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

Rev Sci Instrum. 2010 Nov;81(11):113705. doi: 10.1063/1.3499260.

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.

MeSH terms

  • Fluorescence Resonance Energy Transfer / instrumentation*
  • Microscopy, Confocal / instrumentation*
  • Nitrogen / chemistry
  • Optical Phenomena*
  • Peptides / chemistry
  • Photochemical Processes
  • Polymethyl Methacrylate / chemistry
  • Temperature*

Substances

  • Peptides
  • polyproline
  • Polymethyl Methacrylate
  • Nitrogen