Digital microspectrofluorometry by multichannel scaling and single photon detection

Abstract

Synopsis. A careful analysis of the requirements for microspectrofluorometric measurements has been made. General problems encountered in the measurement of light intensity and spectral distributions are considered, together with some special factors arising when measuring the fluorescence emitted by single cells. Criteria for evaluating microspectrofluorometric apparatus are given, and the relative merits of analog and digital methods are compared.A fully digital instrument using single-photon detection and a multichannel analyser is described. Automatic procedures for obtaining photodecomposition-fime curves and emission spectra are illustrated in detail. Other possible applications of the instrument are also outlined. The experimental results reported show that microspectrofluorometric measurements of high sensitivity are possible, as light intensities down to a few photons per second can be measured.

Pulsed tunable lasers in cytofluorometry

“…Such a low optical density was chosen for the following reasons. (I) to evaluate, as a first approximation, the sensitivity of the system; (2) to stay within the region of linear dependence of fluorescence intensity to optical density (Prenna et al, 1974 a); (3) to reduce the influence of the inner filter effect on the measurements (Prenna et al,z974a). This effect cannot be completely avoided in the present experiment because of the spectral sensitivity of the available photomultiplier.…”

The Feulgen reaction: from pink-magenta to rainbow fluorescent at the Maffo Vialli’s School of Histochemistry

“…This had been the motivation behind improving methods and instruments, eventually transforming cytophotometry into cytofluorometry. 26 , 27 …”

“…Microspectrofluorometers were developed in response to the need to characterize fluorescent substances chemically and physically and to assess their suitability for use in quantitative applications. 26 These instruments are based on fluorescence microscopy and can analyze the spectral distribution of the emitted fluorescence (evaluation of the emission spectrum); they also allow variable excitation lights to continuously explore the light spectrum in all ultraviolet and visible frequencies. In this way, the excitation spectrum of the fluorochrome linked to the biological structure is evaluated.…”

“…The need to investigate the spectral (excitation and emission) properties of new molecules in depth and in situ prompted the Center’s research into microspectrofluorometry. 26 In fact, Prenna was a pioneer in this field also, having oversaw the installation at the Centre of an early Leitz instrument dedicated to such research. 37 For many years, the Palace was also the site of experimentation and interaction between experts from this Company and CNR researchers working on the development of advanced microfluorometric instrumentation.…”

“…Again, a collaboration with the Politecnico of Milan, involving electronics and physics-optics experts, resulted in notable experimental contributions to the introduction of laser sources in cytofluorometry. 25 , 26 The results obtained from the use of power laser sources are especially important both in terms of technological development and application. The studies conducted with pulsed laser sources to obtain new fluorochrome characterization parameters, such as the decay time of the fluorescence pulse, are absolutely unique and of international relevance.…”