Advances in light microscopy have lead to a steadily improving ability to visualize the features of unstained biological specimens over the last fifty years. Most of this progress has represented techniques that are appropriate for transparent specimens. However, for some specimens, such as bacteria on agar, biofilms on metal surfaces, and eukaryotic cells on metallic substrates, such as silicon chips, it is desirable to have modalities of microscopy usable with living tissues that use reflected light. For such specimens the options available include epifluorescence microscopy of immunostained cell surface proteins, lipophilic dyes, or fluorescent analogues of cellular proteins, incident differential interference contrast microscopy, and reflection mode microscopy. All of these techniques have limitations in terms of tissue damage, generality, or contrast available with biological specimens.
The investigators in this project have found that thin biological specimens show substantially improved contrast in the reflection mode of the confocal microscope when grown on specularly reflective substrates, such as metal-shadowed glass. However, the contrast and resolution of images still remain sub-optimal. Based on the reasoning that a modification of the standard reflection confocal microscope, designed to illuminate the specimen with a plane-parallel beam of light, would give substantially improved image contrast, this project will design and construct a modified illumination system for a confocal laser scanning microscope to compensate for the convergence of the incident beam introduced by the microscope objective, and will test this modified reflection confocal microscope with a variety of biological specimens on reflective substrates. In addition, the project will investigate replacement of the reflective substrate with multilayer dichroic substrates, and test their effectiveness in backscatter-enhanced reflection confocal imaging of a variety of specimens, including cells and polytene chromosomes.