Transmission Shift in Fluorescence Microscopy: Adjustable Interference Filters, Theoretical Considerations and Practical Implications

Abstract

In interference filters, transmitted light bands can be continuously shifted to shorter wavelengths and the filter’s full width of half maximum (FWHM) (synonym: half intensity width/HIW) can be reduced when turned into oblique positions so that the incident axial light no longer runs perpendicularly to the filter’s surface. These modifications of the transmitted light can be described by mathematic functions or particular reference lines empirically evaluated for any filter used. Moreover, potential low intensity leaking rays beyond the filter’s principal transmission band can be shifted or eliminated. The optical effects described can be intensified still more when two interference filters are used as doublet. When the angle of incident light deviates from 90 � , the transmitted light is divided into two partial beams being linearly polarized; their planes of oscillation differ by 90 � , their transmission peaks by 3–7%. Thus, the transmitted light can be modulated further with a rotatable polarizer. In fluorescence microscopy, adjustable interference filters can be used for excitation and suppression in the manner described so that all transmitted light spectra can be modulated in tiny steps and optimally adjusted with regard to the properties of the fluorescent specimen. Several astronomy filters were used for practical evaluations of our method. Because of their particular optical design, these filters could be used for excitation with low-intensity halogen light so that any photobleeching (fading) or other irritations of native specimens could be avoided. Photomicrographs could be taken from living motile fluorescent specimens with a flash-free from indistinctness caused by movement. Our method could be successfully carried out with standard laboratory microscopes based on transmitted light as well as with standard industry microscopes fitted with a vertical illuminator for examinations in incident light, and all images could be significantly improved by transmission shift.