System for rapid confocal scanning along the z axis of the microscope.

(A) Schematic diagram of the optical system, constructed around an Olympus IX70 inverted microscope. Two dichroic mirrors (DM1 and DM2) in the light path provided, respectively, for irradiation by near-UV light from a shuttered arc lamp for photolysis of caged InsP₃ and for confocal monitoring of calcium-dependent fluorescence signals. DM1 and the excitation filter F1 were located in a standard epifluorescence cube in the microscope. DM2 and the confocal optics were mounted on an external optical breadboard, and interfaced through the video port of the microscope. Light (lambda 355 nm) from a pulsed UV laser (not shown) was combined with that from the arc lamp by a beam splitter. The objective lens was driven by a piezoelectric translator (PZT) to rapidly focus through a range of up to 35 µm at rates up to 200 Hz. The oocyte was viewed through a coverglass cemented in a small (5-mm diameter) aperture in a rigid Plexiglas chamber, firmly bolted to the microscope stage. DM1, dichroic mirror reflecting lambda  < 400 nm; DM2, dichroic mirror reflecting lambda < 500 nm; F1, bandpass filter lambda  340-400 nm; F2, barrier filter, lambda > 510 nm; Ar ion laser, attenuated 488-nm beam from 100 mW argon ion laser; APD, avalanche photodiode photon counting module.

(B) Traces showing position of the piezo drive during one cycle of the sine-wave scan (top) and corresponding output from the fluorescence detector after addition of sync pulses (bottom). The fluorescence signal was obtained while scanning into an oocyte, and the extreme downward deflection of the scan (trough of sine wave at center of scan cycle) corresponds to the position of the coverglass. Two broad fluorescence peaks are apparent, resulting as the spot scanned from within the oocyte toward the cover glass (left), and again as the objective moved back in the reverse direction (right). The fluorescence deep within the oocyte is low because of light scattering and absorption, and irregular troughs arise as the spot moves through organelles. The fluorescence profiles on the "down" and "up" scans are not perfect mirror images because of lateral hysteresis in the translator. (C) Axial linescan image showing reflectance from a mirrored slide as the microscope focus was advanced by hand in 2-µm increments at roughly 1-s intervals. Each vertical line forming the image comprises one cycle of the scan, and time runs from left to right. (D) Plot of pixel position versus focus displacement for the selected region (arrows) of the image in C. The central portion of the scan was close to linear over a range of ~22 µm.