Confocal reflection microscopy : The other confocal mode

When most biomedical researchers think “confocal microscopy”, they usually have fluorescence imaging in mind. There is a very good reason for this connection. Many biomedical applications of the confocal microscope have utilized its optical sectioning power, combined with the exquisite specificity of immunofluorescence or fluorescence in situ hybridization (FISH) to produce improved images of multiple labeled cells and tissues. Confocal reflection microscopy (CRM) can be used to glean additional information from a specimen with relatively little extra effort, since it calls for minimal specimen preparation and instrument configuration. CRM provides information from unstained tissues, tissues labeled with probes that reflect light, and in combination with fluorescence. Examples of the latter would be for detecting unlabeled cells in a population of fluorescently labeled cells or for imaging the interactions between fluorescently labeled cells growing on opaque, patterned substrata (Figure 1). A major attraction of CRM for biomedical imaging is the ability to image unlabeled live tissue. In fact, CRM has been used to image many different tissues, including brain, skin, bone, teeth, and eye tissue (2). CRM works especially well for imaging the cornea and lens of the eye because they are transparent. For example, optical sections have been collected from as deep as 400 μm into the living cornea and lens using a long working distance water immersion lens (8). There has been a history of using CRM for imaging unstained specimens since it was one of the only modes available to the designers of the early confocal instruments in the time before epifluorescence (9). Both the laser scanning confocal microscope (LSCM) and the spinning disk microscope can be used for CRM. The spinning disk microscope has the advantage that images can be collected in real time, viewed in real color, and lack a reflection artifact that is sometimes present in the LSCM. The artifact appears as a bright spot in the image and is caused by reflection from one or more of the optical elements in the microscope. There are several remedies for this artifact. It can be avoided by scanning away from the optical axis of the microscope and zooming the bright spot out of the frame. It can be removed from the image by digitally subtracting a background image of the spot away from the image of interest. Alternatively, polarizing filters can be added to the instrument to eliminate the reflection from optical elements (12). A traditional biological application of wide-field reflectedlight imaging is for observing the interactions between cells growing in tissue culture on glass coverslips using a technique called interference reflection microscopy (1). Here, the adhesions between the cell and its substratum are visible at the interface of the glass coverslip and the underside of the cell. These regions of cellular adhesion continue to be a research area of great interest (5). The proteins associated with the focal contacts are analyzed using immunofluorescence, and the contacts themselves can be viewed using interference reflection microscopy. Cell-substratum adhesions are viewed in a similar way using CRM (Figure 2). Again, the interface between the coverslip and the cell is imaged (10). This surface can be hard to find in the confocal microscope, but the highly reflective cov-