Development of a confocal scanning microscope for fluorescence imaging and spectroscopy at variable temperatures.

Abstract

We developed and tested a confocal scanning optical microscope that fits into a thermally controlled, commercial research cryostat designed for operation from ambient temperature down to below 4 K. The home-built microscope is a fiber-coupled, self-contained instrument based on readily available mechanical and optical components. Its sample module is sealed in a protective stainless steel tube that minimizes vibrations caused by the flow of cryogenic gas. A high numerical aperture microscope objective specifically designed for cryogenic and high-vacuum applications focuses the excitation light onto the sample, while the core of an optical fiber attached to an avalanche photodiode acts as the confocal detection pinhole. The sample is displaced using a piezotube scanner mounted on top of a three-axis, low-temperature nanopositioner assembly for coarse sample positioning. A broadband polarizing cube beam splitter in the emission path allows for polarization-resolved imaging and spectroscopy. Fluorescence excitation scans are acquired with custom-written software that correlates fluorescence photon counts with the output from a high precision wavelength meter, which is part of a narrow-band, tunable dye laser setup. The imaging and spectral data acquisition capabilities of the microscope were confirmed using a variety of samples and excitation wavelengths at temperatures ranging from 5 K to room temperature.