Microscopy Construction

Second generation
video-rate multi-photon microscope

We are presently constructing an upgraded two-photon microscope system, which will be used by Mark Miller in his new position at the University of Washington for in vivo imaging studies of the immune system.

The microscope utilizes the same basic resonant-scanner system as employed in our earlier confocal and 2-photon systems, but incorporates several improvements:

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.

To see the images at full resolution, click on the image of choice.

Second generation video-rate multi-photon microscope ('2G2P')

Two-Photon filter diagram


PMT head


Control Panel

For more information about the basic design, control circuitry, real-time image corrections,
scan head bracket design, aquisition software, and part list visit

Mike Sanderson's webpage on Real-Time CONFOCAL MICROSCOPY

1146 Mc Gaugh Hall
University of California, Irvine
Irvine, CA 92697-4550
Lab Tel: 949-824-7833
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last updated 01/17/2006. Please send enquiries to ianparkerlabweb@gmail.com