Posted by
Alessandro Esposito on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-3D-Eroded-Object-Masks-commercial-response-tp3272647p3301746.html
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-24-10-3261Concerning 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