Compact modular design. The scanner and detector head are two modules, based on Olympus reflected light illuminators and turret heads. They ‘plug’ onto a regular Olympus BX71 microscope frame using standard Olympus mounts. Construction of these units required some courage, as we reduced most of a $4,000 housing to metal shavings while milling out mounts for the photomultipliers
High-efficiency, 4-channel detector. The photomultiplier detectors are located as close as possible to the back aperture of the objective lens, for increased collection of non-collimated (scattered) fluorescence emission. A primary dichroic in a rotating turret reflects visible wavelengths toward the detector unit, while allowing transmission of IR excitation light from the scanner unit mounted above. Within the detector housing are three further dichroic/filter cubes, mounted on a dovetail slide for easy interchange, that direct light to four photomultipliers.
Simultaneous 4-channel acquisition. Photomultiplier signals, and sync pulses from the scanner unit, are connected to a Raven acquisition board, that permits simultaneous acquisition from 4 channels at pixel clock rates up to 20 MHz.
Dual-laser excitation. The system is driven by two Chameleon femtosecond lasers (Coherent Inc.), which can be independently tuned to separate wavelengths. Selection, and attenuation of the laser beams is controlled by computer-operated Pockels cells. Thus, alternate frames (33 ms) can be captured with differing excitation wavelengths; each frame comprising 4 color channels at about 500 x 500 pixel resolution.