Subject: | |
From: | |
Reply To: | |
Date: | Wed, 27 Mar 1996 10:56:00 EST |
Content-Type: | text/plain |
Parts/Attachments: |
|
|
What Paul Goodwin has said is correct. The SNR for a true deconvolution
system equipped with a cooled CCD camera is much higher than a confocal
microscope equipped with a photomultiplier tube. For a Scanalytics
system, the cooled CCD camera recommended has a dynamic range of 12 bits
or higher-- providing 4095 grey levels or more -- whereas a PMT is
usually 8 bit, with 255 grey levels
.
For most problems where spatial and temporal resolution is important,
wide-field capture of images followed by rigorous deconvolution is a
better solution than the signal rejection technique of confocal
microscopy. In an independent comparison between several confocal
microscope manufacturers and the Exhaustive Photon Reassignment (EPR)
algorithm by Scanalytics (at the AQLM course held at Woods Hole each
spring), the z-axis resolution (as measured by FWHM) of the Scanalytics
system produced superior results to any of the other confocal microscopes
(0.4 um for Scanalytics, 0.8 um for other confocal manufacturers). For
x-y resolution (also FWHM), resolution was equivalent between the two
systems, both systems ultimately limited by the diffraction limitations
of the microscope. The specimen was composed of 0.1 um latex microbeads
with a fluorescein-like spectrum.
However, with ratio imaging, there are other issues to consider. Most
researchers looking at calcium flux are looking at millisecond (or
faster!) events. Some of the best cooled CCD cameras can only read out a
few frames per second, and that over a fairly small area. In addition,
deconvolution usually requires imaging in the z-dimension over several
optical depths (the Scanalytics system requires at least 5 planes for a
restoration). If events are occuring very rapidly, by the time the 5th
plane is imaged, the first plane is probably no longer the same. As a
true quantitative deconvolution (eg. EPR) requires that that all signal
present in a through-focus volume retain correct inter-plane
registration, it becomes necessary to acquire the 5 planes in a shorter
period of time than the event being studied (actually, in half the time
according to Nyquist). As the quantum efficiency and read-out rates of
affordable CCD cameras continues to improve, it is slowly becoming
possible to perform this level of imaging. A word to the wise is to be
extremely skeptical of claims made many manufacturers about the
"resolution" capabilities of their instruments. We are all limited by
photon statistics which, in practice, ultimately govern the
spatial/temporal resolution tradeoffs we must make.
|
|
|