The polarizer determines polarization of the excitation beam, and polarization of the detected fluorescent light. The excitation beam (488 nm, Ar laser) is focused by an objective into a small volume in the cell. The fluorescent light from this volume is detected by a photomultiplier tube (PMT1) in the spectral region 510-550 nm selected by interference filters (channel #1). A pinhole discriminates against the regions above and below the selected volume. The pinhole size is adjusted depending on magnification and numerical aperture of the objective. For example, for a x60 objective with NA=1.4, the pinhole diameter is set at 100 µm. The focused beam scans the sample both in the horizontal plane, located at a fixed depth z, and in the vertical plane. The intensity of fluorescence as the function of coordinates is measured and stored in the computer memory. Each horizontal scan produces a thin "optical slice" with thickness determined by the resolution of FCPM.
Simultaneously with the FCPM image, a standard polarizing-microscope texture of the same sample can be recorded by measuring the intensity of light that passes through the polarizer P, sample, and a crossed analyzer A. The intensity of transmitted light is detected by PMT2, channel #2. Note that the wide view polarizing microscope textures can be obtained in the transmission mode while the FCPM textures - in the reflection mode.
Spectral Separation of Excitation and Fluorescence Light in the FCPM is done with use of the interference filters