Single-Photon Counting Multicolor Multiphoton Fluorescence Microscope
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We present a multicolor multiphoton fluorescence microscope with single-photon counting sensitivity. The system integrates a standard multiphoton fluorescence microscope, an optical grating spectrograph operating in the UV–Vis wavelength region, and a 16-anode photomultiplier tube (PMT). The major technical innovation is in the development of a multichannel photon counting card (mC-PhCC) for direct signal collection from multi-anode PMTs. The electronic design of the mC-PhCC employs a high-throughput, fully-parallel, single-photon counting scheme along with a high-speed electrical or fiber-optical link interface to the data acquisition computer. There is no electronic crosstalk among the detection channels of the mC-PhCC. The collected signal remains linear up to an incident photon rate of 108 counts per second. The high-speed data interface offers ample bandwidth for real-time readout: 2 MByte λ-stacks composed of 16 spectral channels, 256× 256 pixel image with 12-bit dynamic range can be transferred at 30 frames per second. The modular design of the mC-PhCC can be readily extended to accommodate PMTs of more anodes. Data acquisition from a 64-anode PMT has been verified. As a demonstration of system performance, spectrally resolved images of fluorescent latex spheres and ex-vivo human skin are reported. The multicolor multiphoton microscope is suitable for highly sensitive, real-time, spectrally-resolved three-dimensional imaging in biomedical applications.
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- Single-Photon Counting Multicolor Multiphoton Fluorescence Microscope
Journal of Fluorescence
Volume 15, Issue 1 , pp 41-51
- Cover Date
- Print ISSN
- Online ISSN
- Kluwer Academic Publishers-Plenum Publishers
- Additional Links
- Multiphoton microscopy
- single-photon counting
- imaging spectroscopy
- Industry Sectors
- Author Affiliations
- 1. Paul Scherrer Institut, CH-5232, Villigen, Switzerland
- 2. Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts
- 3. Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts