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Multifocal multiphoton microscopy (MMM) at a frame rate beyond 600 Hz

2007; Optica Publishing Group; Volume: 15; Issue: 17 Linguagem: Inglês

10.1364/oe.15.010991

1094-4087

Karsten Bahlmann, Peter T. C. So, Michael T. Kirber, R. Reich, B. B. Kosicki, William H. McGonagle, Karl Bellvé,

Photoacoustic and Ultrasonic Imaging

We introduce a multiphoton microscope for high-speed three-dimensional (3D) fluorescence imaging. The system combines parallel illumination by a multifocal multiphoton microscope (MMM) with parallel detection via a segmented high-sensitivity charge-couple device (CCD) camera. The instrument consists of a Ti-sapphire laser illuminating a microlens array that projects 36 foci onto the focal plane. The foci are scanned using a resonance scanner and imaged with a custom-made CCD camera. The MMM increases the imaging speed by parallelizing the illumination; the CCD camera can operate at a frame rate of 1428 Hz while maintaining a low read noise of 11 electrons per pixel by dividing its chip into 16 independent segments for parallelized readout. We image fluorescent specimens at a frame rate of 640 Hz. The calcium wave of fluo3 labeled cardiac myocytes is measured by imaging the spontaneous contraction of the cells in a 0.625 second sequence movie, consisting of 400 single images.