Journal of Fluorescence

, Volume 15, Issue 1, pp 41–51 | Cite as

Single-Photon Counting Multicolor Multiphoton Fluorescence Microscope

  • Christof Buehler
  • Ki H. Kim
  • Urs Greuter
  • Nick Schlumpf
  • Peter T. C. So
Article

Abstract

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.

KEY WORDS:

Multiphoton microscopy single-photon counting imaging spectroscopy 

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Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Christof Buehler
    • 1
  • Ki H. Kim
    • 2
  • Urs Greuter
    • 1
  • Nick Schlumpf
    • 1
  • Peter T. C. So
    • 2
    • 3
  1. 1.Paul Scherrer InstitutVilligenSwitzerland
  2. 2.Department of Mechanical EngineeringMassachusetts Institute of TechnologyCambridge
  3. 3.Biological Engineering DivisionMassachusetts Institute of TechnologyCambridge

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