Frequency domain time-resolved microscope using a fast-scan CCD camera

Peter T. C. So; Todd E. French; Enrico Gratton

doi:10.1117/12.182713

17 August 1994 Frequency domain time-resolved microscope using a fast-scan CCD camera

Peter T. C. So, Todd E. French, Enrico Gratton

Proceedings Volume 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV; (1994) https://doi.org/10.1117/12.182713
Event: OE/LASE '94, 1994, Los Angeles, CA, United States

Abstract

Time-resolved fluorescence imaging can enhance the contrast of microscope images and it can also provide important information about the micro-environment in cellular systems. We have developed a fluorescence microscope which can measure fluorescence lifetimes over an entire image. Fluorescence lifetimes are measured by using heterodyne frequency domain techniques. Heterodyning is accomplished by using an intensity modulated laser light source and a fast scan CCD camera coupled with a gain modulated microchannel plate as the detector. The high duty cycle of this method allows us to generate a phase resolved image with about five seconds integration time. Operating in the fast scan mode, the systematic uncertainties in lifetime determination caused by photobleaching are less severe than those of slow-scan cameras. The microchannel plate can be modulated at frequencies up to 300 MHz, which allows us to measure lifetimes as short as 500 ps with resolution of 50 ps. The modulation of the microchannel plate only slightly degrades the spatial resolution of the image from the diffraction limit; 0.8 micron resolution is maintained with 500 nm laser excitation.

Citation Download Citation

Peter T. C. So, Todd E. French, and Enrico Gratton "Frequency domain time-resolved microscope using a fast-scan CCD camera", Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); https://doi.org/10.1117/12.182713

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