failed to show whether the MP excitation wavelength was
optimal for EGFP or any of the dyes evaluated. They also did not state
the pinhole size for MPEF or whether they used NDD path for MPEF. For
spinning disk, they used an iXonEM+ DV897 EMCCD, but did not state the
EM gain or even if they used the EM gain register (probably).
On 5/16/2012 6:49 AM, Jan Pala wrote:
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> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Dear listservers,
>
> looking for some application techniques with deep tissue imaging I found
> this article:
>
http://neuronet.jp/pdf/O_101.pdf> Journal of Integrative Neuroscience, Vol. 10, No. 1 (2011) 121–129
> DOI: 10.1142/S0219635211002658
> NIPKOW CONFOCAL IMAGING FROM DEEP BRAIN TISSUES
> YUJI TAKAHARA, NORIO MATSUKI and YUJI IKEGAYA
>
> where the authors describe advantages of spinning disc in comparison with
> standard confocal laser scanning microscope and two-photon microscope.
>
> What do you think about presented results and experimental conditions? For
> me, it is quite strange, see part 3.1:
> 1. the laser power was fixed at 5 mW in all microscope systems - for CLSM
> the power is too high, for multiphoton too low. There is no description
> about the way the power was measured and set
> 2. Real Z-resolution for all the systems
> 3. Results: CLSM penetration depth 80 microns, spinning disc penetration
> depth 150 microns and multiphoton penetration depth 200 microns - it is
> quite unbelieveable to see almost double penetration depth in between
> spinning disc and CLSM.
>
> Thank you in advance.
>
> Best regards,
> Jan Pala
>
>