Microscope image processing techniques

Images of thin section, fungal specimens require attention to a variety of different factors due to the variation of thickness of the thin sections, including lighting and location of the organisms in study. Many of the microorganisms can range in size from 1 to 500 µm, and the smaller organisms are difficult to relocate on a slide, thus diligent notes and location information is crucial. Due to this variation of thickness and the three-dimensionality of fungi and fungal-like organisms within these sections, post image processing is often required.

4.1 Lighting and filters

Due to the variation of thickness of an individual thin section as well as the ample presence of minerals (in contrast to acetate peels where the mineral content was dissolved during the etching process), lighting and filters should be taken into consideration while examining in transmitted light. Digital images can be taken with a Leica DC500 digital camera attachment.

Using a white filter while taking images of thin sections is essential. If a proper filter is not

available, then a sheet a plain white paper can be used as a substitute. The paper can be placed under the slide on the stage of the microscope. The filter or white paper is useful in avoiding a vignetting effect while focal stacking images (see section 4.3.1) (Figure 4h).

4.2 Coordinate system

Because of the variability in the size, microorganisms are often difficult to relocate on a thin section or slide. Thus, one of the most beneficial and crucial steps in documenting fossil fungi is using a standard x-axis and y-axis coordinate system on a microscope stage. This

practice is commonly used in palynological studies where similar to fossil microorganisms, small microscopic objects such as pollen and spores are difficult to find again (e.g., Holt et al., 2011).

Therefore, through the use of the coordinate system on the microscope stage, one is able to find and locate the desired object again without delay even at high magnification. It is also

recommended to either write the coordinates down for specific specimens in a lab notebook or within the file name of the image taken.

4.3.1 Focal stacking techniques

All digital images in this study were taken with a Leica DC500 digital camera attachment and minimally processed using Adobe Photoshop CS6 Version 13.0 x64 (1990-2012, Adobe Systems). Multiple micrographs of the same specimen at different focal planes were compiled to produce composite images (e.g., Bercovici et al., 2009). The images were stacked in Adobe Photoshop CS4 and specific areas were erased to reveal the full three-dimensional view that can be seen through the thin sections. Measurements were taken using ImageJ 1.43u software (Abràmoff et al., 2004).

When taking multiple images at different focal planes, it is important to maintain the same magnification and not to move the stage in the x-axis or y-axis direction. The only

difference for focal planes is using the z-axis setting. Additionally, all of the light setting for the microscope must be consistent and all of the images must be the same pixel dimensions. Last, the order that the images taken is important; thus when naming the digital images, label them in a sequential order from the begging (lowest z-axis plane) to the end of the focal plane sequence (highest z-axis plane).

When importing images into Adobe Photoshop, using the example of 6 images to be focal stacked, import all 6 images into one file but with each individual image as a separate layer; with the first image in the focal stack sequence as the first layer, and the last image in the sequence as the last layer (Figure 5a). Hiding the 3^rd through 6^th layer (and all other layers not in use), there should be only two open or active layers while processing an area (Figure 5b).

Selecting the second image/layer, one can either decrease the opacity of the layer by selecting

“Opacity” and decreasing it using the slider arrows, or the preferred method of changing back and forth between layers by making this visible or invisible. Open the layer that is higher in sequential order, or the second layer in this example and select the eraser tool. The eraser tool should be an appropriate diameter for the area of interest and most importantly, should be: Brush mode, “Hard Round” with 53% Opacity and 57% Flow, these are all options that can be selecting using the eraser tool. This will provide a blurred eraser tool that is best used for blending two or more layers together. Then working on the second layer, begin erasing areas that are visible in the previous layer but not the in second layer, thus revealing the full three-dimensionality through all focal planes (Figures 6a, 6b). When finished with the second layer, make the third layer visible and repeat the previous steps until the last and final layer is finished. Eventually, the full effect of focal stacking will be revealed and a more complete image will be produced

(Figures 7a, 7b). It is important while focal stacking images, to only stitch together images from a sequence of images within a different focal planes of the same object, and not to change the object completely through too much image manipulation or processing.

4.3.2 Adobe Photoshop vs. Helicon Focus

There are specific programs that will automatically stack images together to make composite image results. One of these is the Helicon Focus 4.2.9 x64. Once a sequence of images at different focal planes is imported this program will digitally stitch them together to create a complete composite image. Several images are digitally focal stacked using Helicon Focus software (e.g., Method B, Radius 8, Smoothing 4) in order to study intricate areas of the specimens (e.g., Saupe and Selden, 2009). The positives with this program include the process is automated and quick, thus saving time as compared to stacking by hand in Adobe Photoshop.

There are however, many negative aspects to using Helicon Focus. Unless each image is

manipulated before being imported into Helicon, the user is unable to specifically target an area of interest. Thus other aspects of an image that are not desired would be incorporated into the final product. Additionally, due to variation in lighting between individual images, areas that are vignetted, especially images taking at high magnification, are often highlighted in the final result.

Overall, although Helicon Focus is a useful program for the automated, quick process, and for areas of interest that are not complex, it is not recommended for the study of microorganisms that have a high level of detail in a particular area of study.