Spot Size Engineering in Microscope-Based Laser Spectroscopy

Abstract

Thanks to the development of microscopy and confocal microscopy, diffraction-limited spot sizes are routinely achieved in spectroscopies such as fluorescence and Raman to achieve submicrometer spatial resolution. Because of their high numerical aperture and collection efficiency, objective lenses are also used in many applications even when a high spatial resolution is not required. In such cases, a diffraction-limited spot size is not necessary and a larger spot may in fact be preferable for a number of reasons, for example, to avoid photodamage, to perform spatial averaging, to ensure a uniform excitation profile, and so forth. In this context, we discuss here simple practical approaches to tailor the spot size with a special emphasis on obtaining large spot sizes with an objective lens. In particular, we demonstrate experimentally that Self’s formalism [Self, S. A. Appl. Opt. 1983, 22, 658] for the transformation of Gaussian beams provides an easy yet powerful approach to predict spot sizes in a typical laser-microscopy setup. A simple practical solution for a continuously tunable spot-size is proposed and studied. These discussions are backed up by experimental measurements in a typical Raman microscopy configuration and further illustrated by practical examples of the use of large spot sizes in the contexts of fluorescence, Raman, and surface-enhanced Raman spectroscopy.