Instrumental techniques and the documentation of findings

Abstract

Any microscope is suitable as long as it is fitted with an appropriate light source and suitable filters. Based on current requirements, however, one should aim for a specialised fluorescence microscope. The optimal adjustment of all the component parts to suit the absorption and emission spectrum of the fluorochrome in question, is a pre-requisite for good results. This is especially so for the transmission properties of the excitation and blocking filters and also, secondarily, for the excitation light source which must emit sufficient radiation in the region of the excitation maximum for the fluorescence being investigated. The quality of the microscopic image is determined mainly by the brightness and contrast of the field. The image contrast depends on the relationship between the fluorescence of structures stained specifically or nonspecifically by the conjugate (see 4.4) and by the brightness of the background. Self-fluorescence of optical components, mentioned by Ploem [1973], is no longer an issue today. Moreover, it is self evident that any microscope slides, cover slips, or embedding and immersion media used must also be fluorescence-free.