Journal of Fluorescence

, Volume 15, Issue 1, pp 41–51

Single-Photon Counting Multicolor Multiphoton Fluorescence Microscope


  • Christof Buehler
    • Paul Scherrer Institut
  • Ki H. Kim
    • Department of Mechanical EngineeringMassachusetts Institute of Technology
  • Urs Greuter
    • Paul Scherrer Institut
  • Nick Schlumpf
    • Paul Scherrer Institut
  • Peter T. C. So
    • Department of Mechanical EngineeringMassachusetts Institute of Technology
    • Biological Engineering DivisionMassachusetts Institute of Technology

DOI: 10.1007/s10895-005-0212-z

Cite this article as:
Buehler, C., Kim, K.H., Greuter, U. et al. J Fluoresc (2005) 15: 41. doi:10.1007/s10895-005-0212-z


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.


Multiphoton microscopysingle-photon countingimaging spectroscopy

Copyright information

© Springer Science + Business Media, Inc. 2005