Jose,
Please excuse the delayed answer.
While not competent to answer your questions in detail, might I
suggest a (relatively) simple test of confocal performance?
If we accept that the optics of single-laser-beam confocals are
generally quite similar, the most important variable is "Photon
Efficiency": the fraction of photons leaving the focal plane that
actually contribute to the number stored in the image memory.
Unfortunately, this parameter is a function of a very large number of
other settings and design decisions. As a result, although all
manufactures claim excellent Photon Efficiency, none actually provide
any specifications.
We have to do it ourselves. Let us first agree that it is Detection
Photon Efficiency that we are really interested in. Now the problem
becomes, how can we create a "specimen" that has a repeatable
brightness. I suggest that the best plan is to use the transmission
illumination source as your specimen. All scopes have one, it is
adjustable in intensity and as the area illuminated is much larger
than that measured by the scanned pinhole, it is fairly easy to
measure the light present at the specimen level with a simple light
meter.
Set up the microscope's normal transmission illumination system for
Köhler illumination and adjust the field diaphragm for a known
diameter in the image plane (You can measure it as the diameter of
the bright circle detected by your confocal with the lasers off.).
The arc or incandescent source must be provided with a regulated
power supply and it is a good plan to add a bandpass filter to
normalize for differences in spectral distribution that occur between
different sources and/or different settings of the same source (try
an "interference green" as used for DIC). Once this has been set up,
the only major variables remaining are the illumination power
setting, the amount of metal deposited on the inside of the glass
tube surrounding the source, the bandpass effects of any filters that
remain in the light path, the pinhole diameter and the PMT voltage.
In many instruments, it is relatively easy to remove all of the
dichroic and bandpass filters in the confocal head and let the light
from the image plane pass directly to the PMT (with the META, you
will have to sum the signal from all channels). Under these
conditions, one should get a standard reading with a given objective,
pinhole size and lamp power. As long as the NA of the condenser is
less than that of the objective, you can normalize for all the design
differences up to the specimen by actually measuring the light coming
out of the condenser with any sort of light meter (Use a
carefully-placed black cloth to make sure that you aren't mostly
measuring room light!)
The confocal microscope with the laser off is just a flying spot
detector and only collects photons from one pixel at a time with the
result that the effective intensity (in photon/sec/pixel) is about
250,000x less than if it were measuring the entire field. Even so, it
may be necessary to place ND filters between the source and the
condenser lens to permit the PMT to be operated near its normal
voltage. Line-frequency variations in the filament heating current
may be visible in the data, as will any instability in the plasma of
arc sources. For this reason it may be more convenient to measure the
PMT output with an analog DC voltmeter than via the ADC in the image
memory.
By introducing ND filters below the stage, it is be possible to
reduce the intensity of the light to the level at which
photon-counting is appropriate. On those instruments that have this
ability (basically Bio-Rad), you can easily make measurements to
determine a rough ratio between the actual number of photons being
detected (using photon-counting) and analog-intensity-values stored
in the memory at the same settings of the PMT gain.
I hope that this doesn't sound too intimidating, because it really is
the only fairly reliable way to measure conveniently this most
important factor.
Cheers,
Jim Pawley
>Still on the subject of the Zeiss META, I was wondering if someone
>in the list has actually detailed hands-on info on the system. We're
>in the process of buying a new confocal, and this is an expensive
>option (on top of Zeiss being the most expensive per se). I
>understand the technicalities and the potential for multispectral
>imaging, but would like also to hear practical details, like
>reliability, merging with the software, detection comparisons (Zeiss
>claims it's the same, but going through a grating filter and
>spliting the available photons between an array of PMT's sounds
>hardly like a way of increasing sensitivity) and any other details
>that may make one decide whether or not it's worth the money.
>
>I was kind of hesitating to bring up to the list another "I have to
>buy a new one- HELP!" request, it's a sort of re-current issue that
>I believe most people gets bored with. Anyway since I found a
>METAexcuse, let me extend the quest for help to anyone that has been
>recently passed through the hellish process of having to make the
>usual BioRad-Leica-Zeiss decision. Please mind that I would value
>more realistic oppinions of realistic hands-on, paper-publishing,
>biology users than strictly technical oppinions, I've been getting a
>lot of the latest. Specially wellcomed are:
>
>1. Direct side-by-side confrontation of Bio-Rad and Leica on the
>usual issues (sensitivity, resolution, separation, etc.)? (2100 vs.
>SP2)
>2. If Bio-Rad 2100, what optics? I only hear about Nikon, did
>everyone gave up on trying others on the Bio-Rad?
>3. If Bio-Rad, how well is their software coping with Leica and
>Zeiss? Is it user-friendly enough for a multi-user facility in which
>the regular users considers anoying and time-consuming anything
>further than pushing a button on a screen?
>4. If Bio-Rad how is the engineering vs. the others? Bio-Rad has a
>tradition of having to pull 10 knobs and filters to get the thing
>going, while the others usually integrate much better the optics,
>does that stands in the 2100?
>5. If Leica, does anyone feels limited by the objective inferior
>working distances, specially on live imaging?
>6. If Leica, I've heard some negative comparisons with the 2 others
>(SP2, 510 and 2100) on the issue of sensitivity, does anyone stands
>agaisnt or confirms this?
>7. If Zeiss, any other really strong argument besides the software?
>8. 10 good reasons to pay a lot more money for a Zeiss or a Leica
>over a Bio-Rad?
>
>
>basal config we are comparing for all brands includes:
>- 3 lasers (Red solid state in the BioRad)
>- AOTF
>- 3 PMT's (2+1 arrayPMT in the ZeissMETA)
>
>thanks in advance for any help in any of these points. The matter
>has some urgency, decisions have to be taken quite quickly, so I
>would appreciate quick, even if short answers.
>
>Cheers
>Jose
>
>
>
>**********************************************************
>Jose' A. Feijo', Prof.
>----------------------------------------------------------
>Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>
>tel. +351.21.440.79.41, fax +351.21.440.79.70
>
>and
>
>Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
>PT-1749-016 Lisboa, PORTUGAL
>
>tel. +351.21.750.00.69, fax +351.21.750.00.48
>__________________________________________________________
>e.mail: [log in to unmask]
>URL: http://193.126.26.2/Groups/plant_development.html
>**********************************************************
--
****************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building, FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706 [log in to unmask]
"A scientist is not one who can answer questions
but one who can question answers."
Theodore Schick Jr., Skeptical Enquirer, 21-2:39
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