Shearing Interferometer with Ti-Sapph

[Heping Yuan]

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Hello, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?

Thanks for any help,
Tim

***** 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. *****

Here's a shot in the dark here for you, since I have never tried doing what you asked about.

Short pulse lasers only interfere over a short distance called their coherence length. It might only be 10-30 microns or so depending on the laser's bandwidth. If the path length difference of the two interfering beams is more than this 10-30 microns, then no fringes.

If the interferometer works by beam 1 reflecting off the front of the glass wedge (going through no glass) and beam 2 reflecting off the back surface of the glass at a little different angle (going through the glass twice), then the optical path length of beam 2 is quite a bit more than that of beam 1. Probably by much more than 10-30 microns since the wedge is probably at least 1 mm thick where you use it. I would guess no fringes.

Basically, I am asking whether your interferometer is designed for short pulse lasers. I bet the Thorlabs folks could weigh in on it if you email them.

Anyway, isn't it easier to check collimation by shooting the beam across the room and checking the diameter is constant?

On Tue, Sep 20, 2016 at 9:07 PM, Tim <[hidden email]> wrote:

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Hello, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?

Thanks for any help,
Tim

--

[Mark Cannell-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. ***** You could check the collimation with the Ti-S in CW mode (i.e. not mode locked)?

HTH

Mark

On 21/09/2016, at 5:07 am, Tim <[hidden email]> wrote:

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Hello, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?

Thanks for any help,
Tim

Mark  B. Cannell Ph.D. FRSNZ FISHR
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Faculty of Biomedical Sciences
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[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 Tim,

no experience with shearing interferometry, but few thoughts:

As pointed out, 100 fs pulse has 'physical length' (s=c*t) of some 30 um. With the mentioned interferometer (more than 20 mm path difference according to Thorlabs specs!) you end up with two copies of the pulse separated by 70 ps. They will only interfere if you watch them through extremely narrow-band filter (just a theory, probably not practical at all).

Even in CW, as suggested by Mark (not all Ti:saphs can be easily forced to CW) the coherence length may not be enough to see fringes, depending on the laser.

One solution would be zero path difference interferometer, which can be constructed with common path geometry:

http://www.sciencedirect.com/science/article/pii/S0030402605002226

But I'm not aware of it being available commercially.

There are other options to measure collimation, like wavefront sensors (quite costly) or watching the far field of the beam (needs lots of space or some already collimated optics)...

Good luck!

--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, VA
http://www.kcci.virginia.edu/
tel: [phone]
Annual FRET Workshop: http://kcci.virginia.edu/workshop-2017

---------- Původní zpráva ----------
Od: Joshua Vaughan <[hidden email]>
Komu: [hidden email]
Datum: 21. 9. 2016 0:40:55
Předmět: Re: Shearing Interferometer with Ti-Sapph

***** 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. *****

Here's a shot in the dark here for you, since I have never tried doing what you asked about.

Short pulse lasers only interfere over a short distance called their coherence length. It might only be 10-30 microns or so depending on the laser's bandwidth. If the path length difference of the two interfering beams is more than this 10-30 microns, then no fringes.

If the interferometer works by beam 1 reflecting off the front of the glass wedge (going through no glass) and beam 2 reflecting off the back surface of the glass at a little different angle (going through the glass twice), then the optical path length of beam 2 is quite a bit more than that of beam 1. Probably by much more than 10-30 microns since the wedge is probably at least 1 mm thick where you use it. I would guess no fringes.

Basically, I am asking whether your interferometer is designed for short pulse lasers. I bet the Thorlabs folks could weigh in on it if you email them.

Anyway, isn't it easier to check collimation by shooting the beam across the room and checking the diameter is constant?

On Tue, Sep 20, 2016 at 9:07 PM, Tim <[hidden email]> wrote:

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Hello, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?

Thanks for any help,
Tim

--

Joshua C. Vaughan

Assistant Professor

Department of Chemistry

Box 351700

University of Washington

Seattle, WA 98195
206-543-4644

[-]

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Hi Tim,



  I regularly check the collimation of the Spectra Physics MaiTai Laser
  by running it in CW with a Thorlabs shearing plate.
  Works well.

  Best
  Max

> Am 21.09.2016 15:21 schrieb [hidden email]:
>> ***** To join, leave or search the confocal microscopy listserv, go
>> to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy [1] Post
>> images on http://www.imgur.com [2] and include the link in your
>> posting. ***** Hi Tim,
>> no experience with shearing interferometry, but few thoughts:
>> As pointed out, 100 fs pulse has 'physical length' (s=c*t) of some 30
>> um. With the mentioned interferometer (more than 20 mm path difference
>> according to Thorlabs specs!) you end up with two copies of the pulse
>> separated by 70 ps. They will only interfere if you watch them through
>> extremely narrow-band filter (just a theory, probably not practical at
>> all).
>> Even in CW, as suggested by Mark (not all Ti:saphs can be easily
>> forced to CW) the coherence length may not be enough to see fringes,
>> depending on the laser.
>> One solution would be zero path difference interferometer, which can
>> be constructed with common path geometry:
>> http://www.sciencedirect.com/science/article/pii/S0030402605002226
>>
>> But I'm not aware of it being available commercially.
>> There are other options to measure collimation, like wavefront sensors
>> (quite costly) or watching the far field of the beam (needs lots of
>> space or some already collimated optics)...
>> Good luck!
>>
>> --
>> Zdenek Svindrych, Ph.D.
>> W.M. Keck Center for Cellular Imaging (PLSB 003)
>> University of Virginia, Charlottesville, VA
>> http://www.kcci.virginia.edu/
>> tel: [phone]
>> Annual FRET Workshop: http://kcci.virginia.edu/workshop-2017
>>
>> ---------- Původní zpráva ----------
>> Od: Joshua Vaughan <[hidden email]>
>> Komu: [hidden email]
>> Datum: 21. 9. 2016 0:40:55
>> Předmět: Re: Shearing Interferometer with Ti-Sapph
>>
>>> ***** To join, leave or search the confocal microscopy listserv, go
>>> to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy [1] Post
>>> images on http://www.imgur.com [2] and include the link in your
>>> posting. *****
>>>
>>> Here's a shot in the dark here for you, since I have never tried
>>> doing what you asked about.
>>>
>>> Short pulse lasers only interfere over a short distance called their
>>> coherence length. It might only be 10-30 microns or so depending on
>>> the laser's bandwidth. If the path length difference of the two
>>> interfering beams is more than this 10-30 microns, then no fringes.
>>>
>>> If the interferometer works by beam 1 reflecting off the front of
>>> the glass wedge (going through no glass) and beam 2 reflecting off
>>> the back surface of the glass at a little different angle (going
>>> through the glass twice), then the optical path length of beam 2 is
>>> quite a bit more than that of beam 1. Probably by much more than
>>> 10-30 microns since the wedge is probably at least 1 mm thick where
>>> you use it. I would guess no fringes.
>>>
>>> Basically, I am asking whether your interferometer is designed for
>>> short pulse lasers. I bet the Thorlabs folks could weigh in on it if
>>> you email them.
>>>
>>> Anyway, isn't it easier to check collimation by shooting the beam
>>> across the room and checking the diameter is constant?
>>>
>>> On Tue, Sep 20, 2016 at 9:07 PM, Tim <[hidden email]> wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/w a?A0=confocalmicroscopy [1]
>>>> Post images on http://www.imgur.com [2] and include the link in
>>>> your posting.
>>>> *****
>>>>
>>>> Hello, we recently purchased a shearing interferometer from
>>>> Thorlabs (Part # SI254) to test collimation after beam expansion
>>>> in our two-photon microscope setup. The strange thing is we cannot
>>>> seem to get an interference pattern either before or after beam
>>>> expansion (varying the distance between the lenses in the beam
>>>> expander). We do see the pattern clearly if we try on a simple
>>>> laser diode. Does anyone have experience looking at the shearing
>>>> interferometer pattern with a Ti-Sapphire laser or have any ideas
>>>> what may be causing our problem?
>>>>
>>>> Thanks for any help,
>>>> Tim
>>>
>>> --
>>>
>>> Joshua C. Vaughan
>>>
>>> Assistant Professor
>>> Department of Chemistry
>>> Box 351700
>>> University of Washington
>>> Seattle, WA 98195
>>> 206-543-4644
>>
>>
>> Links:
>> ------
>> [1] http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> [2] http://www.imgur.com

[Michael Giacomelli]

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Hi Tim,

A shearing interferometer is not going to work with an ultrafast
laser.  There is a note in the thorlabs catalog that can help you
figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so
you will need a coherence length at least that long.  Try your laser
in CW mode, although even that may not be quite 2 cm depending on how
stable it is without a mode lock.  Usually though testing for
collimation after a beam expander doesn't require an interferometer
since the beam is large the divergence is low.  Have you tried just
bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
>
> Thanks for any help,
> Tim

[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. *****

The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

Craig

On Wed, Sep 21, 2016 at 8:50 AM, Michael Giacomelli <[hidden email]> wrote:

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*****

Hi Tim,

A shearing interferometer is not going to work with an ultrafast
laser.  There is a note in the thorlabs catalog that can help you
figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so
you will need a coherence length at least that long.  Try your laser
in CW mode, although even that may not be quite 2 cm depending on how
stable it is without a mode lock.  Usually though testing for
collimation after a beam expander doesn't require an interferometer
since the beam is large the divergence is low.  Have you tried just
bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
>
> Thanks for any help,
> Tim

[Heping Yuan]

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Thank you all very much for your great feedback!

Tim

[0000001ed7f52e4a-dmarc-request]

***** 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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

 

best wishes

 

Andreas

 

 

-----Original Message-----
From: Craig Brideau <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Wed, Sep 21, 2016 5:30 pm
Subject: Re: Shearing Interferometer with Ti-Sapph

***** 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. *****

The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

Craig

On Wed, Sep 21, 2016 at 8:50 AM, Michael Giacomelli <[hidden email]> wrote:

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Post images on http://www.imgur.com and include the link in your posting.
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Hi Tim,

A shearing interferometer is not going to work with an ultrafast
laser.  There is a note in the thorlabs catalog that can help you
figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so
you will need a coherence length at least that long.  Try your laser
in CW mode, although even that may not be quite 2 cm depending on how
stable it is without a mode lock.  Usually though testing for
collimation after a beam expander doesn't require an interferometer
since the beam is large the divergence is low.  Have you tried just
bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
>
> Thanks for any help,
> Tim

[Tim Feinstein]

***** 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 Andreas, 

Are you using Huygens Suite?  As I recall Huygens has a limit of roughly 1/2 their recommended resolution (92 nm in this case) beyond which the algorithm just won't proceed.  In the 'parameters' window you will see XY resolution highlighted in red.  I believe this is because a pixel needs to be at least a bit smaller than the majority of the PSF for deconvolution to work.  Their recommended resolution limit of 46 nm is the point where you stop getting any computational benefit from making pixels any smaller.  

I have seen other implementations work with lower resolution (i.e., Fiji plugins) but those are simpler algorithms that don't test whether you are in the resolution range where it is appropriate to deconvolve.  

Best, 

Tim

Timothy Feinstein, Ph.D. 

Research Scientist

University of Pittsburgh Department of Developmental Biology

From: Confocal Microscopy List <[hidden email]> on behalf of "[hidden email]" <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Thursday, September 22, 2016 at 8:22 AM
To: "[hidden email]" <[hidden email]>
Subject: Sampling rate for confocal microscopy

***** 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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

 

best wishes

 

Andreas

 

 

-----Original Message-----
From: Craig Brideau <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Wed, Sep 21, 2016 5:30 pm
Subject: Re: Shearing Interferometer with Ti-Sapph

***** 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. *****

The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

Craig

On Wed, Sep 21, 2016 at 8:50 AM, Michael Giacomelli <[hidden email]> wrote:

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Tim,

A shearing interferometer is not going to work with an ultrafast
laser.  There is a note in the thorlabs catalog that can help you
figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so
you will need a coherence length at least that long.  Try your laser
in CW mode, although even that may not be quite 2 cm depending on how
stable it is without a mode lock.  Usually though testing for
collimation after a beam expander doesn't require an interferometer
since the beam is large the divergence is low.  Have you tried just
bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
>
> Thanks for any help,
> Tim

[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 Andreas,

I suggest ALL of:

* X,Y pixel size = 100 nm

* Z pixel size = 300 nm

(you can evaluate smaller values later, if the below help).

* Re-evaluate the fluorophore(s) you are using.

* optimize mounting medium to minimize photobleaching of the fluorophore(s) you are using, also match R.I. with the immersion medium (R.I. ~1.515).

* optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).

* Evaluate all of the current deconvolution vendors, SVI, AutoQuant, Microvolution, to get demo's (once you've done the other things).

* Use a Booster ... treat your current current (or post-re-evaluation) fluorophore as a hapten. For example, if you are stuck with Alexa Fluor 488, detect with an anti-X and then detect that (FASER has all the reagents):

AntiAlexa Fluor® 488, Rabbit IgG Fraction (A-11094)

https://tools.thermofisher.com/content/sfs/manuals/mp11094.pdf

if you are still using fluorescein (and have optimized pH 8.0 and it is still dim), Molecular Probes has anti-fluorescein but I'll use as examples,

Anti-Fluorescein antibody (ab19491)
http://www.abcam.com/fluorescein-antibody-ab19491.html

Miltenyi Biotec's FASER kits (for APC, "FITC", PE ... Fluorescence Amplification by Sequential Employment of Reagents, check out all the tabs on the product page)

http://www.miltenyibiotec.com/en/products-and-services/macs-flow-cytometry/reagents/support-reagents/faser-kits.aspx

Consider detecting with a 'state of the art' fluorophore, like Brilliant Violet BV421 (BD Biosciences), though may require very low laser power. Be sure to use their buffer that comes with the kits, especially if expanding the experiments to multiple Brilliant's (they tend to aggregate in water, hence their inclusion of the buffer). You could also "change colors" to quantum dots, of which QD625 is the brightest of the Molecular Probes product line (shorter excitation wavelength has higher extinction coefficient and longer effective Stokes shift ... maybe we should call the difference of wavelength used and emission peak wavelength the "Bruchez shift").

May as many of your photons as possible be detected and PSFs symmetrical,

George

On 9/22/2016 7:22 AM, [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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

 

best wishes

 

Andreas

 

 

-----Original Message-----
From: Craig Brideau [hidden email]
To: CONFOCALMICROSCOPY [hidden email]
Sent: Wed, Sep 21, 2016 5:30 pm
Subject: Re: Shearing Interferometer with Ti-Sapph

***** 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. *****

The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

Craig

On Wed, Sep 21, 2016 at 8:50 AM, 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.
*****

Hi Tim,

A shearing interferometer is not going to work with an ultrafast
laser.  There is a note in the thorlabs catalog that can help you
figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so
you will need a coherence length at least that long.  Try your laser
in CW mode, although even that may not be quite 2 cm depending on how
stable it is without a mode lock.  Usually though testing for
collimation after a beam expander doesn't require an interferometer
since the beam is large the divergence is low.  Have you tried just
bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
>
> Thanks for any help,
> Tim

[Tobias Rose]

***** 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. *****

Just quickly:

* optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).

Why median?

Tobias

 

On 9/22/2016 7:22 AM, [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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

 

best wishes

 

Andreas

 

 

-----Original Message-----
From: Craig Brideau [hidden email]
To: CONFOCALMICROSCOPY [hidden email]
Sent: Wed, Sep 21, 2016 5:30 pm
Subject: Re: Shearing Interferometer with Ti-Sapph

***** 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. *****

The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

 

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

 

Craig

 

On Wed, Sep 21, 2016 at 8:50 AM, 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.
*****

Hi Tim,

A shearing interferometer is not going to work with an ultrafast
laser.  There is a note in the thorlabs catalog that can help you
figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so
you will need a coherence length at least that long.  Try your laser
in CW mode, although even that may not be quite 2 cm depending on how
stable it is without a mode lock.  Usually though testing for
collimation after a beam expander doesn't require an interferometer
since the beam is large the divergence is low.  Have you tried just
bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
>
> Thanks for any help,
> Tim

 

-- 

George McNamara, PhD

Houston, TX 77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

https://works.bepress.com/gmcnamara/75/

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Tobias Rose]

***** 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. *****

Sorry. Wrong line cited the last time. I wanted to ask George why he suggests to use the median pixel intensity for frame averaging.

 

Tobias

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Thursday, September 22, 2016 4:10 PM
To: [hidden email]
Subject: Re: Sampling rate for confocal microscopy

 

***** 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 Andreas,

I suggest ALL of:

* X,Y pixel size = 100 nm

* Z pixel size = 300 nm

(you can evaluate smaller values later, if the below help).

* Re-evaluate the fluorophore(s) you are using.

* optimize mounting medium to minimize photobleaching of the fluorophore(s) you are using, also match R.I. with the immersion medium (R.I. ~1.515).

* optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).

* Evaluate all of the current deconvolution vendors, SVI, AutoQuant, Microvolution, to get demo's (once you've done the other things).

* Use a Booster ... treat your current current (or post-re-evaluation) fluorophore as a hapten. For example, if you are stuck with Alexa Fluor 488, detect with an anti-X and then detect that (FASER has all the reagents):

AntiAlexa Fluor® 488, Rabbit IgG Fraction (A-11094)

https://tools.thermofisher.com/content/sfs/manuals/mp11094.pdf

if you are still using fluorescein (and have optimized pH 8.0 and it is still dim), Molecular Probes has anti-fluorescein but I'll use as examples,

Anti-Fluorescein antibody (ab19491)
http://www.abcam.com/fluorescein-antibody-ab19491.html

Miltenyi Biotec's FASER kits (for APC, "FITC", PE ... Fluorescence Amplification by Sequential Employment of Reagents, check out all the tabs on the product page)

http://www.miltenyibiotec.com/en/products-and-services/macs-flow-cytometry/reagents/support-reagents/faser-kits.aspx

Consider detecting with a 'state of the art' fluorophore, like Brilliant Violet BV421 (BD Biosciences), though may require very low laser power. Be sure to use their buffer that comes with the kits, especially if expanding the experiments to multiple Brilliant's (they tend to aggregate in water, hence their inclusion of the buffer). You could also "change colors" to quantum dots, of which QD625 is the brightest of the Molecular Probes product line (shorter excitation wavelength has higher extinction coefficient and longer effective Stokes shift ... maybe we should call the difference of wavelength used and emission peak wavelength the "Bruchez shift").

 

May as many of your photons as possible be detected and PSFs symmetrical,

George

 

On 9/22/2016 7:22 AM, [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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

 

best wishes

 

Andreas

 

 

-----Original Message-----
From: Craig Brideau [hidden email]
To: CONFOCALMICROSCOPY [hidden email]
Sent: Wed, Sep 21, 2016 5:30 pm
Subject: Re: Shearing Interferometer with Ti-Sapph

***** 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. *****

The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

 

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

 

Craig

 

On Wed, Sep 21, 2016 at 8:50 AM, 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.
*****

Hi Tim,

A shearing interferometer is not going to work with an ultrafast
laser.  There is a note in the thorlabs catalog that can help you
figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so
you will need a coherence length at least that long.  Try your laser
in CW mode, although even that may not be quite 2 cm depending on how
stable it is without a mode lock.  Usually though testing for
collimation after a beam expander doesn't require an interferometer
since the beam is large the divergence is low.  Have you tried just
bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
>
> Thanks for any help,
> Tim

 

-- 

George McNamara, PhD

Houston, TX 77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

https://works.bepress.com/gmcnamara/75/

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Giulia De Luca]

***** 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 Andreas,

As imaging specialist at SVI I am happy to answer your question. We always suggest to use our Nyquist Calculator, that you nicely link, to calculate the ideal x, y and z sampling specific for your imaging settings. This will assure that you optimally preserve the spatial frequencies in your images for further image restoration with deconvolution. Such small sampling distances may arise the worry for bleaching. However, because the restoration procedure will improve the Signal to Noise Ratio of the images dramatically, this should not worry you! You can proceed in lowering the laser power or the exposure time/acquisition speed and then proceed with deconvolution afterwards. You will see that the noise will be taken care of in the deconvolution process.

Please do not hesitate to contact us if you have any further questions, we are happy to help you!

Best regards,

Giulia

2016-09-22 17:37 GMT+02:00 Tobias Rose <[hidden email]>:

***** 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. *****

Just quickly:

* optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).

Why median?

Tobias

 

On 9/22/2016 7:22 AM, [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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

 

best wishes

 

Andreas

 

 

-----Original Message-----
From: Craig Brideau [hidden email]
To: CONFOCALMICROSCOPY [hidden email]
Sent: Wed, Sep 21, 2016 5:30 pm
Subject: Re: Shearing Interferometer with Ti-Sapph

***** 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. *****

The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

 

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

 

Craig

 

On Wed, Sep 21, 2016 at 8:50 AM, 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.
*****

Hi Tim,

A shearing interferometer is not going to work with an ultrafast
laser.  There is a note in the thorlabs catalog that can help you
figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so
you will need a coherence length at least that long.  Try your laser
in CW mode, although even that may not be quite 2 cm depending on how
stable it is without a mode lock.  Usually though testing for
collimation after a beam expander doesn't require an interferometer
since the beam is large the divergence is low.  Have you tried just
bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
>
> Thanks for any help,
> Tim

 

-- 

George McNamara, PhD

Houston, TX 77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

https://works.bepress.com/gmcnamara/75/

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Barbara Foster]

***** 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, Giulia,

Great answer!  Cn you send us the link to your Nyquist calculator?

Best regards,
Barbara Foster, President & Chief Consultant
Microscopy/Microscopy Education  ... "Education, not Training"
7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P: 972-924-5310
www.MicroscopyEducation.com

Microscopy/Microscopy Education is a division of The Microscopy & Imaging Place, Inc.


NEW!   Getting involved in Raman or FTIR?
MME is now offering courses in these areas specifically for microscopists! 
Now scheduling courses through the mid 2017.  We can customize a course on nearly any topic, from fluorescence to confocal to image analysis to SEM/TEM. 
Call today for a free training evaluation

t 04:51 AM 9/22/2016, Giulia De Luca 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 Andreas,

As imaging specialist at SVI I am happy to answer your question. We always suggest to use our Nyquist Calculator, that you nicely link, to calculate the ideal x, y and z sampling specific for your imaging settings. This will assure that you optimally preserve the spatial frequencies in your images for further image restoration with deconvolution. Such small sampling distances may arise the worry for bleaching. However, because the restoration procedure will improve the Signal to Noise Ratio of the images dramatically, this should not worry you! You can proceed in lowering the laser power or the exposure time/acquisition speed and then proceed with deconvolution afterwards. You will see that the noise will be taken care of in the deconvolution process.

Please do not hesitate to contact us if you have any further questions, we are happy to help you!

Best regards,

Giulia

2016-09-22 17:37 GMT+02:00 Tobias Rose <[hidden email]>:

***** 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. *****

Just quickly:

* optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).

Why median?

Tobias

Â

On 9/22/2016 7:22 AM, [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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

Â

best wishes

Â

Andreas

Â

Â

-----Original Message-----

From: Craig Brideau [hidden email]

To: CONFOCALMICROSCOPY [hidden email]

Sent: Wed, Sep 21, 2016 5:30 pm

Subject: Re: Shearing Interferometer with Ti-Sapph

***** 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. *****

The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

Â

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

Â

Craig

Â

On Wed, Sep 21, 2016 at 8:50 AM, 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.

*****

Hi Tim,

A shearing interferometer is not going to work with an ultrafast

laser.  There is a note in the thorlabs catalog that can help you

figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so

you will need a coherence length at least that long.  Try your laser

in CW mode, although even that may not be quite 2 cm depending on how

stable it is without a mode lock.  Usually though testing for

collimation after a beam expander doesn't require an interferometer

since the beam is large the divergence is low.  Have you tried just

bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?

>

> Thanks for any help,

> Tim

Â

-- 

 

 

George McNamara,
PhD

Houston, TX
77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

https://works.bepress.com/gmcnamara/75/

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

 

[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 Tobias,

Mean vs median (I'm going to ignore "Kalman" running averaging, a nice way to emphasize photobleaching used on old confocals).

PMT's are excellent at detecting photons, often need to be used at high gain (or to put this another way: gain of zero makes for boring images). A simple example, one pixel for 4 frames:

4000

0

0

0

average is 1000. Median is zero. The "right" answer for this pixel is probably zero, not 1000. Sure, deconvolution software could be coded to recognize single 'hot' pixels (and need to be for sCMOS cameras, since most of the sCMOS camera manufacturers do not do quantitative cleanup), but let's say the adjacent pixels have numbers like this:

0

4000

0

0

resulting in some of them averaging around 1000.

If you want more, there were previous listserv discussions of mean and median in the past. MetaMorph has had Stack Arithmetic: Median since around version 1.0 (released end of 1992), so it is not as if the confocal vendors could not enable "medianing" each pixel (voxel).

George

p.s. I'm ignoring I normally set offset above zero, and gain low enough to not get close to saturation (i.e. 12-bit readout, max 4095).

On 9/22/2016 10:40 AM, Tobias Rose 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. *****

Sorry. Wrong line cited the last time. I wanted to ask George why he suggests to use the median pixel intensity for frame averaging.

 

Tobias

 

From: Confocal Microscopy List [[hidden email]] On Behalf Of George McNamara
Sent: Thursday, September 22, 2016 4:10 PM
To: [hidden email]
Subject: Re: Sampling rate for confocal microscopy

 

***** 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 Andreas,

I suggest ALL of:

* X,Y pixel size = 100 nm

* Z pixel size = 300 nm

(you can evaluate smaller values later, if the below help).

* Re-evaluate the fluorophore(s) you are using.

* optimize mounting medium to minimize photobleaching of the fluorophore(s) you are using, also match R.I. with the immersion medium (R.I. ~1.515).

* optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).

* Evaluate all of the current deconvolution vendors, SVI, AutoQuant, Microvolution, to get demo's (once you've done the other things).

* Use a Booster ... treat your current current (or post-re-evaluation) fluorophore as a hapten. For example, if you are stuck with Alexa Fluor 488, detect with an anti-X and then detect that (FASER has all the reagents):

AntiAlexa Fluor® 488, Rabbit IgG Fraction (A-11094)

https://tools.thermofisher.com/content/sfs/manuals/mp11094.pdf

if you are still using fluorescein (and have optimized pH 8.0 and it is still dim), Molecular Probes has anti-fluorescein but I'll use as examples,

Anti-Fluorescein antibody (ab19491)
http://www.abcam.com/fluorescein-antibody-ab19491.html

Miltenyi Biotec's FASER kits (for APC, "FITC", PE ... Fluorescence Amplification by Sequential Employment of Reagents, check out all the tabs on the product page)

http://www.miltenyibiotec.com/en/products-and-services/macs-flow-cytometry/reagents/support-reagents/faser-kits.aspx

Consider detecting with a 'state of the art' fluorophore, like Brilliant Violet BV421 (BD Biosciences), though may require very low laser power. Be sure to use their buffer that comes with the kits, especially if expanding the experiments to multiple Brilliant's (they tend to aggregate in water, hence their inclusion of the buffer). You could also "change colors" to quantum dots, of which QD625 is the brightest of the Molecular Probes product line (shorter excitation wavelength has higher extinction coefficient and longer effective Stokes shift ... maybe we should call the difference of wavelength used and emission peak wavelength the "Bruchez shift").

 

May as many of your photons as possible be detected and PSFs symmetrical,

George

 

On 9/22/2016 7:22 AM, [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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

 

best wishes

 

Andreas

 

 

-----Original Message-----
From: Craig Brideau [hidden email]
To: CONFOCALMICROSCOPY [hidden email]
Sent: Wed, Sep 21, 2016 5:30 pm
Subject: Re: Shearing Interferometer with Ti-Sapph

***** 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. *****

The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

 

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

 

Craig

 

On Wed, Sep 21, 2016 at 8:50 AM, Michael Giacomelli <[hidden email]> wrote:

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Tim,

A shearing interferometer is not going to work with an ultrafast
laser.  There is a note in the thorlabs catalog that can help you
figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so
you will need a coherence length at least that long.  Try your laser
in CW mode, although even that may not be quite 2 cm depending on how
stable it is without a mode lock.  Usually though testing for
collimation after a beam expander doesn't require an interferometer
since the beam is large the divergence is low.  Have you tried just
bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
>
> Thanks for any help,
> Tim

 

-- 

George McNamara, PhD

Houston, TX 77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

https://works.bepress.com/gmcnamara/75/

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Stephen Smith]

*****
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*****

From my previous life I measured CW TiS coherence lengths of a few cm, which matched the measured line widths of about 0.2 cm-1 or 6 GHz)  This would be pretty easy to find overlap in an interferometer.

The divergence and beam waist will shift between mode locked and CW operation too.

[Giulia De Luca]

***** 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 Barbara, 

Thank you! Please enjoy the Nyquist calculator from the Scientific Volume Imaging website:

https://svi.nl/NyquistCalculator

We also have a Nyquist app for Android devices:

https://svi.nl/NyquistApp

The ideal sampling will be calculated depending on the optics of your microscope. No excuse to undersample your images anymore with these two handy tools ;) 

Best regards, 

Giulia

2016-09-22 19:59 GMT+02:00 Barbara Foster <[hidden email]>:

***** 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, Giulia,

Great answer!  Cn you send us the link to your Nyquist calculator?

Best regards,
Barbara Foster, President & Chief Consultant
Microscopy/Microscopy Education  ... "Education, not Training"
7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P: <a href="tel:[phone]" value="[phone]" target="_blank">972-924-5310
www.MicroscopyEducation.com

Microscopy/Microscopy Education is a division of The Microscopy & Imaging Place, Inc.


NEW!   Getting involved in Raman or FTIR?
MME is now offering courses in these areas specifically for microscopists! 
Now scheduling courses through the mid 2017.  We can customize a course on nearly any topic, from fluorescence to confocal to image analysis to SEM/TEM. 
Call today for a free training evaluation

t 04:51 AM 9/22/2016, Giulia De Luca 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 Andreas,

As imaging specialist at SVI I am happy to answer your question. We always suggest to use our Nyquist Calculator, that you nicely link, to calculate the ideal x, y and z sampling specific for your imaging settings. This will assure that you optimally preserve the spatial frequencies in your images for further image restoration with deconvolution. Such small sampling distances may arise the worry for bleaching. However, because the restoration procedure will improve the Signal to Noise Ratio of the images dramatically, this should not worry you! You can proceed in lowering the laser power or the exposure time/acquisition speed and then proceed with deconvolution afterwards. You will see that the noise will be taken care of in the deconvolution process.

Please do not hesitate to contact us if you have any further questions, we are happy to help you!

Best regards,

Giulia

2016-09-22 17:37 GMT+02:00 Tobias Rose <[hidden email]>:

***** 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. *****

Just quickly:

* optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).

Why median?

Tobias

Â

On 9/22/2016 7:22 AM, [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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

Â

best wishes

Â

Andreas

Â

Â

-----Original Message-----

From: Craig Brideau [hidden email]

To: CONFOCALMICROSCOPY [hidden email]

Sent: Wed, Sep 21, 2016 5:30 pm

Subject: Re: Shearing Interferometer with Ti-Sapph

***** 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. *****

The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

Â

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

Â

Craig

Â

On Wed, Sep 21, 2016 at 8:50 AM, 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.

*****

Hi Tim,

A shearing interferometer is not going to work with an ultrafast

laser.  There is a note in the thorlabs catalog that can help you

figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so

you will need a coherence length at least that long.  Try your laser

in CW mode, although even that may not be quite 2 cm depending on how

stable it is without a mode lock.  Usually though testing for

collimation after a beam expander doesn't require an interferometer

since the beam is large the divergence is low.  Have you tried just

bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[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, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?

>

> Thanks for any help,

> Tim

Â

-- 

 

 

George McNamara,
PhD

Houston, TX
77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

https://works.bepress.com/gmcnamara/75/

http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

 

[0000001ed7f52e4a-dmarc-request]

***** 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. *****

Dear all,

 

Many thanks for the replies, it looks like opinion is divided with George suggesting 100 nm and Giulia from SVI suggesting the 46 nm of the Nyquist calculator. It would be best to do the test to see if there is any improvement with the 40 nm sampling, does anyone know of papers looking into this? I would have thought that there is a sweet-spot and further reducing the pixel size (at the same total acquisition time) would just lower the signal to noise ratio and any useful signal would be buried in noise. But I guess the deconvolution algorithm nicely sums up the pixels to get the signal back. Is there any advantage of acquiring e.g. one big pixel instead of four smaller ones? I am thinking of something similar like minimising read-out-noise of CCD cameras by binning, just for the confocal case, or does the PMT and read out electronics behave like CMOS where there is no advantage of binning?

 

best wishes

 

Andreas

 

-----Original Message-----
From: Giulia De Luca <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Thu, 22 Sep 2016 16:55
Subject: Re: Sampling rate for confocal microscopy

***** 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 Andreas,

As imaging specialist at SVI I am happy to answer your question. We always suggest to use our Nyquist Calculator, that you nicely link, to calculate the ideal x, y and z sampling specific for your imaging settings. This will assure that you optimally preserve the spatial frequencies in your images for further image restoration with deconvolution. Such small sampling distances may arise the worry for bleaching. However, because the restoration procedure will improve the Signal to Noise Ratio of the images dramatically, this should not worry you! You can proceed in lowering the laser power or the exposure time/acquisition speed and then proceed with deconvolution afterwards. You will see that the noise will be taken care of in the deconvolution process.

Please do not hesitate to contact us if you have any further questions, we are happy to help you!

Best regards,

Giulia

2016-09-22 17:37 GMT+02:00 Tobias Rose <[hidden email]>:

***** 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. *****

Just quickly:

* optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).

Why median?

Tobias

 

On 9/22/2016 7:22 AM, [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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

 

best wishes

 

Andreas

 

 

[Guy Hagen]

***** 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. *****

Dear Friends:

The proper way to calculate sampling rates in widefield and confocal microscopy can be found in this paper:

Heintzmann, R. (2005). Band limit and appropriate sampling in microscopy. In Simons, K., S. J. V., Hunter, T., Shotton, et al. (Eds.), Cell biology: a laboratory handbook (pp. 29-36). Amsterdam: Elsevier.

The example given in the paper show that (for NA 1.3, ex=488nm, em=500nm)  47.5 nm is what you want for X,Y sampling, and 93.8nm in Z. Also make sure to set your digitizer to the highest setting, 16 bit mode if possible.

I can provide a PDF of the paper to anyone who contacts me.

cheers,
Guy Hagen

On Sat, Sep 24, 2016 at 9:12 AM, <[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. *****

Dear all,

 

Many thanks for the replies, it looks like opinion is divided with George suggesting 100 nm and Giulia from SVI suggesting the 46 nm of the Nyquist calculator. It would be best to do the test to see if there is any improvement with the 40 nm sampling, does anyone know of papers looking into this? I would have thought that there is a sweet-spot and further reducing the pixel size (at the same total acquisition time) would just lower the signal to noise ratio and any useful signal would be buried in noise. But I guess the deconvolution algorithm nicely sums up the pixels to get the signal back. Is there any advantage of acquiring e.g. one big pixel instead of four smaller ones? I am thinking of something similar like minimising read-out-noise of CCD cameras by binning, just for the confocal case, or does the PMT and read out electronics behave like CMOS where there is no advantage of binning?

 

best wishes

 

Andreas

 

-----Original Message-----
From: Giulia De Luca <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Thu, 22 Sep 2016 16:55
Subject: Re: Sampling rate for confocal microscopy

***** 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 Andreas,

As imaging specialist at SVI I am happy to answer your question. We always suggest to use our Nyquist Calculator, that you nicely link, to calculate the ideal x, y and z sampling specific for your imaging settings. This will assure that you optimally preserve the spatial frequencies in your images for further image restoration with deconvolution. Such small sampling distances may arise the worry for bleaching. However, because the restoration procedure will improve the Signal to Noise Ratio of the images dramatically, this should not worry you! You can proceed in lowering the laser power or the exposure time/acquisition speed and then proceed with deconvolution afterwards. You will see that the noise will be taken care of in the deconvolution process.

Please do not hesitate to contact us if you have any further questions, we are happy to help you!

Best regards,

Giulia

2016-09-22 17:37 GMT+02:00 Tobias Rose <[hidden email]>:

***** 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. *****

Just quickly:

* optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).

Why median?

Tobias

 

On 9/22/2016 7:22 AM, [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. *****

Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

 

best wishes

 

Andreas