Multicolor Laser Scanning Confocal Immunofluorescence Microscopy: Practical Application and Limitations

Advances in fluorescent probe chemistry, economical laser availability, and confocal microscope instrumentation are making the enormous potential of multicolor laser scanning confocal microscopy (LSCM) available to a wider range of biologists. This chapter outlines the requirements for performing multicolor immunofluorescence studies with LSCM. The principles of immunofluorescence histochemistry necessary for the preparation of multilabeled specimens are summarized. The chapter examines the technical aspects of confocal microscope instrumentation that affect its application to multicolor studies. The practical application and limitations of this technology are demonstrated for multicolor LSCM, using the inexpensive, air-cooled argon ion and krypton-argon ion lasers as light sources are presented and various aspects of confocal microscopy that affect the comparison of images acquired with different excitation and/or emission wavelengths are examined. The chapter provides a better understanding of the compromises needed to effect the accurate imaging of specimens stained with multiple fluorophores. The chapter also discusses the applications and limitations of multicolor laser scanning confocal immunofluorescence microscopy.