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• W1966819046 abstract "Multidimensional fluorescence microscopy is finding service in forefront biological studies that require separation ofimages from different fluorophores. For example, commercial microscopes are available with multi-band analogdetectors and user-friendly software for of species with overlapping emission spectra. To extend suchtechniques to ultrasensitive and single-molecule applications, we have developed a custom-built microscope, whichincorporates two tunable-wavelength picosecond dye lasers for pulse-interleaved laser excitation, angle-tuned reflectionof the laser beams from narrow-band Raman notch filters to introduce epi-illumination and provide strong rejection ofscattered laser wavelengths, diffraction-limited confocal imaging with 3-dimensional piezo-scanning, an adjustableprism spectrometer for high-throughput resolution of collected fluorescence into 4 spectral bands, and a 4-channel high-quantumefficiency avalanche diode for sub-nanosecond-resolved single-photon detection. Custom software enablesmulti-band fluorescence correlation spectroscopy and identification of photon bursts for single-molecule detection. Forunmixing of spectrally-overlapping signatures for ultrasensitive molecular imaging applications, we find that maximum-likelihoodanalysis can out-perform least-squares-based linear unmixing in the regime of low photon numbers perspectral/temporal channel. Also, the likelihood surface provides the confidence of the parameter estimates and thecovariance of the species contributions. Monte Carlo simulations show that bias in the results of the analysis, whichstems from the constraint that photon numbers should be positive, becomes more pronounced at low signal levels, forboth maximum-likelihood and least-squares based unmixing." @default.
• W1966819046 created "2016-06-24" @default.
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• W1966819046 date "2007-02-08" @default.
• W1966819046 modified "2023-09-23" @default.
• W1966819046 title "Extension of multidimensional microscopy to ultrasensitive applications with maximum-likelihood analysis" @default.
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• W1966819046 doi "https://doi.org/10.1117/12.702217" @default.
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