A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K.
Rev Sci Instrum
; 81(11): 113705, 2010 Nov.
Article
in En
| MEDLINE
| ID: mdl-21133476
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Temperature
/
Microscopy, Confocal
/
Fluorescence Resonance Energy Transfer
/
Optical Phenomena
Type of study:
Diagnostic_studies
Language:
En
Journal:
Rev Sci Instrum
Year:
2010
Document type:
Article
Affiliation country:
Country of publication: