Microscopy Construction

Total Internal Reflection
Fluorescent Microscopy (TIRFM)

Imaging single-channel Ca21 signals by total internal reflection fluorescence microscopy. (A) Schematic of the TIRFM imaging system. The 488-nm beam from an argon ion laser (50 mW) passes through a 53 beam expander (BE) and is focused by a lens (FL; f ¼ 150 mm) via a dichroic mirror (DM)to a spot at the back focal plane of the microscope objective lens (Olympus TIRFM 603, oil immersion, NA ¼ 1.45). The focusing lens is mounted on a micrometer-driven translation stage, so that the laser beam can be adjusted to enter the periphery of the objective aperture so as to achieve total internal reflection at the interface between the cover glass and the aqueous bathing medium. An adjustable rectangular knife-blade aperture (A) located at a conjugate image plane defines the field of excitation. Fluorescence excited in the specimen by the evanescent wave is collected by the same objective, passes through the dichroic mirror and a barrier filter (BF) blocking the laser wavelength, and is imaged by an intensifier tube coupled through a relay lens to a charge-coupled device camera. An oocyte expressing N-type channels is loaded with fluo-4 dextran, stripped of its vitelline envelope and allowed to adhere (animal hemisphere down) to a cover glass forming the base of the imaging chamber. Its membrane potential is controlled by a two-electrode voltage-clamp. (B) Schematic, illustrating the imaging of near-membrane fluorescent Ca21 signals near an open channel by TIRFM. (C) Single video frame obtained by TIRFM illustrating Ca21 signals from simultaneous opening of three channels within an 80 3 80-mm patch of oocyte membrane in response to depolarization to -15 mV. Increasing [Ca21] is denoted both by ‘‘warmer’’ colors and by height.

Demuro, A. & Parker, I. Imaging the activity and localization of single voltage-gated Ca2+ channels by total internal reflection fluorescense microscopy. Biophys. J. 86, 3250-3259, 2004. [PDF]

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