Cell morphology and its function are closely associated [1]. Therefore, observing the cell morphology is common in cellular biology. However, the height and slope in the z-direction (vertical direction) of cells adhering to a culture dish or plate via the extracellular matrix cannot be measured by ordinary optical and electron microscopy [2].

Typically, confocal microscopy and atomic force microscopy are used to observe the three-dimensional morphology of adherent cells [3-5]. Confocal microscopy is an optical imaging technique used to increase optical resolution by using a spatial pinhole to eliminate out-offocus light in cells that are thicker than the focal plane. It enables the reconstruction of three-dimensional structures of cells from multiple images, although it requires cell staining prior to observation. Atomic force microscopy uses a deflectable cantilever as a force sensor. Cell height is physically measured on the basis of the cantilever deflection, providing a three-dimensional surface geometry of adherent cells. However, the device requires substantial time—typically, several minutes—to scan a wide range of area that covers multiple cells.

Here we describe a simple, alternative technique using a rotating microscope stage to evaluate the threedimensional surface geometry of adherent cells. A dish supporting cultured cells observed using oblique lighting (similar to Mempel et al. [6]) was rotated using a motor. Because of the oblique lighting, the stage rotationdependent movements of the start and end points of a shadow behind the illuminated cells determine the relative height differences between points on the cell surface. Thus, the combination of oblique lighting and a rotating stage allows for the rapid evaluation of the cell surface geometry over a large surface covering multiple cells.

A shadow with enhanced contrast was created by slightly displacing the condenser annulus within the horizontal plane. The shadowing effect from the resultant oblique lighting provided cell images similar in appearance to those taken by differential interference contrast (DIC) microscopy and reflected light oblique transillumination microscopy [6] (Figure 2). The pinion was slowly rotated

within the horizontal plane from 0˚ to 360˚ using the stepping motor by rotating the gear wheel in the opposite direction. The dish fixed on the gear wheel was rotated as well while cell images were acquired (Figure 3). After measuring the center of the circular trajectory from the acquired images, the accurate planar angle of oblique illumination was determined from the trajectory center. The individual images were then rotated using Vision considering the calibrated illumination angles. As a result, sequential images with cells illuminated from different angles were obtained (Figures 4 and 5). The image analysis was performed to uniquely determine the relative heights between arbitrary points along the cell surface from the shadows behind cells created by the oblique illumination. The consequent relative heights were visualized using AVese software, yielding the surface geometry of cells (Figure 6). The highest and lowest points in the image were defined to be 1 and 0, respectively.

Phase-contrast microscopy is a common technique in which small phase shifts in the light passing through transparent cells are converted into contrast changes in the image. In this system, the condenser annulus is cen-

tered with respect to the matching objective phase plate. Here by slightly displacing the condenser annular ring within the horizontal plane, we obtained cell images with an enhanced contrast shadow titled to one particular direction, similar to those obtained by reflected light oblique transillumination microscopy [6] (Figure 2). By tracking the shadow of cells subject to horizontal rotations using a motor (Figures 3-5), we demonstrated that the surface geometry of the cells could be determined (Figure 6).

Confocal microscopy and atomic force microscopy are often used to measure cellular surface geometry [3-5]. While they are highly accurate, they have some limitations. Confocal microscopy requires fluorescent markers introduced or expressed in cells to acquire the images. Atomic force microscopy typically takes a long period of time—several minutes—to obtain such measurements. The present technique using oblique lighting and a rotating stage provides an alternative for estimating the surface geometry of unstained cells. With its semiautomatic image analysis, the evaluation of surface geometry of multiple cells is rapid, within tens of seconds for the steady stage rotation. Practically, this system is less expensive than confocal and atomic force microscopy because the surface geometry evaluation relies on subsequent image analysis after acquiring cell images under steady planar rotation of the stage.

The images obtained by the oblique lighting technique are also similar to those by DIC microscopy, which is widely used to enhance the contrast in unstained cells and observe planar morphology. However, DIC microscopy is not applicable to the observation of living cells in plastic culture dishes because of their birefringence. Thus, the combination of oblique lighting and phasecontrast microscopy may be more versatile and suitable for obtaining three-dimensional morphology of cells placed in any transparent material.

Rotation systems for cell structure observation have been reported by Matsumoto and colleagues [7-10] in which an individual cell is captured using a glass pipette and then rotated about the pipette’s axis to observe its three-dimensional structure. Sophisticated feedback controllers and fluorescence imagers are used in this system, which allow for the acquisition of highly accurate images. In contrast, the present system has no feedback controller and is equipped only with a stepping motor because the surface geometry evaluation relies on subsequent analysis of acquired images. The current system was developed to analyze the surface structure of multiple cells adherent to a flat (glass or plastic) plate. Thus, while our rotation system is unable to identify intracellular structures with high resolution, it is particularly well suited for collecting large amounts of data on epithelial structure and changes in adherent cell height induced by pharmacologic agents [4,5].

In conclusion, the present study proposed a technique for the easy estimation of the surface geometry of adherent cells. Living cell images were obtained by oblique transillumination, while the stage supporting the cells was rotated using a motor and a custom-made gear wheel and pinion. Changes in the shadow formed behind the cells were analyzed to evaluate relative height differences between points along the cell surface. Thus, the present system allows for the measurement of the surface geometry of adherent cells within a short time period over tens of seconds. Absolute values of the cell heights can be obtained with additional calibrations and here are expressed as relative values.

The authors thank Yoshihiko Tamura for his assistance in manufacturing the apparatus and Seiichi Washio and Satoshi Takahashi for helpful discussions. SD gratefully acknowledges the support from the Japan Science and Technology Agency. This work was partly supported by Grants-in-Aid for Scientific Research from the Japan Ministry of Education, Culture, Sports, Science, and Technology (#21680039 and #20001007).