2p dead time

[Guy Cox-2]

*****
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.
*****

I've changed the title since the previous one was going in too many directions.  Jim P mentioned that in conventional 2P microscopy we have ~12ns between pulses so with a typical fluorescence lifetime of 2-4ns we have some dead time.  Actually I'm not sure that's a bad thing since getting another hit on a molecule that's already excited is likely to increase bleaching.  And do remember the lifetime is not when fluorescence stops - just when it falls to 1/e of the original intensity, so there's still some to go.

However I recall seeing at conferences much smaller femtosecond lasers with, naturally, much faster repetition rates.  (The physical size of the laser determines the rep rate since it is the time light takes to go round one circuit of the cavity).  These would completely eliminate the dead time.  So has anyone used these for microscopy?  

                        Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

[George McNamara]

*****
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.
*****

Hi Guy,

Yes:

Ji N(1), Magee JC, Betzig E. High-speed, low-photodamage nonlinear imaging using passive pulse splitters.
Nat Methods. 2008 Feb;5(2):197-202. doi: 10.1038/nmeth.1175.
Erratum in Nat Methods. 2009 Feb;6(2):179.

Pulsed lasers are key elements in nonlinear bioimaging techniques such as
two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a
percent or less of the laser power available can be delivered to the sample
before photoinduced damage becomes excessive. Here we describe a passive pulse
splitter that converts each laser pulse into a fixed number of sub-pulses of
equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices
labeled with GFP and show that, in different power regimes, the splitter can be
used either to increase the signal rate more than 100-fold or to reduce the rate
of photobleaching by over fourfold. In living specimens, the gains were even
greater: a ninefold reduction in photobleaching during in vivo imaging of
Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of
photodamage during calcium imaging of rat hippocampal brain slices.

http://www.ncbi.nlm.nih.gov/pubmed/18204458

Long interpulse has also been published, for example in:

Donnert G(1), Eggeling C, Hell SW. Triplet-relaxation microscopy with bunched pulsed excitation.
Photochem Photobiol Sci. 2009 Apr;8(4):481-5. doi: 10.1039/b903357m.

Obtaining high signal levels in fluorescence microscopy is usually spoiled by the
concomitant population of the dark (triplet) state of the marker, which is often
followed by photobleaching. Recently, we introduced the triplet relaxation
(T-Rex) modality in fluorescence microscopy which led to a major increase in
total signal and dye photostability. The idea behind T-Rex is to avoid the
illumination of fluorophores in the triplet state, e.g. by using pulsed
excitation with interpulse time distances that are long enough for the triplet
state to relax between two pulses. While our previous implementation came at the
expense of extended recording, here we investigate pulsed excitation patterns for
T-Rex illumination implying shorter total recording times. In particular, we
balance signal enhancement and imaging speed by exciting with bunches of quickly
succeeding pulses that are separated by dark periods for triplet relaxation.
Taking the green fluorescent protein and the organic dye Atto532 as examples, we
observe the dependence of photobleaching and total fluorescence gain on the
number of pulses within a bunch. Reaching almost T-Rex conditions this excitation
scheme mimics fast scanning of the illumination beam and has the potential to
improve a whole range of analytical tools that suffer from photobleaching and low
signal levels.

http://www.ncbi.nlm.nih.gov/pubmed/19337661

//

lot's of FP's in a small volume: Robinett et al 1996 had 512 GFP's
(S65T, so about the same as EGFP) in a sub-diffraction limited volume in
a cell nucleus (with background smog from overexpression). I encourage
everyone doing fluorescence microscopy on cells (for core staff, you
should read it and encourage your collaborators to check it out) to read:

http://jcb.rupress.org/content/135/6/1685.long

Specifically figure 4a (back issues of JCB are open access).

Tanenbaum et al recently published hanging 24 GFP's off one molecule ...
and multimerizing that complex - see especially their Cas9-SUNtag
experiments, both in the main text and the supplement (telomere repeats).

http://www.ncbi.nlm.nih.gov/pubmed/25307933

too bad they used EGFP - soooo 1996. Jim Pawley might ride a newer
bicycle than that. Current state of the art green FPs are mNeonGreen
(with bad sales/licensing terms) and mClover3. I am unclear on whether
mRuby3 is brighter or dimmer than each tdTomato monomer.

I recently posted - with a reference list at the end that is pretty
comprehensive spanning Robinett to Tanenbaum and many steps in between --

http://works.bepress.com/gmcnamara/65/

with my proposal to use synthetic tandem repeats instead of the flaky
endogenous repeats (endogenous STRs and VNTRs are usually different
lengths even in the two alleles in one person). As an example of what
"SUNtags" (Tanenbaum) or "binary Tattletales / T-Bow" could get to:

42 synthetic tandem repeats
TALE-(linker-epitope tag)10
"binder"-(mClover3)4

At saturation, this would be: 42 x 10 x 4 = 1,680 FPs. But mClover3 is
about 3x brighter than EGFP, so x 3 = 5,040 "brightness units" compared
to Robinett's "512".

"65" also provides the references for two papers on "FingR", which both
provides a different "binder" than Tanembaum's sdAb (there are more,
such as DARPins, or coiled-coils discussed in "65"), which could be
utilized in my proposal to have a FingR like operator site and repressor
as needed. Also possible to have different inducible promoters driving
the protein components, see "linearizer TetOn" reference in "65".

there are many 'complexes' in cells - not limited to genes. It may also
be possible for the synthetic tandem repeat(s) to produce mRNA(s) --
optionally circular RNAs for stability (no end for exonucleases to chew
on) -- and I was happy to see Coquille ... Rackham 2014 publish on
'artificial PPR scaffold(s) for programmable RNA recognition',

http://www.ncbi.nlm.nih.gov/pubmed/25517350

PPRFR (PPR fluorescent reporter) is not as cute a name as PUF-FR (think
puffer fish logo), but when it helps us spy on cells , will be a lot of
fun (giving credit where due; spying has been used in titles by Roger T
several times: PubMed 15984552, 15680976 and 17191067).

Click or tag ... of course replacing mClover3 in the above design with
protein components of HaloTag, SNAPtag, CLIPtag, FlAsH/ReAsH, etc, would
also work (and may work better than FPs or UnaG, phiLOV etc).

As an enzyme example, see Lam ... Ting 2015 for improved APEX, that
could be used with cell permeable tyramide(s),
http://www.ncbi.nlm.nih.gov/pubmed/25419960
Alice Ting has already posted APEX2 to addgene, for example
http://www.addgene.org/49386/
see also
http://www.addgene.org/static/data/30/79/09d736d4-5d22-11e3-8ba4-000c29055998.pdf


Enjoy,

George
p.s. mClover3 and mRuby3

http://www.googlesciencefair.com/en/projects/ahJzfnNjaWVuY2VmYWlyLTIwMTJyQwsSC1Byb2plY3RTaXRlIjJhaEp6Zm5OamFXVnVZMlZtWVdseUxUSXdNVEp5RHdzU0IxQnliMnBsWTNRWXVLOW9EQQw

http://spie.org/Documents/Courses/Education_Outreach/Science%20Fairs/Emily-Wang-CSSF14-Abstract.pdf

http://2014davidsonfellows.com/emily-wang/

*TALENTED STUDENT FROM PALO ALTO REWARDED FOR UNMATCHED ACHIEVEMENT
**Emily Wang to be Awarded $25,000 as a 2014 Davidson Fellow*

Reno, Nev. -- Twenty bright young people named as 2014 Davidson Fellows
<http://www.davidsongifted.org/fellows/> exemplify the extraordinary
work that can be accomplished by U.S. students who are given
opportunities to excel.  One of these gifted students is 18-year-old
Emily Wang of Palo Alto, Calif.

Emily received $25,000 for her science project, "Illuminating Disease
Pathways: Developing Bright Fluorescent Proteins to Improve FRET
Biosensing." Emily created a biological tool to visualize diseases at
the molecular level. She developed Clover3, a bright green fluorescent
protein, and mRuby3, a bright red fluorescent protein. Clover3-mRuby3
can be used to image neurons to investigate Alzheimer's disease, detect
and track cancer growth, and monitor the activities of diseases in
cells. Clover3-mRuby3 may be used to visualize biological events with
greater clarity than before, which may advance current understanding of
illnesses to create new therapies and medicine to improve human health.



On 1/11/2015 2:42 AM, Guy Cox 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.
> *****
>
> I've changed the title since the previous one was going in too many directions.  Jim P mentioned that in conventional 2P microscopy we have ~12ns between pulses so with a typical fluorescence lifetime of 2-4ns we have some dead time.  Actually I'm not sure that's a bad thing since getting another hit on a molecule that's already excited is likely to increase bleaching.  And do remember the lifetime is not when fluorescence stops - just when it falls to 1/e of the original intensity, so there's still some to go.
>
> However I recall seeing at conferences much smaller femtosecond lasers with, naturally, much faster repetition rates.  (The physical size of the laser determines the rep rate since it is the time light takes to go round one circuit of the cavity).  These would completely eliminate the dead time.  So has anyone used these for microscopy?
>
> Guy
>
> Guy Cox, Honorary Associate Professor
> School of Medical Sciences
>
> Australian Centre for Microscopy and Microanalysis,
> Madsen, F09, University of Sydney, NSW 2006
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[Guy Cox-2]

*****
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.
*****

These are all very interesting, but don't seem to answer my question.

                                                                        Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

From: George McNamara [mailto:[hidden email]]
Sent: Monday, 12 January 2015 2:14 AM
To: Confocal Microscopy List
Cc: Guy Cox
Subject: Re: 2p dead time ... can have lots of FPs in one place (or click, tag, or enzymatically localize dyes)

Hi Guy,

Yes:


Ji N(1), Magee JC, Betzig E. High-speed, low-photodamage nonlinear imaging using passive pulse splitters.

Nat Methods. 2008 Feb;5(2):197-202. doi: 10.1038/nmeth.1175.

Erratum in Nat Methods. 2009 Feb;6(2):179.



Pulsed lasers are key elements in nonlinear bioimaging techniques such as

two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a

percent or less of the laser power available can be delivered to the sample

before photoinduced damage becomes excessive. Here we describe a passive pulse

splitter that converts each laser pulse into a fixed number of sub-pulses of

equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices

labeled with GFP and show that, in different power regimes, the splitter can be

used either to increase the signal rate more than 100-fold or to reduce the rate

of photobleaching by over fourfold. In living specimens, the gains were even

greater: a ninefold reduction in photobleaching during in vivo imaging of

Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of

photodamage during calcium imaging of rat hippocampal brain slices.
http://www.ncbi.nlm.nih.gov/pubmed/18204458

Long interpulse has also been published, for example in:


Donnert G(1), Eggeling C, Hell SW. Triplet-relaxation microscopy with bunched pulsed excitation.

Photochem Photobiol Sci. 2009 Apr;8(4):481-5. doi: 10.1039/b903357m.



Obtaining high signal levels in fluorescence microscopy is usually spoiled by the

concomitant population of the dark (triplet) state of the marker, which is often

followed by photobleaching. Recently, we introduced the triplet relaxation

(T-Rex) modality in fluorescence microscopy which led to a major increase in

total signal and dye photostability. The idea behind T-Rex is to avoid the

illumination of fluorophores in the triplet state, e.g. by using pulsed

excitation with interpulse time distances that are long enough for the triplet

state to relax between two pulses. While our previous implementation came at the

expense of extended recording, here we investigate pulsed excitation patterns for

T-Rex illumination implying shorter total recording times. In particular, we

balance signal enhancement and imaging speed by exciting with bunches of quickly

succeeding pulses that are separated by dark periods for triplet relaxation.

Taking the green fluorescent protein and the organic dye Atto532 as examples, we

observe the dependence of photobleaching and total fluorescence gain on the

number of pulses within a bunch. Reaching almost T-Rex conditions this excitation

scheme mimics fast scanning of the illumination beam and has the potential to

improve a whole range of analytical tools that suffer from photobleaching and low

signal levels.
http://www.ncbi.nlm.nih.gov/pubmed/19337661

//

lot's of FP's in a small volume: Robinett et al 1996 had 512 GFP's (S65T, so about the same as EGFP) in a sub-diffraction limited volume in a cell nucleus (with background smog from overexpression). I encourage everyone doing fluorescence microscopy on cells (for core staff, you should read it and encourage your collaborators to check it out) to read:

http://jcb.rupress.org/content/135/6/1685.long

Specifically figure 4a (back issues of JCB are open access).

Tanenbaum et al recently published hanging 24 GFP's off one molecule ... and multimerizing that complex - see especially their Cas9-SUNtag experiments, both in the main text and the supplement (telomere repeats).

http://www.ncbi.nlm.nih.gov/pubmed/25307933

too bad they used EGFP - soooo 1996. Jim Pawley might ride a newer bicycle than that. Current state of the art green FPs are mNeonGreen (with bad sales/licensing terms) and mClover3. I am unclear on whether mRuby3 is brighter or dimmer than each tdTomato monomer.

I recently posted - with a reference list at the end that is pretty comprehensive spanning Robinett to Tanenbaum and many steps in between --

http://works.bepress.com/gmcnamara/65/

with my proposal to use synthetic tandem repeats instead of the flaky endogenous repeats (endogenous STRs and VNTRs are usually different lengths even in the two alleles in one person). As an example of what "SUNtags" (Tanenbaum) or "binary Tattletales / T-Bow" could get to:

42 synthetic tandem repeats
TALE-(linker-epitope tag)10
"binder"-(mClover3)4

At saturation, this would be: 42 x 10 x 4 = 1,680 FPs. But mClover3 is about 3x brighter than EGFP, so x 3 = 5,040 "brightness units" compared to Robinett's "512".

"65" also provides the references for two papers on "FingR", which both provides a different "binder" than Tanembaum's sdAb (there are more, such as DARPins, or coiled-coils discussed in "65"), which could be utilized in my proposal to have a FingR like operator site and repressor as needed. Also possible to have different inducible promoters driving the protein components, see "linearizer TetOn" reference in "65".

there are many 'complexes' in cells - not limited to genes. It may also be possible for the synthetic tandem repeat(s) to produce mRNA(s) -- optionally circular RNAs for stability (no end for exonucleases to chew on) -- and I was happy to see Coquille ... Rackham 2014 publish on 'artificial PPR scaffold(s) for programmable RNA recognition',

http://www.ncbi.nlm.nih.gov/pubmed/25517350

PPRFR (PPR fluorescent reporter) is not as cute a name as PUF-FR (think puffer fish logo), but when it helps us spy on cells , will be a lot of fun (giving credit where due; spying has been used in titles by Roger T several times: PubMed 15984552, 15680976 and 17191067).

Click or tag ... of course replacing mClover3 in the above design with protein components of HaloTag, SNAPtag, CLIPtag, FlAsH/ReAsH, etc, would also work (and may work better than FPs or UnaG, phiLOV etc).

As an enzyme example, see Lam ... Ting 2015 for improved APEX, that could be used with cell permeable tyramide(s),
http://www.ncbi.nlm.nih.gov/pubmed/25419960
Alice Ting has already posted APEX2 to addgene, for example
http://www.addgene.org/49386/
see also
http://www.addgene.org/static/data/30/79/09d736d4-5d22-11e3-8ba4-000c29055998.pdf


Enjoy,

George
p.s. mClover3 and mRuby3

http://www.googlesciencefair.com/en/projects/ahJzfnNjaWVuY2VmYWlyLTIwMTJyQwsSC1Byb2plY3RTaXRlIjJhaEp6Zm5OamFXVnVZMlZtWVdseUxUSXdNVEp5RHdzU0IxQnliMnBsWTNRWXVLOW9EQQw

http://spie.org/Documents/Courses/Education_Outreach/Science%20Fairs/Emily-Wang-CSSF14-Abstract.pdf

http://2014davidsonfellows.com/emily-wang/



TALENTED STUDENT FROM PALO ALTO REWARDED FOR UNMATCHED ACHIEVEMENT Emily Wang to be Awarded $25,000 as a 2014 Davidson Fellow

Reno, Nev. - Twenty bright young people named as 2014 Davidson Fellows<http://www.davidsongifted.org/fellows/> exemplify the extraordinary work that can be accomplished by U.S. students who are given opportunities to excel.  One of these gifted students is 18-year-old Emily Wang of Palo Alto, Calif.

Emily received $25,000 for her science project, "Illuminating Disease Pathways: Developing Bright Fluorescent Proteins to Improve FRET Biosensing." Emily created a biological tool to visualize diseases at the molecular level. She developed Clover3, a bright green fluorescent protein, and mRuby3, a bright red fluorescent protein. Clover3-mRuby3 can be used to image neurons to investigate Alzheimer's disease, detect and track cancer growth, and monitor the activities of diseases in cells. Clover3-mRuby3 may be used to visualize biological events with greater clarity than before, which may advance current understanding of illnesses to create new therapies and medicine to improve human health.


On 1/11/2015 2:42 AM, Guy Cox 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.

*****



I've changed the title since the previous one was going in too many directions.  Jim P mentioned that in conventional 2P microscopy we have ~12ns between pulses so with a typical fluorescence lifetime of 2-4ns we have some dead time.  Actually I'm not sure that's a bad thing since getting another hit on a molecule that's already excited is likely to increase bleaching.  And do remember the lifetime is not when fluorescence stops - just when it falls to 1/e of the original intensity, so there's still some to go.



However I recall seeing at conferences much smaller femtosecond lasers with, naturally, much faster repetition rates.  (The physical size of the laser determines the rep rate since it is the time light takes to go round one circuit of the cavity).  These would completely eliminate the dead time.  So has anyone used these for microscopy?



                       Guy



Guy Cox, Honorary Associate Professor

School of Medical Sciences



Australian Centre for Microscopy and Microanalysis,

Madsen, F09, University of Sydney, NSW 2006








--







George McNamara, Ph.D.

Single Cells Analyst

L.J.N. Cooper Lab

University of Texas M.D. Anderson Cancer Center

Houston, TX 77054

Tattletales http://works.bepress.com/gmcnamara/42

[Craig Brideau]

*****
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.
*****

I was shopping for ultrabroadband Ti:Saphs a few years ago: I had to choose
between a model with 85MHz rep rate or 1GHz rep rate. Since I was new to
the ultrabroadband game I went with the 85MHz one since it was closer to
the Tsunamis and Chameleons I was used to. My thinking was guided by the
oxygenated living samples my lab tends to work with. It seemed generally
better to have a strong 'hit' (high pulse energy) with a long relaxation
time, especially with an ultrabroadband laser on the order of 8fs. That
said, very little biological imaging has been done with the 1GHz laser but
people who used it made the case that many weaker pulses gave you move
time-average signal so you didn't need to excite as hard. I've been on the
fence for both cases for years. I do have a really high-energy OPA system
with 1MHz rep rate that does seem to be hard on 2PF samples though. It's
mainly meant for other non-linear processes so I the 2P functionality was
just a bonus, but it seems to bleach faster than say a Chameleon.

Craig

On Sun, Jan 11, 2015 at 1:42 AM, Guy Cox <[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.
> *****
>
> I've changed the title since the previous one was going in too many
> directions.  Jim P mentioned that in conventional 2P microscopy we have
> ~12ns between pulses so with a typical fluorescence lifetime of 2-4ns we
> have some dead time.  Actually I'm not sure that's a bad thing since
> getting another hit on a molecule that's already excited is likely to
> increase bleaching.  And do remember the lifetime is not when fluorescence
> stops - just when it falls to 1/e of the original intensity, so there's
> still some to go.
>
> However I recall seeing at conferences much smaller femtosecond lasers
> with, naturally, much faster repetition rates.  (The physical size of the
> laser determines the rep rate since it is the time light takes to go round
> one circuit of the cavity).  These would completely eliminate the dead
> time.  So has anyone used these for microscopy?
>
>                         Guy
>
> Guy Cox, Honorary Associate Professor
> School of Medical Sciences
>
> Australian Centre for Microscopy and Microanalysis,
> Madsen, F09, University of Sydney, NSW 2006
>

[Michael Giacomelli]

*****
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.
*****

Hi Guy,

This is something I've been meaning to test by rep rate
doubling/quadrupling our Chameleon and doing SNR measurements.  Its
hard to say from a theoretical point of view which would be better.
Certainly higher rep rate gives you lower multiphoton absorption
probability per unit power, but you don't make as effective use of the
detector dynamic range. Another thing on my mind is that my
(admittedly unscientific) impression is that images taken at low
numbers of pulses per pixel (1-2) tend to be fairly grainy no matter
how high the illumination power, although I've never been entirely
clear what that mechanism would be or if its somehow specific to my
samples.

Mike

On Sun, Jan 11, 2015 at 3:42 AM, Guy Cox <[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.
> *****
>
> I've changed the title since the previous one was going in too many directions.  Jim P mentioned that in conventional 2P microscopy we have ~12ns between pulses so with a typical fluorescence lifetime of 2-4ns we have some dead time.  Actually I'm not sure that's a bad thing since getting another hit on a molecule that's already excited is likely to increase bleaching.  And do remember the lifetime is not when fluorescence stops - just when it falls to 1/e of the original intensity, so there's still some to go.
>
> However I recall seeing at conferences much smaller femtosecond lasers with, naturally, much faster repetition rates.  (The physical size of the laser determines the rep rate since it is the time light takes to go round one circuit of the cavity).  These would completely eliminate the dead time.  So has anyone used these for microscopy?
>
>                         Guy
>
> Guy Cox, Honorary Associate Professor
> School of Medical Sciences
>
> Australian Centre for Microscopy and Microanalysis,
> Madsen, F09, University of Sydney, NSW 2006

[Zdenek Svindrych-2]

*****
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.
*****

Hi Mike,
>images taken at low numbers of pulses per pixel (1-2) tend to be fairly
grainy

did you synchronize the pixel clock with the laser repetition rate? I guess
you did, but if not, then some pixels receive the dose of two pulses and
others just single pulse. This will create some (non-random) pattern that
degrades your image.

It's generally not an issue when the number of pulses per pixels is high, e.
g. 80 MHz Ti:S and 1 us pixel dwell time gives some 80 +/- 1 pulses per
pixel (when not synchronized), limiting the dynamic range to some 40 dB.
Given the SNR of typical two-photon image is much lower than that, the
beating between the laser clock and the pixel clock can't be observed.
zdenek
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, USA
http://www.kcci.virginia.edu/workshop/index.php



---------- Původní zpráva ----------
Od: Michael Giacomelli <[hidden email]>
Komu: [hidden email]
Datum: 11. 1. 2015 15:28:03
Předmět: Re: 2p dead time

"*****
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.
*****

Hi Guy,

This is something I've been meaning to test by rep rate
doubling/quadrupling our Chameleon and doing SNR measurements. Its
hard to say from a theoretical point of view which would be better.
Certainly higher rep rate gives you lower multiphoton absorption
probability per unit power, but you don't make as effective use of the
detector dynamic range. Another thing on my mind is that my
(admittedly unscientific) impression is that images taken at low
numbers of pulses per pixel (1-2) tend to be fairly grainy no matter
how high the illumination power, although I've never been entirely
clear what that mechanism would be or if its somehow specific to my
samples.

Mike

"

[Michael Giacomelli]

*****
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.
*****

>did you synchronize the pixel clock with the laser repetition rate? I guess
you did, but if not, then some pixels receive the dose of two pulses and
others just single pulse. This will create some (non-random) pattern that
degrades your image.

Yes, I sampled at 240 MHz phase-locked to the Ti:Sapphire using a 3x
multiplier.  However, the comparison wasn't really scientific, and
were complicated by the fact that I was using a resonance scanner and
then dewarping the images.   I haven't had a chance to really look
into it, so it is quite possible that there was some other problem.

Mike

On Sun, Jan 11, 2015 at 4:24 PM, Zdenek Svindrych <[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.
> *****
>
> Hi Mike,
>>images taken at low numbers of pulses per pixel (1-2) tend to be fairly
> grainy
>
> did you synchronize the pixel clock with the laser repetition rate? I guess
> you did, but if not, then some pixels receive the dose of two pulses and
> others just single pulse. This will create some (non-random) pattern that
> degrades your image.
>
> It's generally not an issue when the number of pulses per pixels is high, e.
> g. 80 MHz Ti:S and 1 us pixel dwell time gives some 80 +/- 1 pulses per
> pixel (when not synchronized), limiting the dynamic range to some 40 dB.
> Given the SNR of typical two-photon image is much lower than that, the
> beating between the laser clock and the pixel clock can't be observed.
> zdenek
> --
> Zdenek Svindrych, Ph.D.
> W.M. Keck Center for Cellular Imaging (PLSB 003)
> University of Virginia, Charlottesville, USA
> http://www.kcci.virginia.edu/workshop/index.php
>
>
>
> ---------- Původní zpráva ----------
> Od: Michael Giacomelli <[hidden email]>
> Komu: [hidden email]
> Datum: 11. 1. 2015 15:28:03
> Předmět: Re: 2p dead time
>
> "*****
> 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.
> *****
>
> Hi Guy,
>
> This is something I've been meaning to test by rep rate
> doubling/quadrupling our Chameleon and doing SNR measurements. Its
> hard to say from a theoretical point of view which would be better.
> Certainly higher rep rate gives you lower multiphoton absorption
> probability per unit power, but you don't make as effective use of the
> detector dynamic range. Another thing on my mind is that my
> (admittedly unscientific) impression is that images taken at low
> numbers of pulses per pixel (1-2) tend to be fairly grainy no matter
> how high the illumination power, although I've never been entirely
> clear what that mechanism would be or if its somehow specific to my
> samples.
>
> Mike
>
> "
>

[Craig Brideau]

*****
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.
*****

Hi Mike, there's also the issue of pulse-to-pulse stability. The variations
in peak energy tend to average out when you have many pulses. My 1 MHz rep
rate system performs more evenly at 2 pulses per pixel than 1 pulse, but
the entire source is optimized for pulse-to-pulse stability. A regular
Ti:Saph laser may not have this stabilization, and at 20 or more pulses per
pixel would not need it as the effect averages out very nicely.

Craig


On Sun, Jan 11, 2015 at 2:49 PM, Michael Giacomelli <[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.
> *****
>
> >did you synchronize the pixel clock with the laser repetition rate? I
> guess
> you did, but if not, then some pixels receive the dose of two pulses and
> others just single pulse. This will create some (non-random) pattern that
> degrades your image.
>
> Yes, I sampled at 240 MHz phase-locked to the Ti:Sapphire using a 3x
> multiplier.  However, the comparison wasn't really scientific, and
> were complicated by the fact that I was using a resonance scanner and
> then dewarping the images.   I haven't had a chance to really look
> into it, so it is quite possible that there was some other problem.
>
> Mike
>
> On Sun, Jan 11, 2015 at 4:24 PM, Zdenek Svindrych <[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.
> > *****
> >
> > Hi Mike,
> >>images taken at low numbers of pulses per pixel (1-2) tend to be fairly
> > grainy
> >
> > did you synchronize the pixel clock with the laser repetition rate? I
> guess
> > you did, but if not, then some pixels receive the dose of two pulses and
> > others just single pulse. This will create some (non-random) pattern that
> > degrades your image.
> >
> > It's generally not an issue when the number of pulses per pixels is
> high, e.
> > g. 80 MHz Ti:S and 1 us pixel dwell time gives some 80 +/- 1 pulses per
> > pixel (when not synchronized), limiting the dynamic range to some 40 dB.
> > Given the SNR of typical two-photon image is much lower than that, the
> > beating between the laser clock and the pixel clock can't be observed.
> > zdenek
> > --
> > Zdenek Svindrych, Ph.D.
> > W.M. Keck Center for Cellular Imaging (PLSB 003)
> > University of Virginia, Charlottesville, USA
> > http://www.kcci.virginia.edu/workshop/index.php
> >
> >
> >
> > ---------- Původní zpráva ----------
> > Od: Michael Giacomelli <[hidden email]>
> > Komu: [hidden email]
> > Datum: 11. 1. 2015 15:28:03
> > Předmět: Re: 2p dead time
> >
> > "*****
> > 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.
> > *****
> >
> > Hi Guy,
> >
> > This is something I've been meaning to test by rep rate
> > doubling/quadrupling our Chameleon and doing SNR measurements. Its
> > hard to say from a theoretical point of view which would be better.
> > Certainly higher rep rate gives you lower multiphoton absorption
> > probability per unit power, but you don't make as effective use of the
> > detector dynamic range. Another thing on my mind is that my
> > (admittedly unscientific) impression is that images taken at low
> > numbers of pulses per pixel (1-2) tend to be fairly grainy no matter
> > how high the illumination power, although I've never been entirely
> > clear what that mechanism would be or if its somehow specific to my
> > samples.
> >
> > Mike
> >
> > "
> >
>