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Cosmic ray particles centenary /// "The Chase" 2012

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George McNamara

Cosmic ray particles centenary /// "The Chase" 2012

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Dear listserv,

In celebration of the centenary of the discovery of cosmic ray particles, the
Optics & Photonics News image of the week at

http://www.osa-opn.org/home/gallery/

is an image I acquired using one of my image core microscopes. Caption is:

Image shows 83.33 hours of cosmic ray particles striking a Hamamatsu ORCA-
ER digital CCD camera. Each of the three color channels are 10,000 exposures,
10 seconds each. Camera background has been removed and gamma adjusted
for better contrast. August 7, 2012, marks the 100th anniversary of the
discovery of cosmic rays.
— George McNamara, Analytical Imaging Core Facility, University of Miami.

Obviously, this will be up for only one week. At some point in the future I will
post the image at one of my web sites ...
http://works.bepress.com/gmcnamara/subject_areas.html

For those of you who are fans of "The Chase" - my panoramic version of the
1950's classic movie by David rogers of a neutrophil chasing bacteria, I posted
an updated version - with the original in an inset at bottom left plus segment
4 now explains more about temporal area maps (TAM) and histograms (Tomasz
Zal published this as 'temporal projections' in his 2012 Nature Immunology
article and mentioned it in his recent Leica live cell imaging webcast). See
http://works.bepress.com/gmcnamara/18/

Michael Abanto

Imaging collagen with circularly polarized light

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Hi,
Does anybody have a good protocol for imaging collagen in embedded tissue, stained with picrosirius red? I had no problem imaging collagen with linear polarization, but I wanted to try circular and found it difficult.
I have an Olympus BX61 and a Nikon A1, as well as demo lambda plates for both. Do I need an analyzer specific for circular polarization? And do I need a linear polarizer before the circular - and how would you set that up on the Olympus?
Any tips or help on the set-up would be appreciated,
Mike

Alberto Diaspro

Re: Imaging collagen with circularly polarized light

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maybe this set-up could help
Diaspro, A., Bertolotto, M., Vergani, L., & Nicolini, C. (1991). Polarized light scattering of nucleosomes and polynucleosomes-in situ and in vitro studies. IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, 38(7), 670–678.

Il giorno 09/ago/2012, alle ore 14:15, Michael Abanto <[hidden email]> ha scritto:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
> Does anybody have a good protocol for imaging collagen in embedded tissue, stained with picrosirius red? I had no problem imaging collagen with linear polarization, but I wanted to try circular and found it difficult.
> I have an Olympus BX61 and a Nikon A1, as well as demo lambda plates for both. Do I need an analyzer specific for circular polarization? And do I need a linear polarizer before the circular - and how would you set that up on the Olympus?
> Any tips or help on the set-up would be appreciated,
> Mike

Guy Cox-2

Re: Imaging collagen with circularly polarized light

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To join, leave or search the confocal microscopy listserv, go to:
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The signal will always be weaker with CP light. But it will no longer be orientation dependent. See Pu Xu, Eleanor Kable, Colin J. R. Sheppard, and Guy Cox, 2010 A quasi-crystal model of collagen microstructure based on SHG microscopy. Chinese Optics Letters 8, 213-216

Do be aware that your quarter wave plate must be quarter wave for your excitation wavelength - a visible light one will not work if you are using SH / 2P excitation. (Lambda/4 at 900 nm = lambda/2 at 450nm). The analyser is less critical.

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alberto Diaspro
Sent: Thursday, 9 August 2012 11:01 PM
To: [hidden email]
Subject: Re: Imaging collagen with circularly polarized light

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

maybe this set-up could help
Diaspro, A., Bertolotto, M., Vergani, L., & Nicolini, C. (1991). Polarized light scattering of nucleosomes and polynucleosomes-in situ and in vitro studies. IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, 38(7), 670-678.

Il giorno 09/ago/2012, alle ore 14:15, Michael Abanto <[hidden email]> ha scritto:

G. Esteban Fernandez

Re: Imaging collagen with circularly polarized light

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What I do for picrosirius red is sandwich the slide between two cir.
pol. consumer camera filters (oriented correctly and rotated to
extinction), no other polarizers or filters in place. I put one
somewhere between the light source and the specimen (e.g. on top of
the condenser) and the other somewhere between the specimen and the
camera/eyepieces (e.g. take out the fluor. filters and put it in
there). That worked on different microscopes (Nikon, Olympus, Zeiss)
with the filters at different points in the light path. It worked
with 10x and below (that's what the user needed) and I remember it not
working with 20x/0.8 but did with 20x/0.4, so maybe this approach
doesn't work at high NA, I don't know enough to tell you. If you try
it, maybe stop down the condenser aperture with high NA lenses?

-Esteban

On Fri, Aug 10, 2012 at 8:32 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
> *****
>
> The signal will always be weaker with CP light. But it will no longer be orientation dependent. See Pu Xu, Eleanor Kable, Colin J. R. Sheppard, and Guy Cox, 2010 A quasi-crystal model of collagen microstructure based on SHG microscopy. Chinese Optics Letters 8, 213-216
>
> Do be aware that your quarter wave plate must be quarter wave for your excitation wavelength - a visible light one will not work if you are using SH / 2P excitation. (Lambda/4 at 900 nm = lambda/2 at 450nm). The analyser is less critical.
>
> Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alberto Diaspro
> Sent: Thursday, 9 August 2012 11:01 PM
> To: [hidden email]
> Subject: Re: Imaging collagen with circularly polarized light
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> maybe this set-up could help
> Diaspro, A., Bertolotto, M., Vergani, L., & Nicolini, C. (1991). Polarized light scattering of nucleosomes and polynucleosomes-in situ and in vitro studies. IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, 38(7), 670-678.
>
>
> Il giorno 09/ago/2012, alle ore 14:15, Michael Abanto <[hidden email]> ha scritto:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi,
>> Does anybody have a good protocol for imaging collagen in embedded tissue, stained with picrosirius red? I had no problem imaging collagen with linear polarization, but I wanted to try circular and found it difficult.
>> I have an Olympus BX61 and a Nikon A1, as well as demo lambda plates for both. Do I need an analyzer specific for circular polarization? And do I need a linear polarizer before the circular - and how would you set that up on the Olympus?
>> Any tips or help on the set-up would be appreciated,
>> Mike

Arvydas Matiukas

simple test to compare confocals

In reply to this post by George McNamara

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*****

Dear List,

I am looking for simple and quick test (and sample) to compare
acquisition across different confocals. Specifically, to our LSM510
there was recently added Nikon C2 , and users want to know how
they compare by few practical parameters, e.g. signal sensitivity,
spectral bleedthrough, bleaching, and maybe resolution .

I am aware of papers by Zucker, Pawley, Cole that describe detail
evaluation of confocal performance, and even recently measured
some PSFs. However, I am looking for just simple and quick
test that would allow direct visual comparison of images (simple
analysis like getting intensity histogram is fine) acquired on different
confocals.

Please share your thoughts and/or experience.

Thank you in advance,
Arvydas
***************************

Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
Department of Pharmacology
SUNY Upstate Medical University
766 Irving Ave., WH 3167
Syracuse, NY 13210
tel.: 315-464-7997
fax: 315-464-8014
email: [hidden email]

John Oreopoulos

Re: simple test to compare confocals (semi-commercial response)

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To join, leave or search the confocal microscopy listserv, go to:
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*****

Arvydas,

This question has been posed to the listserver several times over the years. Head to head comparisons of various instruments, while not impossible, are extremely non-trivial. To be absolutely fair, the tests have to take into account many instrument parameters; for example, read through Pawley's "39 steps":

http://www.google.ca/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CEQQFjAA&url=http%3A%2F%2Flabs.pbrc.edu%2Fcellbiology%2Fdocuments%2F39steps.pdf&ei=01wpUODWIqq36gH484GYCg&usg=AFQjCNHwENzXTObG9pAP5cVHyzUkybIErA

Unfortunately, there are no standard samples for fluorescence microscopy, but as you mentioned, there have been several researchers who have tried to come up with something that works well. I would also recommend taking a look at the following publications:

Zwier, J.M., et al., Image calibration in fluorescence microscopy. Journal of Microscopy-Oxford, 2004. 216: p. 15-24.

Zwier, J.M., et al., Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and sipchart-based calibration procedures. Journal of Microscopy-Oxford, 2008. 231(1): p. 59-69.

Murray, J.M., et al., Evaluating performance in three-dimensional fluorescence microscopy. Journal of Microscopy-Oxford, 2007. 228(3): p. 390-405.

Personally, I am a big fan of Mike Model's "concentrated dye solutions" which, like the thin polymer films described in the publications by Zwier et al., offer a simple means to assess field illumination uniformity, (axial) resolution, and intensity across the visible spectrum (3 diagnostics from one type of sample):

Model, M.A. and J.K. Burkhardt, A standard for calibration and shading correction of a fluorescence microscope. Cytometry, 2001. 44(4): p. 309-316.

Model, M.A. and J.L. Blank, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope. Journal of Microscopy-Oxford, 2008. 229(1): p. 12-16.

These dye solutions are cheap and very easy to make, not requiring any special equipment (something very important for any kind of microscope imaging "standard"). We used these types of solutions a year ago for characterizing the illumination uniformity in spinning disk confocal microscopes. More information can be found on the Spectral Applied Research Website here:

http://www.spectral.ca/_files/file.php?fileid=fileChRCVXJwJl&filename=file_2_Borealis_Uniformity_Protocol.pdf
http://www.youtube.com/watch?v=Fn8Q5AYusOI
http://www.spectral.ca/Downloads/index.html

Your question also brings to mind a quote from another very well-written article on the topic:

"This complex relationship between qualitative and quantitative content of fluorescence images also expresses itself in the way that commercial instruments are assessed. On the one hand it is unavoidable that users initially are strongly influenced by the visual quality of the images presented by the instrument. On the other hand the longer term scientific value of the instrument depends crucially both on the quality of the visual information and the effectiveness with which it can be reduced for quantitative analysis... The increased complexity of a fluorescent image (in terms of the number of dimensions that can be measured), and the variability of sample preparation, makes it less easy to assess quantitatively the quality of the image. This may seem a surprising, or at least somewhat bleak, assessment. But consider this: In the 20 years or more since introduction of laser scanning confocal microscopes it has not been feasible, from published data, to assess how these systems compare (in terms of absolute units associated with measurements). No one can assert with confidence that instrument A in use in 1990 has better or worse sensitivity than instrument B operating in 2006. There is, therefore, a paramount need for standardized test samples and procedures for their use."

-taken from:
Dixon, A., T. Heinlein, and R. Wolleschensky, Need for standardization of fluorescence measurements from the instrument manufacturer's view standardization and quality assurance in fluorescence measurements ii. 2008. 6: p. 3-24.

Cheers,

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2012-08-13, at 3:36 PM, Arvydas Matiukas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear List,
>
> I am looking for simple and quick test (and sample) to compare
> acquisition across different confocals. Specifically, to our LSM510
> there was recently added Nikon C2 , and users want to know how
> they compare by few practical parameters, e.g. signal sensitivity,
> spectral bleedthrough, bleaching, and maybe resolution .
>
> I am aware of papers by Zucker, Pawley, Cole that describe detail
> evaluation of confocal performance, and even recently measured
> some PSFs. However, I am looking for just simple and quick
> test that would allow direct visual comparison of images (simple
> analysis like getting intensity histogram is fine) acquired on different
> confocals.
>
> Please share your thoughts and/or experience.
>
> Thank you in advance,
> Arvydas
> ***************************
>
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Core
> Department of Pharmacology
> SUNY Upstate Medical University
> 766 Irving Ave., WH 3167
> Syracuse, NY 13210
> tel.: 315-464-7997
> fax: 315-464-8014
> email: [hidden email]

Kilgore, Jason-2

Re: simple test to compare confocals (semi-commercial response)

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

** semi-commercial response here, too **

Those are some great suggestions, particularly Pawley's guide.

I would just add that, in my own experience, using a solution of dye under the coverslip can still be problematic due to non-uniform volume under the coverslip across its width (due to curvature of the coverslip or slight angle).

Photobleaching can be a serious issue, too.

Fluorescent beads are generally more photostable, though they can still differ a bit from bead-to-bead in intensity.

The best I've found are the plastic "Fluor-Ref" slides, which are extremely photostable and uniform (though they are sometimes too bright or too thick).

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division

T 1 800 955 6288 then option 4, then option 6, or 541 335 0353 . F 541 335 0238
29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
www.invitrogen.com/technicalsupport

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Monday, August 13, 2012 1:59 PM
To: [hidden email]
Subject: Re: simple test to compare confocals (semi-commercial response)

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Arvydas,

This question has been posed to the listserver several times over the years. Head to head comparisons of various instruments, while not impossible, are extremely non-trivial. To be absolutely fair, the tests have to take into account many instrument parameters; for example, read through Pawley's "39 steps":

http://www.google.ca/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CEQQFjAA&url=http%3A%2F%2Flabs.pbrc.edu%2Fcellbiology%2Fdocuments%2F39steps.pdf&ei=01wpUODWIqq36gH484GYCg&usg=AFQjCNHwENzXTObG9pAP5cVHyzUkybIErA

Unfortunately, there are no standard samples for fluorescence microscopy, but as you mentioned, there have been several researchers who have tried to come up with something that works well. I would also recommend taking a look at the following publications:

Zwier, J.M., et al., Image calibration in fluorescence microscopy. Journal of Microscopy-Oxford, 2004. 216: p. 15-24.

Zwier, J.M., et al., Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and sipchart-based calibration procedures. Journal of Microscopy-Oxford, 2008. 231(1): p. 59-69.

Murray, J.M., et al., Evaluating performance in three-dimensional fluorescence microscopy. Journal of Microscopy-Oxford, 2007. 228(3): p. 390-405.

Personally, I am a big fan of Mike Model's "concentrated dye solutions" which, like the thin polymer films described in the publications by Zwier et al., offer a simple means to assess field illumination uniformity, (axial) resolution, and intensity across the visible spectrum (3 diagnostics from one type of sample):

Model, M.A. and J.K. Burkhardt, A standard for calibration and shading correction of a fluorescence microscope. Cytometry, 2001. 44(4): p. 309-316.

Model, M.A. and J.L. Blank, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope. Journal of Microscopy-Oxford, 2008. 229(1): p. 12-16.

These dye solutions are cheap and very easy to make, not requiring any special equipment (something very important for any kind of microscope imaging "standard"). We used these types of solutions a year ago for characterizing the illumination uniformity in spinning disk confocal microscopes. More information can be found on the Spectral Applied Research Website here:

http://www.spectral.ca/_files/file.php?fileid=fileChRCVXJwJl&filename=file_2_Borealis_Uniformity_Protocol.pdf
http://www.youtube.com/watch?v=Fn8Q5AYusOI
http://www.spectral.ca/Downloads/index.html

Your question also brings to mind a quote from another very well-written article on the topic:

"This complex relationship between qualitative and quantitative content of fluorescence images also expresses itself in the way that commercial instruments are assessed. On the one hand it is unavoidable that users initially are strongly influenced by the visual quality of the images presented by the instrument. On the other hand the longer term scientific value of the instrument depends crucially both on the quality of the visual information and the effectiveness with which it can be reduced for quantitative analysis... The increased complexity of a fluorescent image (in terms of the number of dimensions that can be measured), and the variability of sample preparation, makes it less easy to assess quantitatively the quality of the image. This may seem a surprising, or at least somewhat bleak, assessment. But consider this: In the 20 years or more since introduction of laser scanning confocal microscopes it has not been feasible, from published data, to assess how these systems compare (in terms of absolute units associated with measurements). No one can assert with confidence that instrument A in use in 1990 has better or worse sensitivity than instrument B operating in 2006. There is, therefore, a paramount need for standardized test samples and procedures for their use."

-taken from:
Dixon, A., T. Heinlein, and R. Wolleschensky, Need for standardization of fluorescence measurements from the instrument manufacturer's view standardization and quality assurance in fluorescence measurements ii. 2008. 6: p. 3-24.

Cheers,

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2012-08-13, at 3:36 PM, Arvydas Matiukas wrote:

John Oreopoulos

Re: simple test to compare confocals (semi-commercial response)

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Jason,

Mike Model's dye solutions are highly concentrated and are actually very difficult to photobleach. Any dye that does get photobleached is rapidly replaced by dye that diffuses into the coverslip-solution interface region. Because of their very high optical density, they only emit fluorescence from an extremely thin region adjacent to the coverslip (thinner than the axial diffraction limit associated with your microscope optics). Therefore, the actual thickness of the solution sandwiched between the coverslip and the slide does not matter. This is what distinguishes them from a regular dilute solution of dye on a slide. Mike Model is on this listserver, so perhaps he can comment more on his experiences with them.

Having said all that, I'm also a fan of all the Molecular Probes microscope test slides with different sized multi-color fluorescent beads (no commercial interest, just a happy customer).

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2012-08-13, at 5:54 PM, Kilgore, Jason wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> ** semi-commercial response here, too **
>
> Those are some great suggestions, particularly Pawley's guide.
>
> I would just add that, in my own experience, using a solution of dye under the coverslip can still be problematic due to non-uniform volume under the coverslip across its width (due to curvature of the coverslip or slight angle).
>
> Photobleaching can be a serious issue, too.
>
> Fluorescent beads are generally more photostable, though they can still differ a bit from bead-to-bead in intensity.
>
> The best I've found are the plastic "Fluor-Ref" slides, which are extremely photostable and uniform (though they are sometimes too bright or too thick).
>
> Jason
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes Labeling and Detection Technologies
> Cells Systems Division
>
> T 1 800 955 6288 then option 4, then option 6, or 541 335 0353 . F 541 335 0238
> 29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
> www.invitrogen.com/technicalsupport
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
> Sent: Monday, August 13, 2012 1:59 PM
> To: [hidden email]
> Subject: Re: simple test to compare confocals (semi-commercial response)
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Arvydas,
>
> This question has been posed to the listserver several times over the years. Head to head comparisons of various instruments, while not impossible, are extremely non-trivial. To be absolutely fair, the tests have to take into account many instrument parameters; for example, read through Pawley's "39 steps":
>
> http://www.google.ca/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CEQQFjAA&url=http%3A%2F%2Flabs.pbrc.edu%2Fcellbiology%2Fdocuments%2F39steps.pdf&ei=01wpUODWIqq36gH484GYCg&usg=AFQjCNHwENzXTObG9pAP5cVHyzUkybIErA
>
> Unfortunately, there are no standard samples for fluorescence microscopy, but as you mentioned, there have been several researchers who have tried to come up with something that works well. I would also recommend taking a look at the following publications:
>
> Zwier, J.M., et al., Image calibration in fluorescence microscopy. Journal of Microscopy-Oxford, 2004. 216: p. 15-24.
>
> Zwier, J.M., et al., Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and sipchart-based calibration procedures. Journal of Microscopy-Oxford, 2008. 231(1): p. 59-69.
>
> Murray, J.M., et al., Evaluating performance in three-dimensional fluorescence microscopy. Journal of Microscopy-Oxford, 2007. 228(3): p. 390-405.
>
>
> Personally, I am a big fan of Mike Model's "concentrated dye solutions" which, like the thin polymer films described in the publications by Zwier et al., offer a simple means to assess field illumination uniformity, (axial) resolution, and intensity across the visible spectrum (3 diagnostics from one type of sample):
>
> Model, M.A. and J.K. Burkhardt, A standard for calibration and shading correction of a fluorescence microscope. Cytometry, 2001. 44(4): p. 309-316.
>
> Model, M.A. and J.L. Blank, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope. Journal of Microscopy-Oxford, 2008. 229(1): p. 12-16.
>
> These dye solutions are cheap and very easy to make, not requiring any special equipment (something very important for any kind of microscope imaging "standard"). We used these types of solutions a year ago for characterizing the illumination uniformity in spinning disk confocal microscopes. More information can be found on the Spectral Applied Research Website here:
>
> http://www.spectral.ca/_files/file.php?fileid=fileChRCVXJwJl&filename=file_2_Borealis_Uniformity_Protocol.pdf
> http://www.youtube.com/watch?v=Fn8Q5AYusOI
> http://www.spectral.ca/Downloads/index.html
>
> Your question also brings to mind a quote from another very well-written article on the topic:
>
> "This complex relationship between qualitative and quantitative content of fluorescence images also expresses itself in the way that commercial instruments are assessed. On the one hand it is unavoidable that users initially are strongly influenced by the visual quality of the images presented by the instrument. On the other hand the longer term scientific value of the instrument depends crucially both on the quality of the visual information and the effectiveness with which it can be reduced for quantitative analysis... The increased complexity of a fluorescent image (in terms of the number of dimensions that can be measured), and the variability of sample preparation, makes it less easy to assess quantitatively the quality of the image. This may seem a surprising, or at least somewhat bleak, assessment. But consider this: In the 20 years or more since introduction of laser scanning confocal microscopes it has not been feasible, from published data, to assess how these systems compare (in terms of absolute units associated with measurements). No one can assert with confidence that instrument A in use in 1990 has better or worse sensitivity than instrument B operating in 2006. There is, therefore, a paramount need for standardized test samples and procedures for their use."
>
> -taken from:
> Dixon, A., T. Heinlein, and R. Wolleschensky, Need for standardization of fluorescence measurements from the instrument manufacturer's view standardization and quality assurance in fluorescence measurements ii. 2008. 6: p. 3-24.
>
> Cheers,
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
>
> On 2012-08-13, at 3:36 PM, Arvydas Matiukas wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear List,
>>
>> I am looking for simple and quick test (and sample) to compare
>> acquisition across different confocals. Specifically, to our LSM510
>> there was recently added Nikon C2 , and users want to know how
>> they compare by few practical parameters, e.g. signal sensitivity,
>> spectral bleedthrough, bleaching, and maybe resolution .
>>
>> I am aware of papers by Zucker, Pawley, Cole that describe detail
>> evaluation of confocal performance, and even recently measured
>> some PSFs. However, I am looking for just simple and quick
>> test that would allow direct visual comparison of images (simple
>> analysis like getting intensity histogram is fine) acquired on different
>> confocals.
>>
>> Please share your thoughts and/or experience.
>>
>> Thank you in advance,
>> Arvydas
>> ***************************
>>
>>
>> Arvydas Matiukas, Ph.D.
>> Director of Confocal&Two-Photon Core
>> Department of Pharmacology
>> SUNY Upstate Medical University
>> 766 Irving Ave., WH 3167
>> Syracuse, NY 13210
>> tel.: 315-464-7997
>> fax: 315-464-8014
>> email: [hidden email]

Vitaly Boyko

Re: simple test to compare confocals (semi-commercial response)

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi John,

I have one comment - why not integrating Borealis vision to both Pro and consumer grade digital photography - much broader market, isn't it? Is any any reason why it is limited to correction of artefatcs caused by CSU unit?

Cheers,

Vitaly
301-515-7833

________________________________
From: John Oreopoulos <[hidden email]>
To: [hidden email]
Sent: Monday, August 13, 2012 6:04 PM
Subject: Re: simple test to compare confocals (semi-commercial response)

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Jason,

Mike Model's dye solutions are highly concentrated and are actually very difficult to photobleach. Any dye that does get photobleached is rapidly replaced by dye that diffuses into the coverslip-solution interface region. Because of their very high optical density, they only emit fluorescence from an extremely thin region adjacent to the coverslip (thinner than the axial diffraction limit associated with your microscope optics). Therefore, the actual thickness of the solution sandwiched between the coverslip and the slide does not matter. This is what distinguishes them from a regular dilute solution of dye on a slide. Mike Model is on this listserver, so perhaps he can comment more on his experiences with them.

Having said all that, I'm also a fan of all the Molecular Probes microscope test slides with different sized multi-color fluorescent beads (no commercial interest, just a happy customer).

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2012-08-13, at 5:54 PM, Kilgore, Jason wrote:

to assess how these systems compare (in terms of absolute units associated with measurements). No one can assert with confidence that instrument A in use in 1990 has better or worse sensitivity than instrument B operating in 2006. There is, therefore, a paramount need for standardized test samples and procedures for their use."

>
> -taken from:
> Dixon, A., T. Heinlein, and R. Wolleschensky, Need for standardization of fluorescence measurements from the instrument manufacturer's view standardization and quality assurance in fluorescence measurements ii. 2008. 6: p. 3-24.
>
> Cheers,
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
>
> On 2012-08-13, at 3:36 PM, Arvydas Matiukas wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear List,
>>
>> I am looking for simple and quick test (and sample) to compare
>> acquisition across different confocals. Specifically, to our LSM510
>> there was recently added Nikon C2 , and users want to know how
>> they compare by few practical parameters, e.g. signal sensitivity,
>> spectral bleedthrough, bleaching, and maybe resolution .
>>
>> I am aware of papers by Zucker, Pawley, Cole that describe detail
>> evaluation of confocal performance, and even recently measured
>> some PSFs. However, I am looking for just simple and quick
>> test that would allow direct visual comparison of images (simple
>> analysis like getting intensity histogram is fine) acquired on different
>> confocals.
>>
>> Please share your thoughts and/or experience.
>>
>> Thank you in advance,
>> Arvydas
>> ***************************
>>
>>
>> Arvydas Matiukas, Ph.D.
>> Director of Confocal&Two-Photon Core
>> Department of Pharmacology
>> SUNY Upstate Medical University
>> 766 Irving Ave., WH 3167
>> Syracuse, NY 13210
>> tel.: 315-464-7997
>> fax: 315-464-8014
>> email: [hidden email]

George McNamara

Re: simple test to compare confocals

In reply to this post by Arvydas Matiukas

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Convallaria (lily of the valley) cross section slide - simplest to
borrow from your local Zeiss sales rep or service engineer. Basically,
if an Zeiss LSM or Leica SP# gives a decent image, it passes as far as
a Zeiss or Leica service engineer is concerned (unless you are looking
over their shoulder or better driving the scope and see any issues).
I've not seen Nikon service work on a confocal, so this test might be
confocal vendor standard. If you do not have a slide already; bummer
(might be some lily growing outside that you could harvest). The
www.carolina.com web site search engine is so incredibly bad, I was
unable to get a hit just now (amazon.com was no better). You can
probably find it from other prepared slide companies on the Internet.

Practical parameters:

gain 600 (not that all vendors "600' are the same, or even two PMTs in
the same scanhead).
offset so that all pixel values are above zero (i.e. no laser light)
12-bit data mode
low laser power (ND filter or AOTF control level ... need to run the
Argon laser in the "good"power range)
NO AVERAGING (averaging is cheating)
Fastest scan speed available [shortest pixel dwell time] (ideally these
will be identical on both instruments) ... my thanks to Jonathan Boyd of
Leica for demonstrating that "faster is better" (SP5, standard scan
speeds vs resonant scanner, sum images when needed to get same total
dwell time for each mode).
Same size image format for all instruments, i.e. 2048x2048 pixels
highest performance objective lens available, i.e. plan apo 63x/1.4 NA
oil, full resolution - by my math 60 nm pixel size for 1.4 NA
(explanation in previous messages at the listserv).

Note: Zeiss and Nikon oil do not play well together. Need to remove the
oil from the coverglass, clean the coverglass with 70% ethanol, before
oiling for the other scope.

George
p.s. I have not acquired any of my Convallaria slides for Sebastian
Munck's et al PiMP super-resolution calculation method (J Cell Sci 2012,
PubMed 22357945), but am looking forward to doing so in the future. I
have been getting very nice results with 30 nm pixel size on both our
LSM710 and Leica SP5 with 63x/1.4NA, relatively bright initially
specimens, filter size 1.625 (instead of default of 1.1 which is for
somewhat larger pixel size), 16-bit output (so I don't have to remember
where the 32-bit to 16-bit command is located), ~1% photobleaching per
time point. I find it most useful to have one channel time series per
LIF or LSM file. My thanks to Sebastian and Glen M for sending me the
ImageJ plugin.

On 8/13/2012 3:36 PM, Arvydas Matiukas wrote:

Jeremy Adler-4

Re: simple test to compare confocals

In reply to this post by Arvydas Matiukas

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Comparisons between instruments are difficult and tend of focus on
resolution but one area that tends to be overlooked is noise.

A simple noise correction/measurement that we are pushing to
accurately measure colocalization is to compare two images of the same
specimen - the difference is the noise. This can be visualized either
by subtraction or by plotting the intensities of pixels in the two
images against each other - a standard scattergram. The scattergram
makes the level of noise blindingly obvious and the method could
easily be implemented during acquisition, when users have the chance
to do something about it, but currently it is not available in any
commercial software.

Noise clearly depends on acquisition time but ultimately is limited by
photobleaching - a compeletly bleached image has had all the available
information extracted. So a method of comparing microscopes is to
measure the noise for a similar amount of photobleaching.

Quoting Arvydas Matiukas <[hidden email]>:

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

mmodel

Re: simple test to compare confocals (semi-commercial response)

In reply to this post by John Oreopoulos

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Thanks, John, for advocating for concentrated dyes :) I thnk they are also convenient because give you both axial resolution and brightness, as their brightness is reproducible and you can choose between brightly fluorescent solutions (such as rose bengal in DMSO) and less bright ones. But of course the quality of a confocal microscope cannot be reduced to a single parameter. I would guess that for the majority of users the decisive factors would be the ease of use (assuming they operate the microscopes themselves) and the ability to image dim staining. Perhaps you could take a few slides of rather poor quality and see what you get with comparable objectives.

Mike Model

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of John Oreopoulos [[hidden email]]
Sent: Monday, August 13, 2012 6:04 PM
To: [hidden email]
Subject: Re: simple test to compare confocals (semi-commercial response)

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Jason,

Mike Model's dye solutions are highly concentrated and are actually very difficult to photobleach. Any dye that does get photobleached is rapidly replaced by dye that diffuses into the coverslip-solution interface region. Because of their very high optical density, they only emit fluorescence from an extremely thin region adjacent to the coverslip (thinner than the axial diffraction limit associated with your microscope optics). Therefore, the actual thickness of the solution sandwiched between the coverslip and the slide does not matter. This is what distinguishes them from a regular dilute solution of dye on a slide. Mike Model is on this listserver, so perhaps he can comment more on his experiences with them.

Having said all that, I'm also a fan of all the Molecular Probes microscope test slides with different sized multi-color fluorescent beads (no commercial interest, just a happy customer).

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2012-08-13, at 5:54 PM, Kilgore, Jason wrote:

Arvydas Matiukas

Re: simple test to compare confocals

In reply to this post by George McNamara

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear list and George,

Thanks for all the suggestions regarding simple confocal test.
I meant a test that allows to quickly verify that confocal
is functioning normally and stable. One of typical situations
is when I need to prove to a customer that confocal is OK,
and it is the bad sample that produces poor image.
I agree that in detail testing/evaluation mentioned in some replies is useful and
interesting to perform for a new system or after a
major overhaul. I would not be very enthusiastic to do it often (e.g. weekly).

My special thanks to George whose Convallaria slide based simple test
is the best aligned with my own line of thought (which I did not present initially
to avoid any bias and get fresh ideas). I have been routinely doing similar test
on our LSM510 to verify normal and stable performance (green/red fluorescence
at FITC and Texas Red settings).
Now based on George's experience I will use the test to quickly compare
confocals. I 100% agree that it is very important to keep identical imaging
parameters. However, one of caveats may be different emission filters (in terms
of bandwidth and transmission).
I assume this simple and quick test while not being a substitute to
a strict/detail comparison answers the concerns of the Core user who has
to switch between confocals.

Best wishes,
Arvydas

>>> George McNamara <[hidden email]> 8/13/2012 9:36 PM >>>
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Convallaria (lily of the valley) cross section slide - simplest to
borrow from your local Zeiss sales rep or service engineer. Basically,
if an Zeiss LSM or Leica SP# gives a decent image, it passes as far as
a Zeiss or Leica service engineer is concerned (unless you are looking
over their shoulder or better driving the scope and see any issues).
I've not seen Nikon service work on a confocal, so this test might be
confocal vendor standard. If you do not have a slide already; bummer
(might be some lily growing outside that you could harvest). The
www.carolina.com web site search engine is so incredibly bad, I was
unable to get a hit just now (amazon.com was no better). You can
probably find it from other prepared slide companies on the Internet.

Practical parameters:

gain 600 (not that all vendors "600' are the same, or even two PMTs in
the same scanhead).
offset so that all pixel values are above zero (i.e. no laser light)
12-bit data mode
low laser power (ND filter or AOTF control level ... need to run the
Argon laser in the "good"power range)
NO AVERAGING (averaging is cheating)
Fastest scan speed available [shortest pixel dwell time] (ideally these
will be identical on both instruments) ... my thanks to Jonathan Boyd of
Leica for demonstrating that "faster is better" (SP5, standard scan
speeds vs resonant scanner, sum images when needed to get same total
dwell time for each mode).
Same size image format for all instruments, i.e. 2048x2048 pixels
highest performance objective lens available, i.e. plan apo 63x/1.4 NA
oil, full resolution - by my math 60 nm pixel size for 1.4 NA
(explanation in previous messages at the listserv).

Note: Zeiss and Nikon oil do not play well together. Need to remove the
oil from the coverglass, clean the coverglass with 70% ethanol, before
oiling for the other scope.

George
p.s. I have not acquired any of my Convallaria slides for Sebastian
Munck's et al PiMP super-resolution calculation method (J Cell Sci 2012,
PubMed 22357945), but am looking forward to doing so in the future. I
have been getting very nice results with 30 nm pixel size on both our
LSM710 and Leica SP5 with 63x/1.4NA, relatively bright initially
specimens, filter size 1.625 (instead of default of 1.1 which is for
somewhat larger pixel size), 16-bit output (so I don't have to remember
where the 32-bit to 16-bit command is located), ~1% photobleaching per
time point. I find it most useful to have one channel time series per
LIF or LSM file. My thanks to Sebastian and Glen M for sending me the
ImageJ plugin.

On 8/13/2012 3:36 PM, Arvydas Matiukas wrote:

George McNamara

Re: simple test to compare confocals

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Arvydas,

You're welcome. To test instrument stability, run the Convallaria
overnight or over the weekend with the most important laser lines (hint:
all are important, but Zeiss and possibly other some other vendors
software only lets you go to four tracks ... I've done 20 scan tracks on
the Leica SP5 - let me check different AOTF power settings). To avoid
photobleaching the Convallaria slide (too much), use low laser power. My
main test (explained in archived posts) is to send to transmitted light
detector, no averaging, same low gain, condenser field aperture shrunk
down to be in the field and adjusted to be in focus. Use an edge of the
Convallaria slide so you have some blank area. If you are (un)lucky the
building temperature will change during the test (I don't have access to
an USB thermometer data recorder - would not be surprised if hidden in
the instrument logs are occasional temperature readings from inside the
laser enclosures).

If any of the laser lines do fluctuate, don't assume it is the laser
itself (or themselves) - Bob Zucker has suggested the AOTF(s) could be
temperamental by way of temperature fluctuations. Of course at very low
laser output (AOTF at <5%, certainly at 0.5%) there could be unavoidable
fluctuations.

Enjoy,

George

On 8/14/2012 4:23 PM, Arvydas Matiukas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list and George,
>
> Thanks for all the suggestions regarding simple confocal test.
> I meant a test that allows to quickly verify that confocal
> is functioning normally and stable. One of typical situations
> is when I need to prove to a customer that confocal is OK,
> and it is the bad sample that produces poor image.
> I agree that in detail testing/evaluation mentioned in some replies is useful and
> interesting to perform for a new system or after a
> major overhaul. I would not be very enthusiastic to do it often (e.g. weekly).
>
> My special thanks to George whose Convallaria slide based simple test
> is the best aligned with my own line of thought (which I did not present initially
> to avoid any bias and get fresh ideas). I have been routinely doing similar test
> on our LSM510 to verify normal and stable performance (green/red fluorescence
> at FITC and Texas Red settings).
> Now based on George's experience I will use the test to quickly compare
> confocals. I 100% agree that it is very important to keep identical imaging
> parameters. However, one of caveats may be different emission filters (in terms
> of bandwidth and transmission).
> I assume this simple and quick test while not being a substitute to
> a strict/detail comparison answers the concerns of the Core user who has
> to switch between confocals.
>
> Best wishes,
> Arvydas
>
>
>
>
>>>> George McNamara<[hidden email]> 8/13/2012 9:36 PM>>>
>>>>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Convallaria (lily of the valley) cross section slide - simplest to
> borrow from your local Zeiss sales rep or service engineer. Basically,
> if an Zeiss LSM or Leica SP# gives a decent image, it passes as far as
> a Zeiss or Leica service engineer is concerned (unless you are looking
> over their shoulder or better driving the scope and see any issues).
> I've not seen Nikon service work on a confocal, so this test might be
> confocal vendor standard. If you do not have a slide already; bummer
> (might be some lily growing outside that you could harvest). The
> www.carolina.com web site search engine is so incredibly bad, I was
> unable to get a hit just now (amazon.com was no better). You can
> probably find it from other prepared slide companies on the Internet.
>
> Practical parameters:
>
> gain 600 (not that all vendors "600' are the same, or even two PMTs in
> the same scanhead).
> offset so that all pixel values are above zero (i.e. no laser light)
> 12-bit data mode
> low laser power (ND filter or AOTF control level ... need to run the
> Argon laser in the "good"power range)
> NO AVERAGING (averaging is cheating)
> Fastest scan speed available [shortest pixel dwell time] (ideally these
> will be identical on both instruments) ... my thanks to Jonathan Boyd of
> Leica for demonstrating that "faster is better" (SP5, standard scan
> speeds vs resonant scanner, sum images when needed to get same total
> dwell time for each mode).
> Same size image format for all instruments, i.e. 2048x2048 pixels
> highest performance objective lens available, i.e. plan apo 63x/1.4 NA
> oil, full resolution - by my math 60 nm pixel size for 1.4 NA
> (explanation in previous messages at the listserv).
>
> Note: Zeiss and Nikon oil do not play well together. Need to remove the
> oil from the coverglass, clean the coverglass with 70% ethanol, before
> oiling for the other scope.
>
> George
> p.s. I have not acquired any of my Convallaria slides for Sebastian
> Munck's et al PiMP super-resolution calculation method (J Cell Sci 2012,
> PubMed 22357945), but am looking forward to doing so in the future. I
> have been getting very nice results with 30 nm pixel size on both our
> LSM710 and Leica SP5 with 63x/1.4NA, relatively bright initially
> specimens, filter size 1.625 (instead of default of 1.1 which is for
> somewhat larger pixel size), 16-bit output (so I don't have to remember
> where the 32-bit to 16-bit command is located), ~1% photobleaching per
> time point. I find it most useful to have one channel time series per
> LIF or LSM file. My thanks to Sebastian and Glen M for sending me the
> ImageJ plugin.
>
> On 8/13/2012 3:36 PM, Arvydas Matiukas wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear List,
>>
>> I am looking for simple and quick test (and sample) to compare
>> acquisition across different confocals. Specifically, to our LSM510
>> there was recently added Nikon C2 , and users want to know how
>> they compare by few practical parameters, e.g. signal sensitivity,
>> spectral bleedthrough, bleaching, and maybe resolution .
>>
>> I am aware of papers by Zucker, Pawley, Cole that describe detail
>> evaluation of confocal performance, and even recently measured
>> some PSFs. However, I am looking for just simple and quick
>> test that would allow direct visual comparison of images (simple
>> analysis like getting intensity histogram is fine) acquired on different
>> confocals.
>>
>> Please share your thoughts and/or experience.
>>
>> Thank you in advance,
>> Arvydas
>> ***************************
>>
>>
>> Arvydas Matiukas, Ph.D.
>> Director of Confocal&Two-Photon Core
>> Department of Pharmacology
>> SUNY Upstate Medical University
>> 766 Irving Ave., WH 3167
>> Syracuse, NY 13210
>> tel.: 315-464-7997
>> fax: 315-464-8014
>> email: [hidden email]
>>
>>
>>
>

Peter Gabriel Pitrone

Re: simple test to compare confocals

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hello folks,

I don't know how much it will help, but you can look through my TechRMS
thesis on a similar topic: http://goo.gl/qmf2O

Have fun!!
Pete

On Wed, August 15, 2012 2:39 am, George McNamara wrote:
| *****
| To join, leave or search the confocal microscopy listserv, go to:
| http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
| *****
|
| Hi Arvydas,
|
| You're welcome. To test instrument stability, run the Convallaria
| overnight or over the weekend with the most important laser lines (hint:
| all are important, but Zeiss and possibly other some other vendors
| software only lets you go to four tracks ... I've done 20 scan tracks on
| the Leica SP5 - let me check different AOTF power settings). To avoid
| photobleaching the Convallaria slide (too much), use low laser power. My
| main test (explained in archived posts) is to send to transmitted light
| detector, no averaging, same low gain, condenser field aperture shrunk
| down to be in the field and adjusted to be in focus. Use an edge of the
| Convallaria slide so you have some blank area. If you are (un)lucky the
| building temperature will change during the test (I don't have access to
| an USB thermometer data recorder - would not be surprised if hidden in
| the instrument logs are occasional temperature readings from inside the
| laser enclosures).
|
| If any of the laser lines do fluctuate, don't assume it is the laser
| itself (or themselves) - Bob Zucker has suggested the AOTF(s) could be
| temperamental by way of temperature fluctuations. Of course at very low
| laser output (AOTF at <5%, certainly at 0.5%) there could be unavoidable
| fluctuations.
|
|
| Enjoy,
|
| George
|
|
|
| On 8/14/2012 4:23 PM, Arvydas Matiukas wrote:
|> *****
|> To join, leave or search the confocal microscopy listserv, go to:
|> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
|> *****
|>
|> Dear list and George,
|>
|> Thanks for all the suggestions regarding simple confocal test.
|> I meant a test that allows to quickly verify that confocal
|> is functioning normally and stable. One of typical situations
|> is when I need to prove to a customer that confocal is OK,
|> and it is the bad sample that produces poor image.
|> I agree that in detail testing/evaluation mentioned in some replies is
|> useful and
|> interesting to perform for a new system or after a
|> major overhaul. I would not be very enthusiastic to do it often (e.g.
|> weekly).
|>
|> My special thanks to George whose Convallaria slide based simple test
|> is the best aligned with my own line of thought (which I did not present
|> initially
|> to avoid any bias and get fresh ideas). I have been routinely doing
|> similar test
|> on our LSM510 to verify normal and stable performance (green/red
|> fluorescence
|> at FITC and Texas Red settings).
|> Now based on George's experience I will use the test to quickly compare
|> confocals. I 100% agree that it is very important to keep identical
|> imaging
|> parameters. However, one of caveats may be different emission filters
|> (in terms
|> of bandwidth and transmission).
|> I assume this simple and quick test while not being a substitute to
|> a strict/detail comparison answers the concerns of the Core user who
|> has
|> to switch between confocals.
|>
|> Best wishes,
|> Arvydas
|>
|>
|>
|>
|>>>> George McNamara<[hidden email]> 8/13/2012 9:36 PM>>>
|>>>>
|> *****
|> To join, leave or search the confocal microscopy listserv, go to:
|> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
|> *****
|>
|> Convallaria (lily of the valley) cross section slide - simplest to
|> borrow from your local Zeiss sales rep or service engineer. Basically,
|> if an Zeiss LSM or Leica SP# gives a decent image, it passes as far as
|> a Zeiss or Leica service engineer is concerned (unless you are looking
|> over their shoulder or better driving the scope and see any issues).
|> I've not seen Nikon service work on a confocal, so this test might be
|> confocal vendor standard. If you do not have a slide already; bummer
|> (might be some lily growing outside that you could harvest). The
|> www.carolina.com web site search engine is so incredibly bad, I was
|> unable to get a hit just now (amazon.com was no better). You can
|> probably find it from other prepared slide companies on the Internet.
|>
|> Practical parameters:
|>
|> gain 600 (not that all vendors "600' are the same, or even two PMTs in
|> the same scanhead).
|> offset so that all pixel values are above zero (i.e. no laser light)
|> 12-bit data mode
|> low laser power (ND filter or AOTF control level ... need to run the
|> Argon laser in the "good"power range)
|> NO AVERAGING (averaging is cheating)
|> Fastest scan speed available [shortest pixel dwell time] (ideally these
|> will be identical on both instruments) ... my thanks to Jonathan Boyd of
|> Leica for demonstrating that "faster is better" (SP5, standard scan
|> speeds vs resonant scanner, sum images when needed to get same total
|> dwell time for each mode).
|> Same size image format for all instruments, i.e. 2048x2048 pixels
|> highest performance objective lens available, i.e. plan apo 63x/1.4 NA
|> oil, full resolution - by my math 60 nm pixel size for 1.4 NA
|> (explanation in previous messages at the listserv).
|>
|> Note: Zeiss and Nikon oil do not play well together. Need to remove the
|> oil from the coverglass, clean the coverglass with 70% ethanol, before
|> oiling for the other scope.
|>
|> George
|> p.s. I have not acquired any of my Convallaria slides for Sebastian
|> Munck's et al PiMP super-resolution calculation method (J Cell Sci 2012,
|> PubMed 22357945), but am looking forward to doing so in the future. I
|> have been getting very nice results with 30 nm pixel size on both our
|> LSM710 and Leica SP5 with 63x/1.4NA, relatively bright initially
|> specimens, filter size 1.625 (instead of default of 1.1 which is for
|> somewhat larger pixel size), 16-bit output (so I don't have to remember
|> where the 32-bit to 16-bit command is located), ~1% photobleaching per
|> time point. I find it most useful to have one channel time series per
|> LIF or LSM file. My thanks to Sebastian and Glen M for sending me the
|> ImageJ plugin.
|>
|> On 8/13/2012 3:36 PM, Arvydas Matiukas wrote:
|>
|>> *****
|>> To join, leave or search the confocal microscopy listserv, go to:
|>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
|>> *****
|>>
|>> Dear List,
|>>
|>> I am looking for simple and quick test (and sample) to compare
|>> acquisition across different confocals. Specifically, to our LSM510
|>> there was recently added Nikon C2 , and users want to know how
|>> they compare by few practical parameters, e.g. signal sensitivity,
|>> spectral bleedthrough, bleaching, and maybe resolution .
|>>
|>> I am aware of papers by Zucker, Pawley, Cole that describe detail
|>> evaluation of confocal performance, and even recently measured
|>> some PSFs. However, I am looking for just simple and quick
|>> test that would allow direct visual comparison of images (simple
|>> analysis like getting intensity histogram is fine) acquired on
|>> different
|>> confocals.
|>>
|>> Please share your thoughts and/or experience.
|>>
|>> Thank you in advance,
|>> Arvydas
|>> ***************************
|>>
|>>
|>> Arvydas Matiukas, Ph.D.
|>> Director of Confocal&Two-Photon Core
|>> Department of Pharmacology
|>> SUNY Upstate Medical University
|>> 766 Irving Ave., WH 3167
|>> Syracuse, NY 13210
|>> tel.: 315-464-7997
|>> fax: 315-464-8014
|>> email: [hidden email]
|>>
|>>
|>>
|>
|

--
Peter Gabriel Pitrone - TechRMS
Microscopy/Imaging Specialist
Prof. Dr. Pavel Tomancak group
Max Planck Institute for
Molecular Biology and Genetics
Pfotenhauerstr. 108
01307 Dresden

"If a straight line fit is required, obtain only two data points." - Anon.

Emmanuel Gustin

Re: simple test to compare confocals

In reply to this post by Arvydas Matiukas

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Hello,

It is not exactly the same problem, but to track performance of our
confocal over time, I used to try to keep the characteristics of the
resulting image stable, instead of the settings. The images were from
the fluorescent bead slides that are sold by Invitrogen, and we always
adjusted settings so that (with standardized choice of objectives,
pinhole, laser power, scan speed and zoom factors) the image spanned the
range of the A/D convertor. Then I wrote down the PMT settings to see
the evolution of the system over time. The results were reasonably
consistent and indicative of the state of the instrument.

Across different systems you cannot directly compare the numbers, but
you can get an impression of where the settings are in the "usable
range" of PMT and laser power settings for a particular system.

We also use their reference slide that contains muntjac skin fibroblasts
with 3-color staining. A convallaria slide is useful because it is a
fairly thick, bright and stable sample that is very easy to image. The
fibroblast slide takes a bit more time to bring into focus and adjust
(which perhaps gives a more relevant impression of a system's
user-friendliness), but its F-actin staining usually gives a better idea
of a system's resolution than convallaria. It does bleach easier than
convallaria.

Best Regards,

Emmanuel

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Arvydas Matiukas
Sent: Tuesday, 14 August, 2012 22:24
To: [hidden email]
Subject: Re: simple test to compare confocals

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Dear list and George,

Thanks for all the suggestions regarding simple confocal test.
I meant a test that allows to quickly verify that confocal is
functioning normally and stable. One of typical situations is when I
need to prove to a customer that confocal is OK, and it is the bad
sample that produces poor image.
I agree that in detail testing/evaluation mentioned in some replies is
useful and interesting to perform for a new system or after a major
overhaul. I would not be very enthusiastic to do it often (e.g. weekly).

My special thanks to George whose Convallaria slide based simple test is
the best aligned with my own line of thought (which I did not present
initially to avoid any bias and get fresh ideas). I have been routinely
doing similar test on our LSM510 to verify normal and stable performance
(green/red fluorescence at FITC and Texas Red settings).
Now based on George's experience I will use the test to quickly compare
confocals. I 100% agree that it is very important to keep identical
imaging parameters. However, one of caveats may be different emission
filters (in terms of bandwidth and transmission).
I assume this simple and quick test while not being a substitute to a
strict/detail comparison answers the concerns of the Core user who has
to switch between confocals.

Best wishes,
Arvydas

>>> George McNamara <[hidden email]> 8/13/2012 9:36 PM >>>
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Convallaria (lily of the valley) cross section slide - simplest to
borrow from your local Zeiss sales rep or service engineer. Basically,
if an Zeiss LSM or Leica SP# gives a decent image, it passes as far as a
Zeiss or Leica service engineer is concerned (unless you are looking
over their shoulder or better driving the scope and see any issues).
I've not seen Nikon service work on a confocal, so this test might be
confocal vendor standard. If you do not have a slide already; bummer
(might be some lily growing outside that you could harvest). The
www.carolina.com web site search engine is so incredibly bad, I was
unable to get a hit just now (amazon.com was no better). You can
probably find it from other prepared slide companies on the Internet.

Practical parameters:

gain 600 (not that all vendors "600' are the same, or even two PMTs in
the same scanhead).
offset so that all pixel values are above zero (i.e. no laser light)
12-bit data mode low laser power (ND filter or AOTF control level ...
need to run the Argon laser in the "good"power range) NO AVERAGING
(averaging is cheating) Fastest scan speed available [shortest pixel
dwell time] (ideally these will be identical on both instruments) ... my
thanks to Jonathan Boyd of Leica for demonstrating that "faster is
better" (SP5, standard scan speeds vs resonant scanner, sum images when
needed to get same total dwell time for each mode).
Same size image format for all instruments, i.e. 2048x2048 pixels
highest performance objective lens available, i.e. plan apo 63x/1.4 NA
oil, full resolution - by my math 60 nm pixel size for 1.4 NA
(explanation in previous messages at the listserv).

Note: Zeiss and Nikon oil do not play well together. Need to remove the
oil from the coverglass, clean the coverglass with 70% ethanol, before
oiling for the other scope.

George
p.s. I have not acquired any of my Convallaria slides for Sebastian
Munck's et al PiMP super-resolution calculation method (J Cell Sci 2012,
PubMed 22357945), but am looking forward to doing so in the future. I
have been getting very nice results with 30 nm pixel size on both our
LSM710 and Leica SP5 with 63x/1.4NA, relatively bright initially
specimens, filter size 1.625 (instead of default of 1.1 which is for
somewhat larger pixel size), 16-bit output (so I don't have to remember
where the 32-bit to 16-bit command is located), ~1% photobleaching per
time point. I find it most useful to have one channel time series per
LIF or LSM file. My thanks to Sebastian and Glen M for sending me the
ImageJ plugin.

On 8/13/2012 3:36 PM, Arvydas Matiukas wrote:

> bleedthrough, bleaching, and maybe resolution .
>
> I am aware of papers by Zucker, Pawley, Cole that describe detail
> evaluation of confocal performance, and even recently measured some
> PSFs. However, I am looking for just simple and quick test that would
> allow direct visual comparison of images (simple analysis like getting

> intensity histogram is fine) acquired on different confocals.
>
> Please share your thoughts and/or experience.
>
> Thank you in advance,
> Arvydas
> ***************************
>
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Core
> Department of Pharmacology
> SUNY Upstate Medical University
> 766 Irving Ave., WH 3167
> Syracuse, NY 13210
> tel.: 315-464-7997
> fax: 315-464-8014
> email: [hidden email]
>
>