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. 2004 Jun;86(6):3734–3743. doi: 10.1529/biophysj.10x.037630

FIGURE 1.

FIGURE 1

Schematic diagram of instrument for measuring FRAP. Upper part: view from above. Lower part: side view. The 488-nm line of an argon laser (L) is directed by mirrors (M1, M2,…M8) at an optical window (W) (BK7, Melles-Griot, Irvine, CA). The transmitted beam (97% intensity) is used for photobleaching, whereas the reflected beam (2% intensity) is further attenuated by 30× and 8× reflective neutral density filters (ND1 and ND2) and used for monitoring fluorescence recovery. Fast electronic shutters (Vincent Associates, Rochester, NY) in the bleaching (S3) and monitoring (S2) beam paths control exposure of the sample to each beam. Bleaching and monitoring beams are recombined using a cube beam-splitter (BS, Melles-Griot, Irvine, CA) and directed into the epi-illuminator port of a multipurpose Zeiss ACS microscope (Zeiss, Oberkochen, Germany). The Gaussian intensity profile of the incident beam is converted into a quasisquare profile by selecting the central part using a 1.0-mm diameter pinhole (P) located on the excitation image plane of the microscope. The light then passes through the dichroic mirror (DM) and into a 63× air objective (O) to form a uniformly illuminated circular spot of 12 mm radius on the sample (S). Emitted light passes through the DM to reach the photomultiplier tube (PMT) (model #77345, Oriel Instruments, Strafford, CT). To avoid damage to the photocathode during bleaching, a third electronic shutter (S1) positioned before the PMT is closed during bleaching. Typical bleaching times are 40–100 ms. The light reflected from ND2 is collected and directed onto the sample to provide full-field illumination during visual inspection of large, homogeneous layers. An electronic shutter (S4) controls full-field illumination. The four electronic shutters and the PMT are interfaced with a personal computer (Dell Computer, Round Rock, TX) using an I/O board (PCI-MIO-E1, National Instruments, Austin, TX) and the graphical user interface program LabVIEW (National Instruments). Sample temperature is controlled with an aluminum plate (TS) connected to a circulating waterbath. For the sake of clarity, PMT, O, and TS are omitted from the top view, whereas W, M2, M4, ND1, ND2, S2, and S3 are omitted from the front view. Note that P, DM, O, S, TS, S1, and PMT are part of the epifluorescence microscope, not represented.