A high level of fluorescence background signal rejection was achieved for solid and powder samples by using a combination of simple low-resolution spectrograph and ultrafast intensified/gated CCD camera. The unique timing characteristics of CCD camera match exceptionally well characteristics of Ti:sapphire oscillator allowing fast gated light detection at a repetition rate of up to 110 MHz, making this approach superior in terms of duty cycle in comparison with other time-resolved Raman techniques. The achieved temporal resolution was about 150 ps under 785 nm Ti:sapphire laser excitation. At an average excitation power up to 300 mW there was no noticeable sample damage observed. The strong Hexobenzocoronane (HBC) fluorescence with a lifetime about 2.1 ns was efficiently rejected and Raman spectrum revealed. The combination of spectrometer and ultrafast gated CCD camera allows simultaneous study of spectral and temporal characteristics of emitted light for the fluorophores with a fluorescence lifetime in nanosecond range. It is particularly important in biomedical spectroscopy, since the majority of endogenous fluorophores has a relatively short lifetime of about 1-5 ns. This capability opens an exciting possibility to build a universal instrument for solving multitask problems in applied laser spectroscopy.
Observing dynamic reorganization and molecular interactions of cellular components on a precise spatial and temporal scale is not possible using existing microscopic techniques. However, fluorescence lifetimes occur on a nanosecond time scale, are independent of local signal intensity and concentration of the fluorophore, and provide sensitive discrimination of the molecular environment. We designed and implemented a fluorescence lifetime imaging microscope (FLIM) using a picosecond-gated multi-channel plate image intensifier, providing two-dimensional time-resolved images of single cell specimen. BHK21 cells were transfected with vectors for green fluorescent protein (GFP) and placed on an infinity-corrected Olympus epi-fluorescence microscope, coupled to a Coherent tunable femtosecond ti-sapphire pulsed laser and a frequency doubler to select an appropriate excitation wave length. After synchronizing the high-speed gated image intensifier to the excitation laser pulses, time-resolved nanosecond images of fluorescent emission were acquired. These images were processed pixel-by-pixel for single exponential decay to obtain an image based on fluorescence lifetime. Although the nucleus appeared brighter than the cytoplasm by fluorescence intensity measurement, FILM showed a uniform lifetime of the GFP fluorescence in both compartments, indicating that the GFP was in similar molecular environments. This technology also has important applications in fluorescence resonance energy transfer (FRET) imaging.
Conference Committee Involvement (1)
Commercial and Biomedical Applications of Ultrafast Lasers IV
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