The rapid development of fluorescence microscopy together with digital imaging
and, above all, the development of new fluorescent probes of biological activity have
brought a new level of sophistication into quantitative imaging. Most of the measure-
ments are based on the ability to measure accurately the brightness of and the
wavelength emitted from a fluorescent probe within a sample using a digital imaging
system. This is also the basis offlow cytometry, which measures the brightness of
each cell in a population of cells as they pass through a laser beam. Cells can be sorted
into different populations using a related technique,fluorescence-activated cell
sorting.
The brightness of the fluorescence from the probe can be calibrated to the amount
of probe present at any given location in the cell at high resolution. For example, the
concentration of calcium is measured in different regions of living embryos using
calcium indicator dyes, for example fluo-3, whose fluorescence increases in propor-
tion to the amount of free calcium in the cell (Fig. 4.19, see also colour section). Many
probes have been developed for making such measurements in living tissues. Controls
are a necessary part of such measurements since photobleaching and various dyeFig. 4.19Calcium imaging in living cells. A fertilisation-induced calcium wave in the egg of the starfish. The egg
was microinjected with the calcium-sensitive fluorescent dye fluo-3 and subsequently fertilised by the addition
of sperm during observation using time-lapse confocal microscopy with a 40water immersion lens and a
LSCM. An optical section located near the egg equator was collected every 4 s using the normal scan mode
accumulated for two frames, and afterwards the images were corrected for offset and ratioed by linearly
dividing the initial pre-fertilisation image into each successive frame of the time-lapse run. The ratioed images
were then prepared as a montage and outputted with a pseudocolour look-up table in which blue regions
represent low ratios and free calcium levels, and red areas depict high ratios and free calcium levels. Note that
the wave sweeps through the entire ooplasm, rather than being cortically restricted. (Image kindly provided
by Steve Stricker, University of New Mexico, USA.) (See also colour plate.)127 4.5 Measuring cellular dynamics