Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

HI Anda
The PMT 3 actually operatives at less voltage than PMT 2 at an airy disk
of 1  --both are the same type of Hammatsue  PMTs.
That  may imply that there is some  extra scattered light that is
entering this PMT at this voltage.
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, MD 72
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: [log in to unmask]



|---------+------------------------------->
|         |           Anda Cornea         |
|         |           <[log in to unmask]> |
|         |           Sent by: Confocal   |
|         |           Microscopy List     |
|         |           <[log in to unmask]
|         |           UFFALO.EDU>         |
|         |                               |
|         |                               |
|         |           02/02/2005 06:22 PM |
|         |           Please respond to   |
|         |           Confocal Microscopy |
|         |           List                |
|         |                               |
|---------+------------------------------->
  >-------------------------------------------------------------------------------------------------------------|
  |                                                                                                             |
  |      To:       [log in to unmask]                                                                |
  |      cc:                                                                                                    |
  |      Subject:  Re: Pinhole and resolution                                                                   |
  >-------------------------------------------------------------------------------------------------------------|




Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Robert!

How is your signal to noise in PMT-3?  If poor, it would certainly
benefit from increasing the pinhole and result in a better perceived
resolution.

Anda



Anda Cornea Ph.D.
Head of the Imaging and Morphology Core
Oregon National Primate Research Center
Oregon Health & Science University
(503) 690-5293

>>> [log in to unmask] 02/02/05 9:44 AM >>>
Our confocal spectral imaging systems was tested with an inexpensive
multi-ion discharge lamp (MIDL) that contains Hg+, Ar+ and inorganic
fluorophores that emits distinct, stable spectral features.We have
found
that the MIDL characterization verifies not only the accuracy of the
confocal system but the consistency of the confocal spectral imaging
system. The pattern of the lamp shows positional accuracy of spectral
peaks. The FWHM of the peaks appears to represents not only the the
accuracy of the system but also  can be related to the proper
alignment
of the system. We find that this lamp is invaluable for accessing the
performance and reliability of all confocal spectral imaging systems.

Recently we found some interesting data that is in need of an
explanation from the participants of confocal list server group.

DATA:  In our system PMT 1, 2 show superior patterns of the MIDL
spectrum compared to PMT 3 with a pinhole setting equivalent to an
airy
disc of 1. (10 x lens).  Increasing the pinhole size usually degrades
the spectral pattern (PMT 1, 2).  However,  we found that by opening
up
the pinhole to a setting equivalent to an airy disc of 3 and using PMT
3
actually increased (not decreased) the resolution of the MIDL spectral
pattern. The FWHM of the MIDL peaks are less with a higher pinhole
setting using PMT 3 This is in direct contrast to PMT 1,2 which
demonstrated a greater FWHM in the same reference peaks with the
larger
pinholes.

Does anyone have any ideas of how opening a pinhole can increase the
resolution of a spectral system and not decrease it?    Remember there
is NO magic in confocal microscopy only the physics of light to
explain
strange phenomenon.

A  PDF  is  available  on  request,  for  those who are not aware of
our
November  2004  publication  in Cytometry describing the technique
using
the  MIDL  lamp  "Lerner  JL Zucker, R.M.  Calibration and Validation
of
spectroscopic imaging: Cytometry 62A:8-34 2004

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, MD 72
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: [log in to unmask]