Microscopy101
Figure 2 : Digital processing of stacked raw images generated according to the method illustrated in Figure 1 . Fusion of the images and subsequent creation of an anaglyphic photograph may be carried out using the program Picolay [ 8 ].
Collection of data for subsequent construction of 3D (stereo) images can be conducted in two principal ways. Stereo-pair method . T e fi rst method records images of an object from two perspectives with a small angle in between. For this purpose, the object is recorded in its “normal” position by placing the slide with the item to be studied on the sample stage. For the second image, the slide is slightly rotated about the light axis of the microscope (rotation angle: 5 – 10°), and the same object is recorded a second time. For these two images, an identical setup (magnifi cation, light intensity, etc.) must be used. T is method for 3D imaging is most useful for the case of small items exhibiting spherical or close-to-spherical geometry. Image stack method . T e second method for 3D imaging is useful for anisometric objects. Here the selected object is recorded in the same position, but the focus plane is moved step-wise through the object creating an image stack ( Figure 1 ). Photographs with a number of diff erent focus planes within a small transparent object require use of rather high objective magnifi cations (for example, 40×, NA ~ 0.65) to limit the depth of fi eld captured at each focus plane. In principle, distances between the focus planes depend on the size of the object, but
they should be on the order of a few micrometers. For stereo imaging, it is highly recommended to make a stack containing 10 to 20 images. Production of 3D images . For the generation of anaglyphic images viewable with special red-blue glasses, stereo pairs (method 1) or stacked images (method 2) must be processed digitally using appropriate computer soſt ware. A stereo pair of images may be transformed into an anaglyphic photograph via various dedicated computer programs (for
example, Anaglyph Maker;
https://anaglyph-maker.de.soſt onic. com/ ) or with Adobe Photoshop TM [ 3 – 7 ]. In the latter case, tutorials explaining the steps of anaglyph generation are available online (for example,
http://designinstruct.com/photography/3d- anaglyph-photoshop/ ). For processing of stacked images into an anaglyphic photograph, the freely available computer code Picolay (
www.picolay.de ) [ 8 ] is a powerful digital tool. T is program commonly enables the processing of high numbers of raw pictures and off ers numerous opportunities for digital image manipulation ( Figure 2 ).
Results
Radiolaria . In paleontology, 3D images of microfossils can be used for detailed descriptions of shell structures and for a more secure determination of single species. Radiolarians, especially those with globular shells, have become favorite items for 3D imaging in recent years. T is is because they can be easily photographed by applying the methods of stereo pairs or stacked images noted above ( Figure 3 ). T e fossil shells of these microorganisms commonly vary between several tens and several hundred micrometers in size and are therefore
Figure 3 : Anaglyphic photograph of globular Radiolaria with typical spines that provide buoyancy of these unicellular microorganisms. By using low magnifi cation (4×, NA ~ 0.10), generation of stereo pairs was possible by method 1. Microscope: NIKON Eclipse, 40×, NA ~ 0.65. Image width = 200 µm. Semi-images are copyright of and used with permission of Wim van Egmond,
micropolitan.org.
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Figure 4 : Detailed view of a rat neuron and associated dendritic processes stained with silver. The 3D image was dendritic from an image stack containing 10 pictures. Microscope: ZEISS Primotech, 40×, NA ~ 0.65. Image width = 200 µm. Semi-images are copyright of and used with permission of Eberhard Raap,
micropolitan.org.
www.microscopy-today.com • 2017 July
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