In Microscopy Today, January 2011, Piper and Chmela published an article called “Advanced Techniques for Observation and Photomicrography of Subcellular Structures in Diatom Shells.” This paper clamors for some comments of a fundamental nature.
The techniques presented in the paper are called “advanced,” but it is difficult to understand why this should be so. The paper describes observations with the conventional bright-field techniques—including oblique illumination—used since the late nineteenth century. The optics used were a 120/0.9 water immersion and a planapochromatic oil immersion 160/1.4, both somewhat unusual as regards their magnification, but not “advanced” in the sense that they would be optically superior to the 50/1.00 water or oil immersions and 100/1.4 or 63/1.4 (plan)apochromatic oil immersions that have been on the market for decades. As will be shown, any such “advanced” performance of the lenses used is not borne out by the photomicrographs presented.
Where the paper is completely mistaken is in the description, interpretation, and iconographic documentation of the fine structure of the diatom exoskeleton. As regards description and interpretation, consider the following quote:
“Within the respective frustules, the bright stripes result from small perforations separated in very short distances that cannot be resolved in light microscopy. The neighboring dark stripes correspond to small zones that are not perforated…”
The actual situation is as follows:
• In the particular species illustrated, the valve displays fine “lines” (striae) consisting of rows of fine “dots” (puncta). The statement that these puncta cannot be resolved with the light-microscope is false, as will be discussed further on.
• The puncta are indeed perforations of the valve (as evidenced by the electron microscope), and thus the statement that they form “the bright stripes” intuitively seems convincing. In reality, it is erroneous: whether the rows of puncta appear dark or bright solely depends on how the microscope is focused—it is an optical effect. The standard manner in which diatomists have always illustrated the puncta (in both drawings and photomicrographs) is the “black dot focus,” which gives the best contrast.
It is difficult to understand how the authors concluded that the “perforations”—the puncta—cannot be resolved in the light microscope. For a century and a half, resolving the “dots” has been a popular sport among microscopists, and thousands of diatom test slides have been marketed to allow them to test their equipment and their mastery of the microscope. An early, and famous, description of resolution into puncta is that by Quekett [Reference Quekett1], who resolved Pleurosigma angulatum into puncta in 1848 (sic!). The reverend W. Smith published good illustrations of resolved species of this difficult genus a few years later [Reference Smith2], and in the later Victorian era a massive collection of perfectly resolved diatom images was already available (for example see references [Reference Schmidt3, Reference Tempère, Brun, Bergon, Cleve, Dutertre, Grove, Miquel and Peragallo4]).
The diatom “Surirella gemma” discussed and illustrated by Piper and Chmela is a good example of the errors in their paper. Since twenty years, its correct name and authorship have been Petrodictyon gemma (Ehrenberg) D.G. Mann 1990. Resolution of its striae into puncta was obtained in 1870 [Reference Frison5], and for the past century it has been a standard test for an objective with an NA around 1.0.
It is, therefore, difficult to understand how the images in Figure 5 of the paper in question have been obtained with modern—let alone “advanced”—optics. In fact, in sixty years of diatom studies, I cannot remember having seen such an exceedingly poor illustration of this diatom. To justify this criticism, I have intentionally made a photomicrograph of Petrodictyon gemma (Figure 1) with techniques that might be called “retro” instead of “advanced,” as follows: perfectly central ordinary bright-field illumination; white light, completely unfiltered to avoid masking the residual optical shortcomings of the optics used; “dry” condenser, effective NA circa 0.9; and the objective was a Reichert achromatic oil immersion 100/1.3 manufactured around 1912.
Figure 1: The diatom Petrodictyon gemma fully resolved in unfiltered white light with an oil immersion objective manufactured a century ago.
The valve of this diatom is always strongly vaulted, digital stacking would be required to obtain a sharply focused image over the entire valve, but the differences in focus nicely illustrate the “white dot” versus “black dot” settings. Resolution of the striae into puncta is excellent—the results of the “advanced” techniques used for the Piper and Chmela paper were obviously inferior to those that can be obtained with an objective of a century ago.
