New directions in single-molecule imaging and analysis

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   Fig. 1.

   Numbers of papers indexed in the PubMed database with “single molecule” in the title (image courtesy of Taekjip Ha); exponential growth with doubling time of 2.2 years.

   
   
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   Fig. 2.

   Explorations of the sliding behavior of Rep on a single-stranded DNA segment attached to a surface. (A) Schematic of the labeling arrangement for FRET measurements. Traces (B, 22°C; C, 37°C) of donor (green) and acceptor (red) fluorescence signals for a single Rep molecule are shown. [Reproduced with permission from ref. 25 (Copyright 2005, MacMillian Publishers, Ltd.).]

   
   
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   Fig. 3.

   Overview of superresolution imaging. (A) Schematic of a tightly focused laser beam (blue) of diffraction-limited diameter of ≈200 nm irradiating a cell. One molecule is in the focal volume, which emits fluorescence (red). (B) Wide-field fluorescence image of a bacterial cell (red) containing a single protein fusion between the bacterial actin MreB and EYFP (mountain). Acquisition time, 100 ms. (Scale bar, 0.5 μm.) (C) Fluorescence PALM image of PA-GFP molecules on a glass substrate, with green regions showing the approximate blur region of diffraction-limited imaging and yellow dots showing the actual detected positions of the single molecules. [Reproduced with permission from ref. 78 (Copyright 2006, Biophysical Society).] (D) Confocal (Left) and STED (Right) images of a neurofilament in a human neuroblastoma cell labeled by immunofluorescence. [Reproduced with permission from ref. 88 (Copyright 2006, National Academy of Sciences).]

   
   
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   Fig. 4.

   Trapping single molecules in solution with the ABEL trap. (A) Schematic side view of the ABEL trap showing that the microfluidic cell sits above the oil-immersion objective of an inverted fluorescence microscope. Confinement in the z direction along the axis of the microscope is produced by the thin gap between the upper transparent structure and a flat coverslip. Four electrodes are placed in the solution far away from the central trapping region. (B) Top view of the microfluidic cell, showing the trapping region ≈10 × 10 μm in size in the center. Four deep milled channels extend out in the +/− x and +/− y directions. The four sharply pointed raised regions serve to define the thickness of the trap in the z direction normal to the page. (C) Measured (lower right) and pseudofree (center) trajectories of 13 trapped particles of TMV. [Reproduced with permission from ref. 113 (Copyright 2006, National Academy of Sciences).] (D) Position probability distribution of a single fluorescently labeled molecule of the chaperonin, GroEL, trapped in buffer. The standard deviation is shown.