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

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2 Author(s)
Hirschfeld, V. ; Institute of Physics, University of Lübeck, Ratzeburger Allee 160, Lübeck, Germany ; Hubner, C.G.

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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.

Published in:

Review of Scientific Instruments  (Volume:81 ,  Issue: 11 )