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.

Development of an Inverted Epifluorescence Microscope for Long-Term Monitoring of Bacteria in Multiplexed Microfluidic Devices

“…The epifluorescence set up enables part of the light emitted by the illuminated sample to be collected by the same magnification objective [ 23 ]. The collimated beam travels backwards through the same optical path, passing through the dichroic mirror.…”

Microsphere‐Aided Super‐Resolution Scanning Spectral and Photocurrent Microscopy for Optoelectronic Devices

“…[1,2] In commonly-used optical microscopy, DOI: 10.1002/adom.202300172 Abbe's diffraction limit is generally recognized as an unbridgeable barrier in optical imaging due to the loss of highfrequency spatial information in the far field. Various technologies have been developed to overcome the far-field diffraction limit, such as stimulated emission depletion microscopy, [3] confocal microscopy, [4][5][6] and scanning near-field optical microscopy (SNOM). [7] Despite the great performance of these techniques, their applications have been limited because of the system complexity, low optical throughput, or pretreatment requirement.…”

Scanning Super-Resolution Imaging in Enclosed Environment by Laser Tweezer Controlled Superlens

“…However, the diffraction limit prevents conventional optical microscopy of structural features at the nanoscale smaller than $200 nm. To obtain more accurate optical imaging results, many technologies have been developed for this purpose, such as stimulated emission depletion microscopy (1,2), confocal microscopy (3)(4)(5), near-field scanning optical microscopy (6), or photoactivation localization microscopy (7). These technologies solved the problem of super-resolution imaging effectively, but fluorescence imaging is not label free and could harm cell activities and hence would restrict technological applications.…”