MicroscopyEducation
biology labs. Since stomata open and close in response to vari- ous stimuli, a number of experiments can be configured in class demonstrations or as science projects designed by indi- vidual students.
Materials and Methods Microtopographic representations of leaves. Much
of the light microscopy literature shows stomata in a two- dimensional format. Scanning electron microscopy (SEM) can show stomata in highly detailed images with great depth of field. Although SEM images are of high quality, the prepara- tion required for electron microscopy limits the rate at which images can be produced. Te images in Figures 3–6 were taken using light micros-
copy with the addition of focus stacking soſtware [9] to rapidly produce all-in-focus (high depth-of-field) images of planar and oblique views. Figures 4a and 4b are red-cyan stereo anaglyphs that show oblique representations of the surface. Heights of various features were measured above or below a local datum in a planar view using the light microscope without focus stacking (Figure 4b). Koehler illumination was used. Figures 3, 5, and 6 show stomata from three different plants that grow in and around San Francisco, California. Specimen preparation. To see stomata and measure the
sizes of their openings, leaf surfaces may be examined by mak- ing varnish replicas or by examining the epidermis of the leaf aſter it has been peeled or shaved from the leaf (Figure 2). In some cases, stains and other chemical treatments are used [10]. Tere are many videos and written instructions for these meth- ods available on the Internet. Te specimens shown below were all taken from the undersides of leaves. In this work, leaf specimens were acquired and prepared
during sunlight hours. Stomata open and close very slowly. No effort has been made to capture specimens with the stomata in any particular state; however, the study of stomata under vari- ous stimuli or environmental conditions could form the basis of school laboratory projects. A homemade stage microtome was used to obtain single
transverse cross sections of leaves. Cross-sectional views were taken by embedding leaf sections in paraffin and then cut- ting thin sections perpendicular to the plane of the leaf. Te sections were approximately 125 μm thick. Te sections were placed on a microscope slide and surveyed for stomata. See
http://paedia.com/
Ficus_vid.html for more information about the stage microtome. Microscopy equipment. Tis study employed a Nikon
Eclipse LV100 microscope with a trinocular port and a z-axis micrometer indicator. Nikon LU Plan Fluor objective lenses were used. A Point Grey Grasshopper camera was mounted on the trinocular port and controlled by a personal computer with the Windows 10 operating system. Images from the cam- era were stored on the computer’s hard drive. Image originals were 2448×2048 pixels, 8 bits/pixel, except for Figure 4, which was 1224×1024 pixels, 8 bits/pixel. Image acquisition. To perform focus stacking, a series
of images (a stack) with overlapping focal depths is required. Te images in the stack are then processed mathematically to produce an all-in-focus result. Most of the images in this work were taken through a 50×objective with a depth of field
14
Figure 4: Agave “Blue Glow” leaf surface that has been peeled from a leaf. (a) Red (left eye)-cyan (right eye) anaglyph showing an oblique view of a stoma on the leaf underside. (b) Depth indications are shown in micrometers. Depth indications were obtained from a planar view using a microscope’s stage z-axis micrometer
of 1.2 μm. Te following steps were used to acquire and pro-
cess an image: 1. Prepare a slide with a cast, peeled, or shaved plant epidermis. 2. Locate a stoma of interest. 3. Focus on a point above the stoma and note a first reading (in μm) of the stage micrometer.
4. Focus on a point below the stoma and note a second reading of the stage micrometer.
5. Subtract the first and second readings and divide by 1.2 μm, the depth of field of the objective lens. Te result is the number of depths of field between the first and second readings. Focus stacking requires that the depths of field of images overlap, so at least one image must be added to the result. I usually add more than one extra image. Extra images in a stack are pro- cessed, but their contributions are redundant and have no effect on the final image. For example, if the result of this calculation is 10 images, I will include 15 images in a stack in order to ensure overlap of depths of field.
6. With the stage micrometer at one extreme of focus, acquire a first image. Advance the stage to the next calculated position and take a second image. Continue doing this until all the images in a stack are collected and stored on the computer’s hard drive.
7. Start the focus stacking soſtware and import a stack of images into it. 8. Start the rendering process in the soſtware. When rendering is complete, the soſtware presents a final, all-in-focus image that can be saved. Addi- tional processing creates orthogonal and stereo images.
www.microscopy-today.com • 2019 January
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