Spectral: Single channel vs Multi-channel
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Edelmann, Richard E. Dr. |
Spectral: Single channel vs Multi-channel
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Following the discussions of Spectral detection vs filter based
detection, I have had some offline discussions and have come up with a few questions that I am hoping to hear discussed. There are two types of "Spectral" imaging detectors: One uses a prism or diffraction grating and variable width thin slit aperture to select the wavelengths that enter the PMT (Basically using the prism/grating and thin slit window to serve the role of the emission bandpass filter). The other "spectral" imaging is Multi-channel spectral imaging. In which the single PMT is replaced with a detector "array". The total range of wavelengths which are sent to the detector array and the specific range a wavelengths each channel collects is user determined. There are clear flexibility advantages to the spectral detectors vs the filter detectors. And there are analytical / imaging advantages of the multi-channel detectors in having detailed information more specific as to which wavelengths the signal is in - limited by the wavelength resolution of the detector. ( And yes similar information can be gotten by "Scanning" the single channel spectral detector, but at the cost of time). BUT other than cost - what is/are the disadvantages of the single channel spectral vs the multi-channel? There is a "Binning" of the wavelengths effect of the single channel detector which results in a stronger signal (which can be done essentially after the fact with the multi-channels). Is there a actual signal detection difference between the single and multi- channel detectors? Is there more amplification noise in the "array's" vs the tube PMT's? Is there some other factor which effects the signal detection? Maintenance? Calibration? Lifetime? All response are welcome - if you want to respond directly rather than to the list that is fine as well. Thank you! Richard E. Edelmann, Ph.D. Electron Microscopy Facility Director 364 Pearson Hall Miami University, Oxford, OH 45056 Ph: 513.529.5712 Fax: 513.529.4243 E-mail: [hidden email] http://www.emf.muohio.edu "RAM disk is NOT an installation procedure." |
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As you said, time is very important. Multi-channel detector can get a spectrum in one time, that means you can observe the shift of spectrum real-time, ex. in fluorescence. For single channel detector, the "scanning" rely on mechanical shift ----that's too slow! Get different wavelength in different time.
But maybe PMT is more sensitive than "array" usually. Or you can get a spectral EMCCD, ICCD. So, depend on your need for velocity.
Tian Tian,
State key lab of bioelectronics, Southeast University, China, titi_wudi@163.com |
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Craig Brideau |
Re: Spectral: Single channel vs Multi-channel
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We went with multi-channel after a lot of consideration. Our theory was that since dyes photobleach, you have a limited number of photons emitted from your sample. Once they are gone, you can't get them back. If you use a slit-based system you are throwing away photons that you can never get back again. With the multi-channel system you collect everything at once, which is why we went with the Nikon system. We also found that getting complete spectra has been very useful for what we want to do.
Craig On Sun, Apr 12, 2009 at 9:26 AM, qiqi_1983 <[hidden email]> wrote: As you said, time is very important. Multi-channel detector can get a spectrum in one time, that means you can observe the shift of spectrum real-time, ex. in fluorescence. For single channel detector, the "scanning" rely on mechanical shift ----that's too slow! Get different wavelength in different time. |
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Christian-103 |
Re: Spectral: Single channel vs Multi-channel
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In reply to this post by Edelmann, Richard E. Dr.
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If we look at the rival technologies in their simplest form
we have:
Leica: All detection is spectral. Conventional
PMTs are used so sensitivity is maximised. Any desired wavelength range
can be put to each detector so there is never any need to buy filters.
Acquiring a spectrum involves sequentially scanning a slit through the deisred
range, acquiring an image at each point, which is therefore very slow but the
spectrum is high-resolution.
Zeiss 510: Spectral detection uses a 32 PMT array, so
that acquiring a spectrum is quite fast, but relatively low res (32
channels). Any group of channels can be binned to give a wavelength
range within the resolution of the 32 channels. However the 32 channel PMT
array is less sensitive than conventional single PMTs,so an
alternative detection path goes through conventional filters to
conventional PMTs. This gives high sensitivity but has all the limitations
of filter based detection.
Essentially, when it comes to acquiring a spectral image,
it's a trade-off between speed on one side and sensitivity and resolution on the
other. It will depend on your experiment which is
best.
Othe systems do tricks such as sending only part of the
spectrum to a 32-channel array, thus increasing the resolution, but also putting
more optical elements in the way.
Guy
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Sent: Monday, 13 April 2009 5:26 AM To: [hidden email] Subject: Re: Spectral: Single channel vs Multi-channel
No virus found in this incoming message. No virus found in this outgoing message. |
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Armstrong, Brian |
Re: Spectral: Single channel vs Multi-channel
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Yes, I think the account of the Leica SP5 AOBS
system is accurate and could be considered a true spectral system. The LSM 510 is no longer available and the
new LSM 710 now selects the spectral lambda via prisms instead of filters. Moreover,
in the 710 You should be able to discern 32 separate
“colors” with this system and with much greater sensitivity
than the LSM510. Cheers, Brian D Armstrong PhD Light Microscopy Core Manager Beckman Research Institute City of Dept of Neuroscience Duarte, CA 91010 626-256-4673 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox If we look at the rival technologies in
their simplest form we have: Leica: All detection is
spectral. Conventional PMTs are used so sensitivity is maximised.
Any desired wavelength range can be put to each detector so there is never any
need to buy filters. Acquiring a spectrum involves sequentially scanning
a slit through the deisred range, acquiring an image at each point, which is
therefore very slow but the spectrum is high-resolution. Zeiss 510: Spectral detection uses a
32 PMT array, so that acquiring a spectrum is quite fast, but relatively low
res (32 channels). Any group of channels can be binned to give a
wavelength range within the resolution of the 32 channels. However the 32
channel PMT array is less sensitive than conventional single PMTs,so an
alternative detection path goes through conventional filters to
conventional PMTs. This gives high sensitivity but has all the
limitations of filter based detection. Essentially, when it comes to acquiring a
spectral image, it's a trade-off between speed on one side and sensitivity and
resolution on the other. It will depend on your experiment which is best. Othe systems do tricks such as sending
only part of the spectrum to a 32-channel array, thus increasing the
resolution, but also putting more optical elements in the way.
Guy Optical
Imaging Techniques in Cell Biology From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Christian
No virus
found in this incoming message. No virus found in this outgoing message.
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Zucker.Robert |
Re: Spectral: Single channel vs Multi-channel
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In reply to this post by Edelmann, Richard E. Dr.
I have worked extensively with the following spectral systems (Leica,
Zeiss, Nikon and PARISS) and have tested each of them for spectral accuracy, speed, sensitivity, and software. There are 4 companies that make spectral confocal systems (Leica, Zeiss, Nikon and Olympus) plus there are a number of non confocal systems including CRI: Nuance; LightForm: PARISS; and Applied Spectral Imaging: Sky. The fastest, most sensitive instrument, PARISS, is a prism based wide-field system, which yields data between 400-900nm almost instantaneously with a demonstrated spectral resolution of 1 nm. The limitation is the depth of penetration. Diffraction grating systems: The Zeiss 710 has a 9.8nm bandpass that can be reduced to 3nm or 5nm with sequential scanning. The Nikon A1 or C1Si systems have the ability to do 32 channels instantaneously at a spectral resolution of 2.5nm 5nm or 10nm. However, reducing the bandpass size reduces light throughput. The properties of the wavelength dispersive device and the wavelength detector become critical in the detection of emitted light and are factors to consider. Part of the problem for diffraction gratings is that they have a maximum efficiency at a single wavelength, then at shorter and longer wavelengths efficiency falls off fast. This is compounded by the spectral sensitivity of some PMTs that struggle much below 500nm, and above 700 nm. Wide-field spectral systems get around the PMT QE curve by using CCD cameras that offer good efficiency up to 900 nm. Prisms are considerably more efficient than diffraction gratings presenting a fairly flat wavelength versus intensity response. However, the speed to acquire 250 nm from a sequential Leica system is about 2 minutes. In my opinion, many experiments cannot be performed effectively using sequential scanning. Software--All the companies have propriety software, but not all programs are equally effective for spectral unmixing. Bottom line--this is not an easy choice--consider speed, spectral accuracy, software features, and confocal/non confocal companies and company reputation. Choose wisely. Best wishes Bob Robert M. Zucker, PhD U.S. Environmental Protection Agency Office of Research and Development National Health and Environmental Effects Research Laboratory. Reproductive Toxicology Division Telephone: 919-541-1585 Fax: 919-541-4017 e-mail: [hidden email] Mail address: USEPA,ORD,NHEERL,RTD Developmental Biology Branch ( MD 67) Research Triangle Park, North Carolina, 27711 Shipping address: 2525 E.NC Highway 54 Durham, NC, 27713 |------------> | From: | |------------> >--------------------------------------------------------------------------------------------------------------------------------------------| |"Richard E. Edelmann" <[hidden email]> | >--------------------------------------------------------------------------------------------------------------------------------------------| |------------> | To: | |------------> >--------------------------------------------------------------------------------------------------------------------------------------------| |[hidden email] | >--------------------------------------------------------------------------------------------------------------------------------------------| |------------> | Date: | |------------> >--------------------------------------------------------------------------------------------------------------------------------------------| |04/11/2009 09:13 AM | >--------------------------------------------------------------------------------------------------------------------------------------------| |------------> | Subject: | |------------> >--------------------------------------------------------------------------------------------------------------------------------------------| |Spectral: Single channel vs Multi-channel | >--------------------------------------------------------------------------------------------------------------------------------------------| Following the discussions of Spectral detection vs filter based detection, I have had some offline discussions and have come up with a few questions that I am hoping to hear discussed. There are two types of "Spectral" imaging detectors: One uses a prism or diffraction grating and variable width thin slit aperture to select the wavelengths that enter the PMT (Basically using the prism/grating and thin slit window to serve the role of the emission bandpass filter). The other "spectral" imaging is Multi-channel spectral imaging. In which the single PMT is replaced with a detector "array". The total range of wavelengths which are sent to the detector array and the specific range a wavelengths each channel collects is user determined. There are clear flexibility advantages to the spectral detectors vs the filter detectors. And there are analytical / imaging advantages of the multi-channel detectors in having detailed information more specific as to which wavelengths the signal is in - limited by the wavelength resolution of the detector. ( And yes similar information can be gotten by "Scanning" the single channel spectral detector, but at the cost of time). BUT other than cost - what is/are the disadvantages of the single channel spectral vs the multi-channel? There is a "Binning" of the wavelengths effect of the single channel detector which results in a stronger signal (which can be done essentially after the fact with the multi-channels). Is there a actual signal detection difference between the single and multi- channel detectors? Is there more amplification noise in the "array's" vs the tube PMT's? Is there some other factor which effects the signal detection? Maintenance? Calibration? Lifetime? All response are welcome - if you want to respond directly rather than to the list that is fine as well. Thank you! Richard E. Edelmann, Ph.D. Electron Microscopy Facility Director 364 Pearson Hall Miami University, Oxford, OH 45056 Ph: 513.529.5712 Fax: 513.529.4243 E-mail: [hidden email] http://www.emf.muohio.edu "RAM disk is NOT an installation procedure." |
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Christian-103 |
Re: Spectral: Single channel vs Multi-channel
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In reply to this post by Edelmann, Richard E. Dr.
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Christian-103 |
Re: Spectral: Single channel vs Multi-channel
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In reply to this post by Edelmann, Richard E. Dr.
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