Chemiluminescence has become a standard tool in biomedical research. Chemil
uminescent probes are used for immunoassays, nucleic acid identification, r
eporter gene assays, measuring enzyme activity, and the detection of ions a
nd small molecules such as Ca2+, ATP, NO, O-2(-), and H2O2. Along with the
development of new chemiluminescent probes, significant progress has been m
ade in techniques to measure chemiluminescence. Ultra-sensitive photometers
or luminometers have become widely available and can be obtained with auto
matic injectors and microplate readers. In addition, imaging photon detecto
rs have been developed that allow the imaging of chemiluminescence from gel
s, blots, and microplates. Imaging photon detectors have also been attached
to microscopes and allow imaging of chemiluminescent probes and reporter g
enes in cells and tissues. Specific methods of photon collection, storage,
and analysis have been developed for microscopic imaging of chemiluminescen
ce. Two of these methods are discussed in detail. The first is a method of
data storage that allows days of continuous imaging without creating oversi
zed files. The second is a method for calibrating photon imaging microscope
s using a low-light standard. Such calibration will be helpful for comparin
g the performance of various photon imaging systems and for comparing data
obtained in different laboratories.