Basics of Confocal Reflection Microscopy

When many biomedical research think "confocal microscopy", they usually have fluorescence imaging in mind. This is a very good reason for this seemingly obvious connection. A majority of the common 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 multiply-labeled cells and tissues.

Confocal reflection microscopy can be utilized to gather additional information from a specimen with relatively little extra effort, since the technique requires minimum specimen preparation and instrument re-configuration. In addition, information from unstained tissues is readily available with confocal reflection microscopy, as is data from tissues labeled with probes that reflect light. The method can also be utilized in combination with more common classical fluorescence techniques. Examples of the latter application are detection of unlabeled cells in a population of fluorescently labeled cells and for imaging the interactions between fluorescently labeled cells growing on opaque, patterned substrata, as illustrated in Figure 1.

The digital image presented in Figure 1(a) illustrates a glial cell growing on a silicon substratum that is patterned with small 1-micrometer high pillars and imaged using confocal reflection microscopy. The cell is immunofluorescently labeled for vinculin (red) and glial fibrillary acidic protein (GFAP, green). Figure 1(b) depicts neurons growing on a similar silicon substratum and labeled with an antibody to microtubule associated protein 2 (MAP-2) and a fluorescein-labeled secondary antibody. Again, the surface of the silicon substratum was imaged with confocal reflection microscopy.

A major attraction of confocal reflection microscopy for biomedical imaging is the ability to image unlabeled living tissue. In fact, the technique has been utilized to image a variety of different tissues, including brain, skin, bone, teeth, and eye tissue. Confocal reflection microscopy 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 micrometers into the living cornea and lens using long working distance water immersion objectives.

Imaging unstained specimens with confocal reflection microscopy was very common for designers of early confocal instruments prior to the emergence of epifluorescence techniques. Both the laser scanning confocal microscope (LSCM) and the Nipkow spinning disk microscope can be utilized in confocal reflection mode. 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. This 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 the reflection artifact. It can be avoided by scanning the specimen in a region away from the optical axis of the microscope and zooming the bright spot out of the frame. Alternatively, the reflection can be removed from the image by digitally subtracting a background image of the spot or by flat-field correction. Another method for removing the troublesome artifact is to apply polarizing filters to the instrument in order to eliminate the reflection from optical elements.

A traditional biological application of widefield reflected light imaging is for observing the interactions between cells growing in tissue culture on glass coverslips using a technique termed interference reflection microscopy. 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. The proteins associated with the focal contacts are analyzed utilizing immunofluorescence, and the contacts themselves can be viewed using interference reflection microscopy.

Cell-substratum adhesions are viewed in a similar manner using confocal reflection microscopy, as illustrated in Figure 2. Again, the interface between the coverslip and the cell is imaged. This surface can be difficult to locate in the confocal microscope, but the highly reflective coverslip can be employed as an aid in focusing. The cell-substratum interface can be located by focusing in fast scan mode, and the adhesions appear just after the bright flash of the coverslip when penetrating the cell layers. Care should be taken not to overload the photomultiplier tubes from the very bright coverslip surface when focusing in this manner.

Presented in Figure 2 are interference reflection digital images captured with confocal reflection microscopy techniques. Figure 2(a) illustrates a single optical section of a chicken heart fibroblast cell imaged at the cell-substratum interface. The dark streaks at the periphery of the cell are focal contacts. An x-z section of a cell similar to the one shown in Figure 2(a) is depicted in Figure 2(b). Note that the coverslip in Figure 2(b) is extremely bright. This appears as a flash when focusing the microscope (red arrow), and the cell-substratum interface is imaged directly after the flash.

Recently, using a related technique, improved images of filopodia were collected from PC12 cells (rat pheochromocytoma) by growing the cells on a more reflecting substrate. The technique, termed backscatter-enhanced reflection confocal microscopy, produces images that resemble those collected using traditional differential interference contrast (DIC) microscopy. Using this method, unstained cells can be observed growing on opaque substrata, such as silicon chips, a technique that is impossible with conventional transmitted light DIC imaging.

One advantage of using reflected light, rather than fluorescence, for live-cell imaging is the absence of photobleaching artifacts in the reflected light mode. Attention should still be paid to the threat of photodamage to the living specimen, however, and all of the precautions for imaging live cells should be taken. Confocal reflection microscopy has been successfully utilized to follow cell migration through a collagen matrix and collagen fibrillogenesis in vitro, using time-lapse imaging of z-series and subsequent three-dimensional reconstruction (termed 4-D imaging). These methods take advantage of the high reflectance or albedo of the collagen biopolymer.

A drawback of confocal reflection microscopy (when compared with confocal fluorescence imaging) is the lack of specific probes that differentially reflect light for multiple label experiments. It is difficult to improve upon the multiple-wavelength probes that are available for immunofluorescence and live-cell imaging. Probes that can be used in reflected light mode for single label experiments include gold particles, peroxidase labels, and silver grains.

As an example, confocal reflection microscopy offers a significant improvement over conventional brightfield or darkfield microscopy for imaging silver grains in autoradiograms of specimens prepared for in-situ hybridization (Figure 3). The out-of-focus signal from the silver grains throughout the emulsion is effectively eliminated from the in-focus image of the silver grains associated with the riboprobe by optical sectioning the emulsion using confocal reflection microscopy.

Illustrated in Figure 3 are several digital images collected from confocal reflection microscopy experiments with silver grains. The specimen is peripheral blood cells from an HIV-infected individual prepared for in-situ hybridization and stained with Giemsa. Figure 3(a) illustrates the preparation under standard brightfield illumination, while Figure 3(b) shows the same field in darkfield illumination with a significant amount of out-of-focus debris. The results with confocal reflection microscopy (of the same viewfield) are presented in Figure 3(c). Note that the out-of-focus debris is not imaged in this mode. For comparison purposes, specimens can also be imaged with differential interference contrast, in addition to brightfield and darkfield illumination.

Because the reflected light confocal image and the transmitted (brightfield and darkfield) images are collected simultaneously using the same scanning laser beam (in Figure 3), they are in register with one another and can be digitally merged into a single image. The transmitted light brightfield image records the total cell population either as a grayscale image or a real-color image (provided a real-color transmitted light detector is fitted to the microscope). The darkfield illumination image incorporates a signal from the entire population of silver grains in the complete emulsion, including any contaminating dust particles on the glass surfaces. The confocal reflection microscopy image, in contrast, records only those cells labeled with the probe (Figure 3(c)), and hence is a more accurate measure of the labeled cells than the transmitted light images. Moreover, it is more amenable to quantification of the probe, because the image consists of discrete regions of saturated pixels (signal from the probe) on a black background, and is somewhat reminiscent of a fluorescent image in this respect.

The confocal reflection microscopy images are digitally merged with the transmitted light images for display purposes and estimate the number of labeled cells in the total cell population. In most confocal systems, the transmitted light image is collected in one of the channels of the RGB image (usually the blue channel), and a reflected light image, or one or more fluorescent images, is collected simultaneously with it, usually into the red and green channels. Displaying the transmitted light image in this manner produces a colored background, as illustrated in Figure 4(a), instead of the more familiar gradient of gray levels of a typical brightfield or DIC image, or the black background of a darkfield image. A more realistic version of the transmitted light image can be obtained by pasting it onto a different layer from the reflected light image using a digital image editing program such as Adobe PhotoShop (shown in Figure 4(b)).

Confocal reflection microscopy is usually employed in addition to fluorescence to add context to fluorescence images, which can be rather abstract when viewed in isolation (especially confocal fluorescence images, which can consist of a few white pixels on a sea of black). In the examples presented above, the combination of the specific labeling of immunofluorescence and the optical sectioning power of the confocal microscope can actually hamper the interpretation of the images themselves, unless they are digitally merged with a reflected light, transmitted light, or counter-stained fluorescence image.

Although confocal reflection microscopy has limited applications in biomedical imaging, it can sometimes provide additional information from specimens that reflect light or have significant fluctuations of refractive index at certain boundaries. This useful technique, along with transmitted light imaging, may be worth a try when imaging fluorescently labeled specimens with a confocal microscope.

This article originally appeared in BioTechniques: Vol. 32, 274 (2002).


Contributing Authors

Stephen W. Paddock - Laboratory of Molecular Biology, Howard Hughes Medical Institute, University of Wisconsin, Madison, Wisconsin 53706.

This article originally appeared in BioTechniques: Vol. 32, 274 (2002).