A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K

V. Hirschfeld*, C. G. Hübner

*Corresponding author for this work
9 Citations (Scopus)

Abstract

We developed a cryostat suitable for a laser scanning confocal microscope which allows for a short working distance and thus the usage of an objective with a high numerical aperture ensuring high collection efficiency. The in situ preparation of a thin layer of amorphous water is realized in a part of the cryostat, a Dewar vessel, which is put onto a custom-made, liquid-nitrogen immersed spin-coater. First tests on the setup are performed on a perylenemonoimide/polymethyl methacrylate model system using a standard oil objective and a dry objective at ambient temperature as well as a dry objective at liquid nitrogen temperature (77 K). Fluorescence resonance energy transfer (FRET) measurements on doubly labeled, freeze-quenched polyproline chains show the applicability of the new method on biomolecules. The alternating laser excitation (ALEX) is modified to a line-scanning process (slow ALEX) to optimize the sorting of the labeled molecules. Photophysics and photochemistry at liquid nitrogen temperature are investigated.

Original languageEnglish
Article number113705
JournalReview of Scientific Instruments
Volume81
Issue number11
ISSN0034-6748
DOIs
Publication statusPublished - 01.11.2010

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