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Hi Guy,
I do not see any slits in the AODs based LOTOS MPEF system published by
Konnerth's lab:
LOTOS-based two-photon calcium imaging of dendritic spines in vivo.
<http://www.ncbi.nlm.nih.gov/pubmed/22976353>
*Chen* X, Leischner U, Varga Z, Jia H, Deca D, Rochefort NL,
*Konnerth* A.
Nat Protoc. 2012 Oct;7(10):1818-29. doi: 10.1038/nprot.2012.106.
Epub 2012 Sep 13.
PMID:
22976353
Neurons in the mammalian brain receive thousands of synaptic inputs
on their dendrites. In many types of neurons, such as cortical
pyramidal neurons, excitatory synapses are formed on fine dendritic
protrusions called spines. Usually, an individual spine forms a
single synaptic contact with an afferent axon. In this protocol, we
describe a recently established experimental procedure for measuring
intracellular calcium signals from dendritic spines in cortical
neurons in vivo by using a combination of two-photon microscopy and
whole-cell patch-clamp recordings. We have used mice as an
experimental model system, but the protocol may be readily adapted
to other species. This method involves data acquisition at high
frame rates and low-excitation laser power, and is termed low-power
temporal oversampling (LOTOS). Because of its high sensitivity of
fluorescence detection and reduced phototoxicity, LOTOS allows for
prolonged and stable calcium imaging in vivo. Key aspects of the
protocol, which can be completed in 5-6 h, include the use of a
variant of high-speed two-photon imaging, refined surgery procedures
and optimized tissue stabilization.
The methods section does discuss a resonant scanner configuration.
Hi Ben,
You wrote,
Cons:
High pitched frequency noise emitted form the scanner (which gets louder at lower zoom = larger amplitude)
The scanner lasts about 4-6 hours at its lowest zoom (1.25x) before giving an error and shutting down.
We couldn't ever get the bidirectional scan phase to line up perfectly (although we got it close)
No frequency control, you are stuck at the resonant frequency of the scanner
Lots of shot noise in live scan mode (although all high speed scanners would have this).
High pitched noise - well duh! The resonant scanner does work like a
pitchfork. I recommend you buy earplugs or noise canceling headsets for
everyone who works in the room.
Error and shutdown:
option A: complain to Leica that you have a bad instrument, and have
them replace whatever is bad.
option B: shut down the system every three hours.
option C: "Don't do that!" - if you avoid lowest zoom, sounds like
you can go longer.
No frequency control: let me repeat: well duh!
Lots of shot noise ... HyD's have about 2x quantum efficiency of the
standard Leica PMTs. If you purchased standard PMTs, find the money to
go HyD's. Also think about using denoising methods to get the most out
of your data. For example, my suggestion that using the median for each
pixel of an odd number of acquisitions (and others on te listserv have
made alternative suggestions to this). Denoising techniques can have a
huge impact on the usefulness of data - especially high speed, photon
limited, image acquisitions. As a current example, Bewersdorf and
colleagues recently published on how to fully characterize sCMOS sensor
noise (which is different than PMT or HyD noise), to enable sCMOS to
kick EMCCD's butt for single molecule localization:
Video-rate nanoscopy using sCMOS camera-specific single-molecule
localization algorithms. <http://www.ncbi.nlm.nih.gov/pubmed/23708387>
Huang F, Hartwich TM, Rivera-Molina FE, Lin Y, Duim WC, Long JJ,
Uchil PD, Myers JR, Baird MA, Mothes W, Davidson MW, Toomre D,
*Bewersdorf J*.
Nat Methods. 2013 Jul;10(7):653-8. doi: 10.1038/nmeth.2488. PMID:
23708387
Newly developed scientific complementary metal-oxide semiconductor
(sCMOS) cameras have the potential to dramatically accelerate data
acquisition, enlarge the field of view and increase the effective
quantum efficiency in single-molecule switching nanoscopy. However,
sCMOS-intrinsic pixel-dependent readout noise substantially lowers
the localization precision and introduces localization artifacts. We
present algorithms that overcome these limitations and that provide
unbiased, precise localization of single molecules at the
theoretical limit. Using these in combination with a multi-emitter
fitting algorithm, we demonstrate single-molecule localization
super-resolution imaging at rates of up to 32 reconstructed images
per second in fixed and living cells.
http://www.nature.com/nmeth/journal/v10/n7/full/nmeth.2488.html#supplementary-information
http://www.nature.com/nmeth/journal/v10/n7/extref/nmeth.2488-S1.pdf
In particular, I recommend looking at Supplemental Figure 8 (pdf page
10) - supplemental files are free online.
enjoy,
George
On 7/6/2013 12:40 AM, Guy Cox wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> An AOD (as used in the old Noran systems) is highly chromatic and so cannot be used in fluorescence. AOD based fluorescence systems (Lasertek, Noran) have used the equivalent of slit, rather than spot, detection.
>
> Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Smith, Benjamin E.
> Sent: Saturday, 6 July 2013 12:14 AM
> To: [log in to unmask]
> Subject: Re: AOD v Resonant scanner
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hey Gary,
> We have the resonant scanner installed on our Leica SP8, and it certainly has some ups and downs:
>
> Pros:
> Video scan rates (8000 Hz)
> No discernible image distortion
> Zooms to 48x
> Pairs well with hybrid detectors (in line accumulate mode)
> Can be used with a pinhole aperture, resulting in higher axial resolution
>
> Cons:
> High pitched frequency noise emitted form the scanner (which gets louder at lower zoom = larger amplitude)
> The scanner lasts about 4-6 hours at its lowest zoom (1.25x) before giving an error and shutting down.
> We couldn't ever get the bidirectional scan phase to line up perfectly (although we got it close)
> No frequency control, you are stuck at the resonant frequency of the scanner
> Lots of shot noise in live scan mode (although all high speed scanners would have this).
>
> Another thing to consider is I've known AOTFs to burn out over time, but have not yet had a galvanometer burn out, so I would imagine a resonant scanner would last much longer than an AOTF that is rapidly cycling through a frequency regime. My guess is this, and the increased axial resolution, is why manufacturers stick with resonant scanners.
>
> You can also find a good discussion on the challenges of various high speed scan technologies here: http://www.microscopyu.com/articles/confocal/resonantscanning.html
>
> Hope this helps,
> Ben Smith
> ________________________________________
> From: Confocal Microscopy List [[log in to unmask]] on behalf of Laevsky, Gary S. [[log in to unmask]]
> Sent: Friday, July 05, 2013 8:26 AM
> To: [log in to unmask]
> Subject: AOD v Resonant scanner
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi All,
>
> This would be for a MP application, so descanning is not an issue.
>
> I would think resonant galvos would be superior on the throughput side, whereas an AOD would win on speed and control (imagine, a trade-off).
>
> Thanks in advance for your feedback.
>
>
>
>
> Best,
>
> Gary
>
>
>
> Gary Laevsky, Ph.D.
> Confocal Imaging Facility Manager
> Dept. of Molecular Biology
> Washington Rd.
> Princeton University
> Princeton, New Jersey, 08544-1014
> (O) 609 258 5432
> (C) 508 507 1310
>
>
--
George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
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