the December 3, 2004 UK-California Stem Cell Symposium,
here at CHLA, I got to thinking about how many dimensions
can be examined in a light microscopy experiment. The list
that I came up with can be summarized as:
XY dimensions of a 2D image
Z dimension (axial; multiple planes of focus
Time (timepoints within a dataset; also developmental or
Intensity (amplitude of signal = photon flux ... possibly
due to changes in absorption)
(transmission, reflectance, excitation, emission spectra)
For fluorescence spectra, see http://www.mcb.arizona.edu/ipc/fret/default.htm
Tau: Fluorescence lifetime
Theta: Rotational lifetime (how molecules spin)
Polarization (birefringence; fluorescence polarization
Number of replicates (sample size) needed for statistical
to buy and run all of the equipment needed.
propose there is a direct correlation between the quantity
of $ and the number of other dimensions you can work in.
The limit being 0,0.
my purposes, Lambda () includes color, i.e. RGB color or
1,2,3 etc number of images.
for which I do not have good symbols for:
phase - as in Zernike phase contrast (or Nomarski or Smith
how (or whether) to distinguish between
brightfield/DIC/phase etc and fluorescence.
how (or whether) to make distinctions between 1, 2, 3 etc
photon excitation (fluorescence) as well as between 2nd,
3rd, etc harmonic generation (SHG, THG, etcHG).
multiple stage positions and/or stage tiling (acquire
images from greater than field of view)
measurements (changes in "specimen dimensions",
dry mass (re: Graham Dunn DRIMAP - is this a measurement
image or a dimension?)
retardance (i.e. Polscope birefringence imaging).
multichannel ratio - i.e. ratiometric FRET or Fura-2
lens type(s) - i.e. plan apochromat, plan fluorite, plan
achromat, (non-plan) lenses, and the new Super-apo, VC
(violet corrected) apo's etc.
Magnification? - it is very easy to change magnification
on many microscopes, for example, the electronic zoom on a
confocal laser scanning microscope. Too low or high a magnification,
and you will fail to satisfy Nyquist sampling theorem.
Note that there are times when you should "fail
Nyquist", such as in using high zoom to make objects
very large for sub-pixel centroid finding (i.e. single
Resolution? - All microscope objective lenses are engraved
on the barrel with both magnification and numerical
aperture (NA). If a study does not report lens type,
magnification, and NA -- and while we're at it, intermediate
magnification and pixel dimensions - how is anyone
supposed to (1) know how well the microscope should be
performing, and (2) replicate the experiment. For that
matter, if the settings were not documented, how does
someone know they are repeating an experiment even on the
same hardware? Hopefully resolution is kept constant (at
the optimal settings, i.e. Koehler illumination for
standard brightfield microscopy), but I wouldn't count on
Illumination "dimensions"? Multi-line lasers
change relative output depending on power used. Argon ion
lasers have little output at 458 nm at minimum power,
respectable output at higher power (at highest power they
tend to die quickly, after which they of course have zero
power until more $ is pumped into them); Xenon flash lamps
have distinct spectra at low vs high power; arc lamps
frost over with age (mostly an Intensity
Detector "dimensions"? Quantum efficiency,
detector temperature, exposure time(s), all impact light
collection efficiency, background noise, and signal to
noise ratio (SNR). To paraphrase Clinton/Gore 1992:
"it's the SNR stupid" that dictates
dimensions? What happens if you run out of a production
lot of antibodies or serum in the middle of an experiment?
Clearly, temperature, humidity, osmolality, perfusion
rate, oxygen tension (partial oxygen pressure), CO2
concentration, ionic composition, and more, are potential
variables. These could vary on purpose (your experiment:
pre vs post stimulus) or unintentionally (room drafts,
temperature changes, etc).
additions (or subtractions?) from this list are welcome.
Please email comments to me at firstname.lastname@example.org
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