Alessandro wrote:
"Now the LiMo system. Its limiting factor is still the dead time of the detector, but it can detect more photons/laser pulse and this is the reason why can be faster. Having only 4 gates it provides a lower photon-efficiency and less access to heterogeneous decays, but overall performs fast and is a simple and cost-effective system. Guy: is it possible that the comment on photon- efficiency refers to the lack of the requirement of detecting max 1 photon/pulse? "
Well, Alessandro comes from the lab where the LIMO was developed, so it may seem impertinent for a mere paying customer to comment! But the ability to detect more photons per pulse - limited only by detector speed - is a key point. Having only 4 gates means that any photons arriving after about 9ns are lost but unless you have very long-lifetime fluorophores that is relatively trivial. But the other key factor is that the LIMO collects after every pulse, whereas the B&H versions around at the time my discussions took place simply didn't. A lot of laser pulses were missed. This may have improved - but I still don't find people saying they can get good images with 1-4s frame time on a B&H system.
I do emphasize that I'm not denigrating the B&H - I've used it, and published results from it. The LIMO will not do multi-exponential fits, and these can be very useful. (Actually - Nikon NB) my mathematically-inclined colleagues tell me that with 4 points you should be able to distinguish single and dual exponential decays, so maybe the software could be upgraded?
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
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Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Alessandro Esposito
Sent: Wednesday, 22 July 2009 6:01 PM
To: [log in to unmask]
Subject: Re: time-domain FLIM-FRET with fixed samples
Dear all, dear Vitaly,
I used B&H with a PMT or with a MCP-PMT, a LIFA, a LiMo and a custom biult FD-FLIM based on LaVision MCP-based (Kantech) camera.
As other mentioned is very difficult to compare systems in a precise manner, however, some trends are quite evident. There is a very good comparison TD vs FD done by Gratton a few years ago:
http://dx.doi.org/10.1117/1.1586704
and we published a work comparing acquisition throughput of various techniques from a theoretical point of view:
http://www.opticsinfobase.org/abstract.cfm?URI=josaa-24-10-3261
Concerning the mentioned systems, B&H sells a variaty of systems that can provide very slow or quite fast options. With same electronics, if one will use an MCP-PMT as detector will get a very good IRF (<60ps), but comparatevly slow acquisition times. I remember waiting 10 mins to get data with enough photons/pixel to detect in a reliable manner FRET between Cerulean/Venus.
If one uses PMT (probably also the new hybrid PMT, but I still did not use them), will get IRFs 200ps broad, but much faster acquisition times. With a bright sample, 1 min acquisition time start to be possible.
The BIG problem with these systems is pulse-pile up (linked to the dead time of detector/electronics) and the requirement to measure, in average, only one photon per laser pulse (typical for TCSPC). B&H solved the latter providing multi-detector systems, though this will come at a cost.
I do not have direct experience with the LaVision Biotech systems, but it seems interesting and, if I remember correctly, may provide a very good dynamic range because of the implementation of how they split light on their multi-detector system. With a "conventional" TCSPC system you will have to reduce the excitation light intensity at the level where no pixel in the image suffer of pulse pile-up. I think the new implementation by LaVision Biotech may overcome this, but I hope they could post here to explain that.
Now the LiMo system. Its limiting factor is still the dead time of the detector, but it can detect more photons/laser pulse and this is the reason why can be faster. Having only 4 gates it provides a lower photon-efficiency and less access to heterogeneous decays, but overall performs fast and is a simple and cost-effective system. Guy: is it possible that the comment on photon- efficiency refers to the lack of the requirement of detecting max 1 photon/pulse?
Among the scanning systems I would like to remind there is also picoQuant, but I do not have direct experience with their systems.
Wide-fields: LaVision sells a time-gated MCP-based system and Lambert Instruments the FDFLIM LIFA. Wide-field is faster because collects more light from the sample. However, both existing TD and FD wide-field systems are based on gating/modulating an MCP which causes large losses. Still they are fast and the original limitations of FD have been quite overcome in recent times. LIFA is relatevely fast, cost effective and user friendly, but if you have a dim sample for which you need higher spatial resolution, TCSPC could be the way to go.
I'll post a different message concerning fixed samples, but allow me a bit of advertizement :) and a last note on wide-field systems.
There is a time- and space- correlated MCP system available by Europhoton.de. It is a wide-field detector, though all photons are acquired sequentially and therefore is quite slow, but sensitive.
Also, a new genertation of wide-field systems could be available in a few years based on solid-state technologies (http://dx.doi.org/10.1117/1.2208999,
http://www.opticsinfobase.org/oe/abstract.cfm?id=86274) which should overcome problems that current WF systems suffer from.
That is all for now, I hope this long post was somehow more useful than boring!
Alessandro Esposito
www.quantitative-microscopy.org
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