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Microscopy101


Stereoscopic Photography in Transmitted Light Microscopy Robert Sturm


University of Salzburg, Department of Physics and Biophysics, Hellbrunnerstr. 34, A-5020 Salzburg, Austria Robert.Sturm@stud.sbg.ac.at


Abstract: The present contribution describes two techniques for the effi cient generation of scientifi cally useable 3D images. The fi rst method is the production of stereo pairs by photographing a microscopic object from two slightly different perspectives, achieved by rotating the sample slide by 5–10°. The second method involves the generation of an image stack, where the focus plane is stepped through a transparent object. At each position of the focus plane a separate image is recorded. Stereo pairs or stacked images are subsequently converted to anaglyphs using specifi c computer software. Applicability of the described techniques is demonstrated with several examples.


Introduction Scientifi c photography is frequently employed to produce realistic undistorted depictions of the spatial extent of an object. T e spatial dimensioning of an object can be surmised only partially from a single image; whereas, three- dimensional (3D) information can be stored with the help of two semi-images (now called a stereo pair) that portray the object from two slightly diff erent perspectives. If these semi-images are separately directed to the eyes, so that the leſt eye exclusively perceives the leſt image and the right eye is confronted with the right image, the primary visual cortex initiates a fusion of the pictures using its specifi c “spatial sense.” Viewing of these two-dimensional (2D) images together thus evokes the identical reaction in the brain as would emerge from a glance at the object itself [ 1 , 2 ]. Stereoscopic viewing . T e stereoscopic principle was developed over many years starting in the 17th century and climaxing in the second half of the 19th century, when photog- raphy advanced to a routine method. In the latter period, the classical stereoscopic technique using two semi-images placed side by side was enhanced by alternative presentation methods. For example, the well-known anaglyphic method uses the fi lter colors red and green, red and cyan, or red and blue for the pair of images. By application of bicolored glasses with an identical combination of fi lter colors, each eye perceives the corresponding image, resulting in the generation of a spatial impression [ 3 – 7 ].


Both the classical stereoscopic technique and the anaglyphic method have been used in scientifi c photog- raphy from the beginning of the 20th century. During recent decades, stereophotography received more attention in microscopy because a comprehensive description of small objects demands it [ 8 – 10 ]. Stereo microscopy . In scanning electron microscopy (SEM), 3D photography is facilitated by tilting the sample holder.


46


T is method has been used to produce millions of 3D images of small objects ranging in size from 50 nm to 5 mm [ 7 , 11 ]. In light microscopy, however, this optical procedure is frequently stretched to its limits, because viewing of embedded objects from two diff erent perspectives is possible only to a limited extent [ 4 – 7 ]. Transmitted light microscopy is commonly used for the study of transparent objects between 1 µm and 100 µm in size. T e present article describes two simple methods for the production of high-quality stereo images using the transmitted light microscope along with suggestions for computer soſt ware enabling the fast preparation of anaglyphs of stereo pairs.


Material and Methods


Typical items examined in stereo by transmitted light include microfossils (for example, radiolarians, diatoms, etc.) and microorganisms (for example, bacteria, animal and plant cells, etc.). As outlined in detail in previous publications [ 4 – 7 ], these objects are usually embedded in a highly viscous substance with high light refraction (for example, Canada Balsam, n = 1.53) to accentuate their contours under the microscope.


Figure 1 : Generation of stereo images with the transmitted light microscope. A transparent object (e.g., a radiolarian) is recorded at different focus planes (1–3), where the distance between two focus planes (dashed lines at the top) is a few micrometers.


doi: 10.1017/S1551929517000621 www.microscopy-today.com • 2017 July


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