Laser scanning confocal microscope with cryostat. Laser lines: 488 and 594 nm; M—Mirror; LP—Long pass filter; RP—Retarding plates; AL—Achromatic lens; DC—Dichroic mirror; SM—Scanning mirrors; TL—Telecentric lens; VP—Vacuum pump; F—Filter.
Liquid nitrogen spin-coater and sample cryostat. (a) The spin-coater (black) contains the motor and is evacuated by a vacuum pump. The rotating hollow driveshaft extending into the Dewar vessel carries the coverslip held by suction. Liquid nitrogen at the bottom of the Dewar cools the coverslip. After spreading and freezing of a small drop of sample solution the Dewar is transferred to the steel cylinder in (b). (b) The coverslip covered with liquid nitrogen serves for both sample support and optical window in the cryostat. The space between the Dewar and the steel cylinder is evacuated. The dry objective is fit airtight into the steel cylinder. Technical drawings are available on request.
Detection efficiency of the oil immersion objective at 300 K and the dry objective at 300 K and at 77 K shown with PMI in PMMA. (a)–(c) Superimposed images detected in the two channels with horizontally (red) or vertically (green) polarized light. (d)–(f) Histograms of the maximum intensity in both channels of one molecule per millisecond. (g)–(i) Line profiles corresponding to the white lines in the images in (a)–(c). Top row at ambient temperature with the oil immersion objective (NA 1.4), excitation intensity of ; second row at ambient temperature with the dry objective (NA 0.8), excitation intensity of ; bottom row at 77 K with the dry objective (NA 0.8), excitation intensity of .
Stoichiometry/FRET imaging with ALEX of proline 20 at 77 K. (a) Sum image of the intensities obtained in the donor and in the acceptor channel upon donor excitation at 488 nm. The color scale indicates the intensity per millisecond and pixel. (b) Stoichiometry image. The intensities of both the donor and the acceptor channel upon donor excitation are shown in green. The red spots indicate molecules detected under excitation with the 594 nm line in both channels. Consequently, molecules appearing yellow are doubly labeled and are used for the calculation of energy transfer efficiencies shown in (c). (c) Calculated energy transfer efficiencies for the doubly labeled molecules. According to the color scale high transfer efficiency is shown in red while low transfer efficiency is shown in blue. Background signal was eliminated by setting a threshold criterion.
(a) Superimposed image of the donor and the acceptor channel of proline 6 at 77 K showing photobleaching of the donor (1), photobleaching of the acceptor (2), or blinking (3). (b) Transient intensity [line 1 (red online) corresponds to the acceptor channel, line 2 (green online) to the donor channel] of a single proline 6 molecule at 77 K excited with showing change in energy transfer efficiency (at 0.42 s) and then donor bleaching or dark state formation. (c) Distribution of the combined survival times of both chromophores at 77 K for proline 6 (left side) and proline 20 (right side) excited with or .
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