Perfect oblique illumination

Oblique illumination certainly is the oldest method to enhance contrast in microscopy and is a technique of interference contrast. This work provides an overview about its origins, historical development and theory. Oblique illumination was a key to the theory of microscopic resolution. During development of the modern illumination of the microscope, oblique illumination was abandoned, and rediscovered in the second half of the 20th century. Oblique illumination with aperture diaphragms provides improved relief contrast, but does not change resolution. Fourier optics explains well oblique illumination and its unwanted effects resulting from non-optimal adjustment, like broad lights and shadows. The work describes optimal adjustment of oblique

illumination with best results in digital photomicrography.

Keywords: Oblique illumination – resolution of the microscope – Fourier optics – diffraction – interference contrast

In preparation...

Interpretation of acridine orange staining of ciliates living in freshwater and in the soil

Acridine orange staining is found to be specific for the different genera of the Cili- ates (Ciliophora). Minimal laboratory equipment and protocol are presented for reproducible AO staining of the ciliates living in freshwater and in the soil. Live-Cell-Imaging can be obtained by using digital single lens reflex cameras (DSLRs) with video function. Staining of DNA supports identification of species by showing macronuclei und micronuclei. Localization of active DNA by differentiation of dsDNA und ssDNA is possible during mitosis by staining with AO. Phagosomes, lysosomes and acidosomes will be stained depending on pH from green to red supporting visualization of processes during endocytosis and exocytosis. Organelles in regions of the cilia and close to cell membrane will be stained in red for certain genera. This is compared with previous examination of the trichocyst system of the genus Paramecium.

DOI: 10.5414/MKX0058

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Fluorescence co-staining by Hoechst 33342 and acridine orange to determine ciliate species (phylum: Ciliophora)

The work describes a new method of co-staining with the fluorochromes Hoechst 33342 (Ho342) and acridine orange (AO). The method of co-staining is simple to use and, therefore, recommended especially to systematically determine the various species of ciliates (phylum: Ciliophora). The method specifically stains nuclei (in general: DNA) in blue color and also stains phagosomes, lysosomes, acidosomes and in certain cases trichocysts in colors ranging from green to red. Fine structures along the ciliary patterns can be shown by using AO to stain ciliary vacuoles and granules. Correct interpretation of the staining results is discussed in detail. The method allows life-cell-imaging, may be applied to determine any species of ciliates and does not suffer from artifacts. Hence co-staining with Ho342 and AO replaces supravital staining using methyl green-pyronin. The method is successfully applied to determine ciliate species during multi-year monitoring of the natural reserve “Verbrannte Maar“ / „Hellenmaar“ (SU-044) near Bornheim (Rhine), Germany.

Bauer T. Fluoreszenz-Doppelfärbung mit Hoechst 33342 und Acridinorange zur Bestimmung der Arten von Ciliaten (Phylum: Ciliophora). Mikroskopie. 2019; 1: 2-19.

DOI: 10.5414/MKX00192A

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