A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K.
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.
Institute of Physics, University of Lübeck, Ratzeburger Allee 160, Lübeck, Germany.
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Hirschfeld
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Hübner
D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D018613:Microscopy, Confocal / Q000295:instrumentation*; D009584:Nitrogen / Q000737:chemistry; D055620:Optical Phenomena; D010455:Peptides / Q000737:chemistry; D055668:Photochemical Processes; D019904:Polymethyl Methacrylate / Q000737:chemistry; D013696:Temperature
D010455:Peptides; C011083:polyproline; D019904:Polymethyl Methacrylate; D009584:Nitrogen