Optical microscopy conjugate planes

A method to detect diffusion coefficients of single molecules in solution is confocal microscopy. A confocal microscope uses point illumination, which is achieved by a pinhole in front of the objective (in an optically conjugated plane to the sample). The illuminated voliune is in the order of 1 femtoliter and. 

Compared to conventional microscopy, confocal microscopy delivers superior optical sectioning by using a pinhole in a plane conjugate with the focal (xy) plane. It allows a crisp 3D image to be built from a stack of 2D images even for somewhat turbid samples, but each 2D image is acquired by scanning, which imposes limits on acquisition speed. The technique has been described in detail before [82]. 

Fig. 2 Schematic diagram of laser optical tweezers (LOT) system. (Top panel) An infrared laser beam was steered to trap and move pm-size particles in the focal plane. At a sufficiently large laser intensity, the gradient force dominates over the scattering force. (Lower panel) Membrane tethers are extracted from live cells. Dielectric beads (0.5 pm in diameter) were conjugated with antibodies against integiins and attached to the cell membrane. LOT traps one bead and pulls it away from the cell, as indicated by the arrow. Thin membrane tethers extending from the beads to the cell body appear. Using conventional brightfield microscopy, the tether length and its diameter are measured which, in turn, provide an estimate of the plasma membrane tension.

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