Resonant scanning preamplifer bandwidth

[Heping Yuan]

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Hello list users. I had a question about analog to digital conversion in resonant scanning two
photon microscopes. I have seen older discussions involving some users of the open source
ScanBox and ScanImage programs and they advocate for using sampling rates that match
with the Ti-Sapphire laser repetition rate (80 MHz). However, they also suggest using
preamplifiers with cutoffs that match this sampling rate (80 MHz). Should the preamplifier
not be at most half of the sampling rate to avoid aliasing? I believe there may be more
subtle details I am not aware of.

Thank you for your time.

Heping

[Craig Brideau]

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Hi Heping, the analog bandwidth of the amplifier will smear out fast photon
events if it is too slow. This can lead to sampling errors if a single
photon event gets spread across multiple digital sampling windows when
presented to the A/D.

Craig
On Jan 4, 2016 10:16 PM, "Heping Yuan" <[hidden email]> wrote:

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> *****
>
> Hello list users. I had a question about analog to digital conversion in
> resonant scanning two
> photon microscopes. I have seen older discussions involving some users of
> the open source
> ScanBox and ScanImage programs and they advocate for using sampling rates
> that match
> with the Ti-Sapphire laser repetition rate (80 MHz). However, they also
> suggest using
> preamplifiers with cutoffs that match this sampling rate (80 MHz). Should
> the preamplifier
> not be at most half of the sampling rate to avoid aliasing? I believe
> there may be more
> subtle details I am not aware of.
>
> Thank you for your time.
>
> Heping
>

[Michael Giacomelli]

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Hi Heping.

The idea is to sample synchronously with the excitation so that you
don't get beating between the sample and excitation clocks (imagine a
situation where one sample gets two pulses, then next zero pulses, the
next 2 pulses, ...) not necessarily to Nyquist sample the excitation
itself.

As for analog bandwidth, it should be set high enough to sample the
motion of the excitation spot across the sample.  Since most systems
use a resonant scanner with a bidirectional line rate of no more than
16kHz and a laser with an 80 MHz rate, that's 5000 pulses per line,
but probably no more than 1024 pixels.  Even accounting for the fact
that the scanner is moving faster in the center of the scan than the
edges, there is typically still at least 2 excitation pulses per
pixel, which means the analog bandwidth of the image will be around
half that of the excitation (~40Mhz) at the center and less on the
edges.  Therefore you'll be ok with an amp with at least that much
bandwidth and an 80 MHz sampling rate, at least for many systems.

Keep in mind though that you don't want too much bandwidth either,
there is no advantage to amplifying frequencies that are not in your
signal, and you'll quickly start to pick up FM radio stations if you
go above 80MHz.

Mike

On Mon, Jan 4, 2016 at 11:53 PM, Heping Yuan <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello list users. I had a question about analog to digital conversion in resonant scanning two
> photon microscopes. I have seen older discussions involving some users of the open source
> ScanBox and ScanImage programs and they advocate for using sampling rates that match
> with the Ti-Sapphire laser repetition rate (80 MHz). However, they also suggest using
> preamplifiers with cutoffs that match this sampling rate (80 MHz). Should the preamplifier
> not be at most half of the sampling rate to avoid aliasing? I believe there may be more
> subtle details I am not aware of.
>
> Thank you for your time.
>
> Heping

[Peter Rupprecht]

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Hi,
Sampling at 80 MHz does resolve the beating pattern issue, but there is another advantage. If you sample at 80 MHz locked to the excitation pulse and do not significantly broaden the fluorescence decay (2-4 ns), you do not integrate over the noise that would otherwise be smeared into the signal when using e.g. a 10 or 20 MHz bandwidth. If you sample only the (2-4 ns of) signal, and not the noise of the remaining time, you can also increase the gain of the PMT to much higher regimes than for low-bandwidth. This makes a direct comparison of different configurations difficult, since the noise can "look" different ....
I tried this out for my system and realized that 80 MHz (Femto) and 60 MHz (Thorlabs) preamplifiers have not sufficient bandwidth (at least the preamps I used) to show this effect. For a 240 MHz bandwidth, however, (Femto), this effect was very prominent, but the low intrinsic gain of the 240 MHz preamp setting was not optimal. -- Avoiding the beating pattern works for any preamp bandwidth.
To the original question: "Matching" the preamplifier with the sampling rate ...  hm. The bandwidth of a preamplifier is not a cutoff, but a smooth transition, and the "80 MHz" value indicates only a certain point for this frequency curve. And I have to say that for a preamp I'm using (Femto DHCP), the actual bandwidth is very likely lower than the indicated one.
Peter
    Michael Giacomelli <[hidden email]> schrieb am 16:10 Dienstag, 5.Januar 2016:
 
Hi Heping.

The idea is to sample synchronously with the excitation so that you
don't get beating between the sample and excitation clocks (imagine a
situation where one sample gets two pulses, then next zero pulses, the
next 2 pulses, ...) not necessarily to Nyquist sample the excitation
itself.

As for analog bandwidth, it should be set high enough to sample the
motion of the excitation spot across the sample.  Since most systems
use a resonant scanner with a bidirectional line rate of no more than
16kHz and a laser with an 80 MHz rate, that's 5000 pulses per line,
but probably no more than 1024 pixels.  Even accounting for the fact
that the scanner is moving faster in the center of the scan than the
edges, there is typically still at least 2 excitation pulses per
pixel, which means the analog bandwidth of the image will be around
half that of the excitation (~40Mhz) at the center and less on the
edges.  Therefore you'll be ok with an amp with at least that much
bandwidth and an 80 MHz sampling rate, at least for many systems.

Keep in mind though that you don't want too much bandwidth either,
there is no advantage to amplifying frequencies that are not in your
signal, and you'll quickly start to pick up FM radio stations if you
go above 80MHz.

Mike

On Mon, Jan 4, 2016 at 11:53 PM, Heping Yuan <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello list users. I had a question about analog to digital conversion in resonant scanning two
> photon microscopes. I have seen older discussions involving some users of the open source
> ScanBox and ScanImage programs and they advocate for using sampling rates that match
> with the Ti-Sapphire laser repetition rate (80 MHz). However, they also suggest using
> preamplifiers with cutoffs that match this sampling rate (80 MHz). Should the preamplifier
> not be at most half of the sampling rate to avoid aliasing? I believe there may be more
> subtle details I am not aware of.
>
> Thank you for your time.
>
> Heping

[Vitaly Boyko]

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Dear All,
I am looking for a nuclear dye that is on the other side of the spectra, 700-900 nm emission range (as we are planing to use ATTO 390 for immunostainings instead of dapi, Hoechst). Any suggestions/guidance will be appreciated. TO-PRO-5 is no longer available.
Thank you.
Vitaly

[George McNamara]

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DRAQ7
Manufacturer web page is
http://www.biostatus.com/DRAQ7

In the US available from several distributors ... Google DRAQ7.

Plan H, N or NPC: you could instead use an Cy5.5 etc anti-histone. Even
better: anti-Lamin, just label the nuclear envelope; if you are a
pointillist, anti-nuclear pore complex protein, see
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916659/figure/F3/
Less is more: the fewer pixels or voxels you obscure with counterlabel,
the cleaner your background and higher signal to background. To coin a
phrase" don't waste voxels on counterstains".
For paraformaldehyde fixed cells, I see nice cytoplasmic structures
(maybe mitochondria) prominently in the green channel, also most longer
channels.

On 1/8/2016 9:14 AM, Vitaly Boyko wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> *****
>
> Dear All,
> I am looking for a nuclear dye that is on the other side of the spectra, 700-900 nm emission range (as we are planing to use ATTO 390 for immunostainings instead of dapi, Hoechst). Any suggestions/guidance will be appreciated. TO-PRO-5 is no longer available.
> Thank you.
> Vitaly
>

--



George McNamara, Ph.D.
Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab)
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42
http://works.bepress.com/gmcnamara/75
https://www.linkedin.com/in/georgemcnamara