displayed as series ofZ-projections or stereo movies. Multidimensional experiments
can present problems for handling large amounts of data since gigabytes of infor-
mation can be collected from a single 4D imaging experiment.
4.5 Measuring cellular dynamics
Understanding the function of proteins within the context of the intact living cell is
one of the main aims of contemporary biological research. The visualisation of
specific cellular events has been greatly enhanced by modern microscopy. In addition
to qualitatively viewing the images collected with a microscope, quantitative infor-
mation can be gleaned from the images. The collection of meaningful measurements
has been greatly facilitated by the advent ofdigital image processing. Subtle changes
in intensity of probes of biochemical events can be detected with sensitive digital
detectors. These technological advancements have allowed insight into the spatial
aspects of molecular mechanisms.
Relatively simple measurements include counting features within a 2D image or
measuring areas and lengths. Measurements of depth and volume can be made in 3D,
4D and 5D data sets. Images can be calibrated by collecting an image of a calibration
grid at the same settings of the microscope as were used for collecting the images
during the experiment. Many image processing systems allow for a calibration factor
to be added into the program, and all subsequent measurements will then be
comparable.
Single wavelength
time-lapse Multiple wavelength
Single wavelength Z series
Z
focus
time wavelength
t 1
t 2
t 3
t 4
X
(a) (b) (c)
Y
Fig. 4.18Multidimensional imaging. (a) Single wavelength excitation over time or time-lapseX,Yimaging;
(b)Z-series orX,Y,Zimaging. The combination of (a) and (b) is 4D imaging. (c) Multiple wavelength imaging.
The combination of (a) and (b) and (c) is 5D imaging.
126 Microscopy
