Sensitivity and AOBS

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Ken Pluomis Ken Pluomis
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Sensitivity and AOBS

Dear all,

 

We are in the process of purchasing a confocal microscope to serve a broad range of biological applications. We are considering the Leica SP5 and the Zeiss 710. Both companies are claiming “our confocal provides the highest sensitivity”, but they are unable to provide some proofs of that. Could you provide me some practical feedback?

 

Particularly we are a bit confused about the AOBS. Leica people claim more than 95% transmittance, while Zeiss people are saying that the transmittance is much worse than any conventional beam splitter. Again nobody is providing any kind of evidence of such statements. Do you have any experience/information on this subject?

 

Thanks.

 

Dr. Ken. G. Pluomis


CONFOCAL TL Andresen CONFOCAL TL Andresen
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Re: Sensitivity and AOBS

Hi Ken,

 

I have just been through exactly the same process. I did not succeed in finding out actual proof of who was right but ended up buying the SP5, which was mainly due to a better deal.

 

We tested the microscopes severely but could not find clear differences.

 

One of the things I would have liked to know is that Zeiss claims that their new dual beamsplitter suppresses the laser light to a much higher degree than the AOBS (known as the OD number). I asked Leica for data on the AOBS but they have never gotten back to me on this even though I asked many times. They also say it doesn’t matter because the laser light is removed later by the spectral cut off. But this is what Zeiss claims to be the biggest advantage, and I could not find out if it makes sense or not.

 

I would be very happy to know if you find actual differences.

 

Best regards, Thomas

 

Thomas L. Andresen
Senior Scientist
Phone direct +45 4677 5480
[hidden email] 
www.cbio.dk

Department of Micro and Nanotechnology
Technical University of Denmark – DTU
Building 124, P.O. Box 49
DK-4000 Roskilde, Denmark
Tel +45 4677 4714
Fax +45 4677 4791
www.nanotech.dtu.dk

 

 


From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ken Pluomis
Sent: Thursday, October 02, 2008 8:56 PM
To: [hidden email]
Subject: Sensitivity and AOBS

 

Dear all,

 

 

We are in the process of purchasing a confocal microscope to serve a broad range of biological applications. We are considering the Leica SP5 and the Zeiss 710. Both companies are claiming “our confocal provides the highest sensitivity”, but they are unable to provide some proofs of that. Could you provide me some practical feedback?

 

Particularly we are a bit confused about the AOBS. Leica people claim more than 95% transmittance, while Zeiss people are saying that the transmittance is much worse than any conventional beam splitter. Again nobody is providing any kind of evidence of such statements. Do you have any experience/information on this subject?

 

Thanks.

 

Dr. Ken. G. Pluomis

 

vb-2 vb-2
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a horizontal table for sample/slide preparation/storage

Dear List,
 
I would greatly appreciate if you could refer to a supplier of a high precision horizontal table (in a box-like configuration) for sample/slide preparation and/or storage.
 
It is a kind of an obvious thing in the NEW ERA of "PALM and STORM along thinking" or simply surface imaging (TIR microscopy ...).
 
Thank you very much in advance,
 
Vitaly
 
NCI-Frederick,
301-846-6575
 
Craig Brideau Craig Brideau
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Re: Sensitivity and AOBS

In reply to this post by CONFOCAL TL Andresen
The key thing I would look for is a simple optical path and what light detector they are using.  Photomultiplier tubes come in many different flavors with lots of different spectral ranges, sensitivities, and quantum efficiencies.  Depending on what wavelength you are looking for your detector can have a large impact.
In terms of filtering out the laser light from the fluorescence signal; there are quite good band pass and long pass filters out there and they are commonly available.  Rather than worrying about 10% plus or minus in the filtering I'd be more concerned about the actual detectors used and how the light is coupled into them.

Craig


On Thu, Oct 2, 2008 at 1:15 PM, <[hidden email]> wrote:

Hi Ken,

 

I have just been through exactly the same process. I did not succeed in finding out actual proof of who was right but ended up buying the SP5, which was mainly due to a better deal.

 

We tested the microscopes severely but could not find clear differences.

 

One of the things I would have liked to know is that Zeiss claims that their new dual beamsplitter suppresses the laser light to a much higher degree than the AOBS (known as the OD number). I asked Leica for data on the AOBS but they have never gotten back to me on this even though I asked many times. They also say it doesn't matter because the laser light is removed later by the spectral cut off. But this is what Zeiss claims to be the biggest advantage, and I could not find out if it makes sense or not.

 

I would be very happy to know if you find actual differences.

 

Best regards, Thomas

 

Thomas L. Andresen
Senior Scientist
Phone direct +45 4677 5480
[hidden email] 
www.cbio.dk

Department of Micro and Nanotechnology
Technical University of Denmark – DTU
Building 124, P.O. Box 49
DK-4000 Roskilde, Denmark
Tel +45 4677 4714
Fax +45 4677 4791
www.nanotech.dtu.dk

 

 


From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ken Pluomis
Sent: Thursday, October 02, 2008 8:56 PM
To: [hidden email]
Subject: Sensitivity and AOBS

 

Dear all,

 

 

We are in the process of purchasing a confocal microscope to serve a broad range of biological applications. We are considering the Leica SP5 and the Zeiss 710. Both companies are claiming "our confocal provides the highest sensitivity", but they are unable to provide some proofs of that. Could you provide me some practical feedback?

 

Particularly we are a bit confused about the AOBS. Leica people claim more than 95% transmittance, while Zeiss people are saying that the transmittance is much worse than any conventional beam splitter. Again nobody is providing any kind of evidence of such statements. Do you have any experience/information on this subject?

 

Thanks.

 

Dr. Ken. G. Pluomis

 


Julio Vazquez Julio Vazquez
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Re: Sensitivity and AOBS

In reply to this post by Ken Pluomis
=
Hello Ken, 

I have struggled with this type of question in the past, and I have to admit from the start I don't have any answer. While getting some definite answer from someone will be valuable, I now feel this is probably a non-issue (at least for me):

There are so may factors affecting the final sensitivity of a confocal system, that trying to find out if one particular component is better in this or that system is probably meaningless.... chances are they are both performing just fine. To test the relative performance of the two systems, the only way I can see would be to have a suitable sample (preferably fine structures of low-medium staining intensity), try to get an acceptable image on both systems (at some level of image quality based on resolution and signal to noise), and see how the images on both systems degrade over multiple frames when you run a time lapse. This of course would assume that both systems are perfectly adjusted and optimized, and are performing to the best of their abilities, which in itself is probably not a trivial thing to achieve or determine.

In my opinion, both systems will be just fine for 95% of your applications. Then, there may be a few applications here or there for which one system may have an edge, although it is hard for me to imagine that you would come across any experiment that you could do on one system but not on the other. If you are anticipating really demanding experiments, then the best would be to try them on either system and see which one works best.

I had to make a similar choice a few years ago between the Leica SP2 AOBS and the Zeiss LSM 510. We went with the 510 (for various reasons, not necessarily linked to performance).  In retrospect, this is what I thought were the main issues we encountered:

The 510 is filter-based. This means that for fast imaging, you need to have a multi-band dichroic and set your detection channels in such a way that you do not move any hardware between channels. By doing this, you sacrifice sensitivity (because of narrower detection bands). If you want to maximize sensitivity, you need to switch mirrors and filters, and you loose speed. Since you can't predict which combinations of dyes you will be using, there are times when you won't have the choice and will need to use the slower scan mode (with filters and mirrors switching). With a filterless system, you won't have to make those choices. This has not been a critical issue for us, though... by that I mean we have been able to live with those limitations and compromises. However, a more important issue has been that since we are in a multi-user facility, we are sometimes worried that users will have a configuration that requires mirror/filter switching, and there is a chance they try to run some fast scans (line scans, for instance), which will put incredible stress on the hardware.  You won't run into this type of issues with a filterless system. Other than that, the 510 has performed fine for us, even though it has been a bit temperamental (hardware and software-wise). We don't own an SP2 (or SP5), so I don't know how happy we would have been with that, and I haven't looked carefully into the improvements the 710 brings. Other practical differences may be the speed at which you can run wide-band spectral scans on the two systems, so if you are considering doing fast spectral scans, maybe check how both systems perform in that respect. Another issue of practical importance will be the support you get from both companies in your area.  Once you have purchased your instrument, it's unlikely you'll ever worry about the sensitivity of the AOBS again...

my $ 0.02  (which are worth about $ 0.012 as we speak, and still going down...)

Julio.

--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024


=

On Oct 2, 2008, at 11:55 AM, Ken Pluomis wrote:

Dear all,

 

We are in the process of purchasing a confocal microscope to serve a broad range of biological applications. We are considering the Leica SP5 and the Zeiss 710. Both companies are claiming “our confocal provides the highest sensitivity”, but they are unable to provide some proofs of that. Could you provide me some practical feedback?
 
Particularly we are a bit confused about the AOBS. Leica people claim more than 95% transmittance, while Zeiss people are saying that the transmittance is much worse than any conventional beam splitter. Again nobody is providing any kind of evidence of such statements. Do you have any experience/information on this subject?
 
Thanks.
 
Dr. Ken. G. Pluomis



RICHARD BURRY RICHARD BURRY
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Re: Sensitivity and AOBS

In reply to this post by Craig Brideau
In addition to the filters and the PMTs, one issue is the light scatter within the confocal head.  The best is a light path that is clean and there is no defracted light reaching the PMT.  In previous Zeiss and Leica instruments this has been a problem.  In the 710 confocals, Zeiss has greatly reduced this problem, but I have no experience with the Leica.
 
Dick Burry

----- Original Message -----
From: Craig Brideau <[hidden email]>
Date: Thursday, October 2, 2008 4:27 pm
Subject: Re: Sensitivity and AOBS
To: [hidden email]


> The key thing I would look for is a simple optical path and what light detector they are using.  Photomultiplier tubes come in many different flavors with lots of different spectral ranges, sensitivities, and quantum efficiencies.  Depending on what wavelength you are looking for your detector can have a large impact.
> In terms of filtering out the laser light from the fluorescence signal; there are quite good band pass and long pass filters out there and they are commonly available.  Rather than worrying about 10% plus or minus in the filtering I'd be more concerned about the actual detectors used and how the light is coupled into them.

> Craig


> On Thu, Oct 2, 2008 at 1:15 PM, <<A href="javascript:main.compose('new','t=thomas.andresen@risoe.dk')">thomas.andresen@...> wrote:

> Hi Ken,

 

> I have just been through exactly the same process. I did not succeed in finding out actual proof of who was right but ended up buying the SP5, which was mainly due to a better deal.

 

> We tested the microscopes severely but could not find clear differences.

 

> One of the things I would have liked to know is that Zeiss claims that their new dual beamsplitter suppresses the laser light to a much higher degree than the AOBS (known as the OD number). I asked Leica for data on the AOBS but they have never gotten back to me on this even though I asked many times. They also say it doesn't matter because the laser light is removed later by the spectral cut off. But this is what Zeiss claims to be the biggest advantage, and I could not find out if it makes sense or not.

 

> I would be very happy to know if you find actual differences.

 

> Best regards, Thomas

 

> Thomas L. Andresen
> Senior Scientist
> Phone direct +45 4677 5480
<A title=mailto:thomas.andresen@risoe.dk href="javascript:main.compose('new','t=thomas.andresen@risoe.dk')" target=1>> thomas.andresen@... 
> www.cbio.dk

Department of Micro and Nanotechnology
> Technical University of Denmark – DTU
> Building 124, P.O. Box 49
> DK-4000 Roskilde, Denmark
> Tel +45 4677 4714
> Fax +45 4677 4791
> www.nanotech.dtu.dk

 

 



> From: Confocal Microscopy List [mailto:<A href="javascript:main.compose('new','t=CONFOCALMICROSCOPY@LISTS.UMN.EDU')" target=1>CONFOCALMICROSCOPY@...] On Behalf Of Ken Pluomis
> Sent: Thursday, October 02, 2008 8:56 PM
> To: <A href="javascript:main.compose('new','t=CONFOCALMICROSCOPY@LISTS.UMN.EDU')" target=1>CONFOCALMICROSCOPY@...
> Subject: Sensitivity and AOBS



 

> Dear all,

 

 

> We are in the process of purchasing a confocal microscope to serve a broad range of biological applications. We are considering the Leica SP5 and the Zeiss 710. Both companies are claiming "our confocal provides the highest sensitivity", but they are unable to provide some proofs of that. Could you provide me some practical feedback?

 

> Particularly we are a bit confused about the AOBS. Leica people claim more than 95% transmittance, while Zeiss people are saying that the transmittance is much worse than any conventional beam splitter. Again nobody is providing any kind of evidence of such statements. Do you have any experience/information on this subject?

 

> Thanks.

 

> Dr. Ken. G. Pluomis



 










> Spam
> Not spam
> Forget previous vote



Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814  Cell 614.638.3345  Fax 614.247.8849


Jeremy Adler Jeremy Adler
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Re: Sensitivity and AOBS

In reply to this post by Ken Pluomis
A very basic question is
 
what fraction of the emitted photons are detected ?
 
An answer covers most of the critical microscope components.
 
On the whole confocal microscopes are not short of excitation light, its detected photons that are in short supply.
 
If you get answer from you suppliers you might share the information.
 
Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories F5
Stockholm University
Stockholm 106 91
Sweden

________________________________

From: Confocal Microscopy List on behalf of Ken Pluomis
Sent: Thu 02-Oct-08 20:55
To: [hidden email]
Subject: Sensitivity and AOBS



Dear all,

 

We are in the process of purchasing a confocal microscope to serve a broad range of biological applications. We are considering the Leica SP5 and the Zeiss 710. Both companies are claiming "our confocal provides the highest sensitivity", but they are unable to provide some proofs of that. Could you provide me some practical feedback?

 

Particularly we are a bit confused about the AOBS. Leica people claim more than 95% transmittance, while Zeiss people are saying that the transmittance is much worse than any conventional beam splitter. Again nobody is providing any kind of evidence of such statements. Do you have any experience/information on this subject?

 

Thanks.

 

Dr. Ken. G. Pluomis
George Ring George Ring
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Re: Sensitivity and AOBS

In reply to this post by Ken Pluomis
Hi, Ken,
 
A  couple of years ago we had demos of the SP5 and 510 Meta at the same time.  We could look at a single sample on one instrument and then compare
the images acquired by the second instrument.  The SP5 blew the 510 away as far as sensitivity.  There were samples that the 510 could not detect at all for which the SP5 produced lovely images.  So the SP5 with AOBS is definitely more sensitive than the filter based 510.  I don't know about the 710, though.  If you can get Leica and Zeiss to demo their instruments side by side you can get a really good comparison of the performances of both machines.  The Leica rep used to love these side-by-side comparisons when he was up against the 510.  I don't know if they're as anxious to go up against the 710.
 
Good Luck,
 
George
 

>>> Ken Pluomis <[hidden email]> 10/2/08 2:55:31 PM >>>

Dear all,

 

We are in the process of purchasing a confocal microscope to serve a broad range of biological applications. We are considering the Leica SP5 and the Zeiss 710. Both companies are claiming “our confocal provides the highest sensitivity”, but they are unable to provide some proofs of that. Could you provide me some practical feedback?

 

Particularly we are a bit confused about the AOBS. Leica people claim more than 95% transmittance, while Zeiss people are saying that the transmittance is much worse than any conventional beam splitter. Again nobody is providing any kind of evidence of such statements. Do you have any experience/information on this subject?

 

Thanks.

 

Dr. Ken. G. Pluomis


Ken Pluomis Ken Pluomis
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Re: Sensitivity and AOBS

In reply to this post by Ken Pluomis
Dear Dr. Sudipta Maiti
I would really appreciate you could send me this paper as well as the program.
 
Many thanks.
 
Dr. Ken G. Pluomis
 

 

----- Original Message ----
From: Sudipta Maiti <[hidden email]>
To: [hidden email]
Sent: Friday, October 3, 2008 5:35:50 AM
Subject: Re: Sensitivity and AOBS

One of the best ways I know to do this is described in our paper:

Quantitative measurement of the resolution and sensitivity of confocal microscopes using line-scanning fluorescence correlation spectroscopy
Microscopy Research and Technique
Volume 66, Issue 4, Date: 1 March 2005, Pages: 198-202
J. Balaji, S. Maiti

It may sound a bit daunting to the uninitiated, but it is pretty simple, really. All it takes is to record an image of a bead solution for a few seconds. The data can be analyzed with a program that I can send off the list.

Sudipta

On Thu, 2 Oct 2008 12:02:49 -0700, Ken Pluomis wrote


> Dear all,
>
>
>  
> We are in the process of purchasing a confocal microscope to serve a broad range of biological applications. We are considering the Leica SP5 and the Zeiss 710. Both companies are claiming [WINDOWS-1252?]“our confocal provides the highest [WINDOWS-1252?]sensitivity”, but they are unable to provide some proofs of that. Could you provide me some practical feedback?
>  
> Particularly we are a bit confused about the AOBS. Leica people claim more than 95% transmittance, while Zeiss people are saying that the transmittance is much worse than any conventional beam splitter. Again nobody is providing any kind of evidence of such statements.. Do you have any experience/information on this subject?
>  
> Thanks.
>  
> Dr. Ken. G. Pluomis


Dr. Sudipta Maiti
Associate Professor
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: 91-22-2280-4610
alternate e-mail: [hidden email]
url: www.tifr.res.in/~biophotonics


Goodhouse, Joseph G. Goodhouse, Joseph G.
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Re: Sensitivity and AOBS

In reply to this post by George Ring
    One thing you  need to measure for these comparisons is the amount of excitation power that is coming to your specimen.  This can vary due to the different optics but they should be set to the same level on each system.  You need a tunable sensitive light meter that can read in the uWatt range placed above the objective.  Scan in a spot or line mode rapidly to prevent meter blanking.
 

Joe Goodhouse
Confocal Core Lab Manager
Dept. of Molecular Biology
Princeton University
609-258-5432

Visit us at http://www.molbio1.princeton.edu/facility/confocal/  

 


From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George Ring
Sent: Friday, October 03, 2008 8:37 AM
To: [hidden email]
Subject: Re: Sensitivity and AOBS

Hi, Ken,
 
A  couple of years ago we had demos of the SP5 and 510 Meta at the same time.  We could look at a single sample on one instrument and then compare
the images acquired by the second instrument.  The SP5 blew the 510 away as far as sensitivity.  There were samples that the 510 could not detect at all for which the SP5 produced lovely images.  So the SP5 with AOBS is definitely more sensitive than the filter based 510.  I don't know about the 710, though.  If you can get Leica and Zeiss to demo their instruments side by side you can get a really good comparison of the performances of both machines.  The Leica rep used to love these side-by-side comparisons when he was up against the 510.  I don't know if they're as anxious to go up against the 710.
 
Good Luck,
 
George
 

>>> Ken Pluomis <[hidden email]> 10/2/08 2:55:31 PM >>>

Dear all,

 

We are in the process of purchasing a confocal microscope to serve a broad range of biological applications. We are considering the Leica SP5 and the Zeiss 710. Both companies are claiming “our confocal provides the highest sensitivity”, but they are unable to provide some proofs of that. Could you provide me some practical feedback?

 

Particularly we are a bit confused about the AOBS. Leica people claim more than 95% transmittance, while Zeiss people are saying that the transmittance is much worse than any conventional beam splitter. Again nobody is providing any kind of evidence of such statements. Do you have any experience/information on this subject?

 

Thanks.

 

Dr. Ken. G. Pluomis


Mario-2 Mario-2
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Re: Sensitivity and AOBS

Re: Sensitivity and AOBS
Hi All,

Joe's suggestion is an important part of determining the overall quantum detection efficiency of any confocal or wide field microscope, the main question then being how many fluorescence photons are captured and detected per the number of photons that reach the excitation volume in the sample. About 10+ years ago I went to the trouble of performing this measurement with a BioRad 1024 equipped with an inverted Nikon using a 1.4 NA objective.

As a reference sample I used a either a 1 or? 10 uM fluorescein (pH 8.0, 50% glycerol: 50% PBS) sample in freshly prepared diphenylenediamine antifade and used a focus position 10 um from coverslip into the solution. I had previously found that intensity measurements using fluorescein solutions ranging from 0.1 uM up to 100 uM gave a decent straight line. Of course, different total dwell times were used and the data normalized accordingly.

To make this story shorter, taking into account the Q.E. and e of fluorescein at pH 8.0, the laser power delivered at 488 nm, the measured fluorescence signal, pinhole size set to realize optimal x,y,z resolution, I measured an overall Q.D.E. of ~0.0004, which was similar to the results given in an article in the 2nd Edition of J.P.'s "....Handbook of Confocal Microscopy..." for an earlier version of the BioRad confocal (R.I.P.). The 1024 model detected only about 1 photon in about 2500 emitted photons. The Nikon objective did its job meaning that with an NA of 1.4 it managed to captured about 30% of emitted photons; the rest of the losses, a factor of 0.0013, resulted from the other optical and detection components of the system. Clearly, there was a very large potential for improvement in sensitivity.

Finally, for present day microscopes, a crude working comparison between instruments should not be too difficult. Just make reference sample(s) that emit at a couple of wavelengths that are reasonably stable such as Alexa 488 nm and a couple of others at longer wavelengths for which the Q.E.'s and extinction coefficients are known, or which you can measure (there are a lot of options for ref. samples). Make sure that you use objectives that are nominally rated for the same NA (I always measure the PSF for any objective I buy, especially at the high end) and make sure that the settings for pinholes are properly configured so you are working at the Rayleigh resolution and that you normalize for differences in dwell time. Always check the statistical noise of the output signal. Even when the signals are strong the noise profile can tell you whether you are truly getting the dynamic range suggested by the peak signal intensities. You can have outputs that appear nearly identical in intensities but one system may give you 32 levels of statistically useful values whereas another may give you 64 or better.

Although there are many highly qualified and expert microscope vendor representatives, there are many sales people that lack the expertise to explain the technical differences that pertain to their instruments. It's caveat emptor folks.

Final comment: In this instance, since I have been a third party consultant for various pharmaceutical companies purchasing new microscopy systems, I can't claim to be a disinterested party.

Mario


    One thing you  need to measure for these comparisons is the amount of excitation power that is coming to your specimen.  This can vary due to the different optics but they should be set to the same level on each system.  You need a tunable sensitive light meter that can read in the uWatt range placed above the objective.  Scan in a spot or line mode rapidly to prevent meter blanking.
 
Joe Goodhouse
Confocal Core Lab Manager
Dept. of Molecular Biology
Princeton University
609-258-5432
Visit us at http://www.molbio1.princeton.edu/facility/confocal/  

-- 
________________________________________________________________________________
Mario M. Moronne, Ph.D.

ph (510) 528-8076