Biological imaging using fast laser scanning heterodyne differential phase confocal microscopes

Abstract

Differential phase microscopy has proved invaluable in the study of live, unstained, thin biological samples because of its ability to image changes in refractive index and topography. Similarly, because of its optical sectioning capability, confocal microscopy is now a well‐established technique in the study of relatively thick live biological samples. This paper describes the development and application of two differential phase heterodyne confocal microscopes, and compares their performance. The use of these systems for imaging in‐vitro cell and tissue cultures is considered and compared with confocal reflection microscopy. It is demonstrated that the differential phase capability reveals subcellular structural information not readily seen in the confocal reflection images. This technique opens up the possibility of imaging thick unstained live tissues, avoiding cell damage and artefacts associated with staining procedures. Furthermore, the differential phase images can be used to provide a visual frame within which stained features can be located.