Nick -- I agree with much of what you have said. But on one point you are in
error. The width of the beam is not as big an issue as you have made it for
pulse-width time-of-flight discrimination of doublets. If the laser beam
width is subtracted from the pulse width time-of-flight signal in real time
using a good biased amplifier, resolution is not a problem. I have
personally sized objects smaller than the wavelength of light (possible
since edge detection is all that is necessary, not actual imaging) with a 15
micron beam width. The limits of detection depend only on how good your
hydrodynamic focusing is to deliver all of the cells or objects to the same
position in the beam at a steady velocity. For successful doublet correction
the only limit is that the heterogeneity of nuclear sizes (if you do
fluorescence pulse width time-of-flight)or cell sizes (if you do light
scatter pulse width time-of-flight). The implementation in commercial
systems is the problem. Whereas we spend a few dollars more on a good
10-turn potentiometer for our high-resolution pulse-width time-of-flight
systems some manufacturers (I won't name them) use cheap potentiometers
(that the service reps actually adjust with a screwdriver!). This is a
classic case of saving a few dollars on some critical components and, if you
will excuse the analogy with what I have said above, screwing the customer.
So this technology has been around for almost 20 years, largely unused
and ignored, even on some of the commercial instruments. We actually sized
nuclei around the cell cycle (much harder than doublet detection)a number of
years ago (Rudolph, N.S., Ohlsson-Wilhelm, B.M., Leary, J.F., Rowley,
P.T.: "Single-Cell Analysis of the Relationship Between Transferrin
Receptor, Proliferation and Cell Cycle Phase in K562 Cells" Cytometry 6:
151-158 (1985)).
Happy holidays!
Best regards (to you and everyone
out there on the listserve),
Jim Leary
-----Original Message-----
From: Nicholas Terry [mailto:ntflow@odin.mdacc.tmc.edu]
Sent: Tuesday, December 21, 1999 10:13 AM
To: cyto-inbox
Subject: Re: A DNA analysis question -additional
At 08:16 AM 12/20/99 -0600, I wrote:
>
><musing quietly>
>If your hardware supports it integral versus peak is much more useful than
>width v. area. Wide beams don't help either (grateful for his 5 micron
>beam). Saving these options sometimes an extra marker (appropriate cyclins)
>can help.
(Putting hornets back in nest - returning worms to can). Lots of mail on
this one. Sorry I should have been more specific. Peak signals can be very
helpful in doublet discrimination but only if you have narrow beam
excitation optics. IMHO adequate DNA measurements can only be made if the
cross section of your interogating beam is substantially narrower than the
diameter of the objects that you are measuring.
(Waiting for flames.)
Under these conditions 2 G1 cells stuck together will have a very similar
integral (yes - mostly the same as area) signal as a true single G2/M cell.
The peak signal from a G2/M cell will however be significantly greater than
that from a G1 cell and from the sequential peak signals from a G1G1
doublet. But, the latter is only visible if the exciting beam is narrow.
In comparisons between area/width and integral (area)/peak discriminating
ability on an instrument with 5 micron excitation optics the integral peak
analysis gave the best discrimination. It is even possible to recognize
doublets that are not progressing linearly through the beam due to
turbulence or other factors.
How anyone manages to discriminate doublets from true G2 cells with a 40
micron beam, or nuclei with a 15-20 micron beam is beyond me. (tactfully).
(Pondering the likelihood that there is a greater proportion of tetraploid
tumors in the literature than in reality.)
Happy Christmas/holidays and New Year to all!
Nick
Nicholas Terry, M.A., Ph.D.,
Experimental Radiation Oncology - 066,
The University of Texas M. D. Anderson Cancer Center,
1515 Holcombe Blvd., Houston, Texas 77030.
'Phone: 713-792-3424,
'Fax: 713-794-5369.
http://drad52.mdacc.tmc.edu.
ntflow@odin.mdacc.tmc.edu.
Nicholas Terry, M.A., Ph.D.,
Experimental Radiation Oncology - 066,
The University of Texas M. D. Anderson Cancer Center,
1515 Holcombe Blvd., Houston, Texas 77030.
'Phone: 713-792-3424,
'Fax: 713-794-5369.
http://drad52.mdacc.tmc.edu.
ntflow@odin.mdacc.tmc.edu.
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