Design and development of a rapid acquisition laser-based fluorometer with simultaneous spectral and temporal resolution

We report the design, development, and characterization of a sensitive, time-resolved fluorescence spectrometer capable of measuring fluorescence spectra and transient decays simultaneously, with data acquisition times less than 1 s. The spectrometer, a portable fluorescence lifetime spectrometer (FLS), was designed to be compatible with both laboratory and clinical research studies on biological systems, and was applied to the study of several biological fluorophores in vitro and human tissue in vivo. The instrument consisted of a nitrogen laser pumping a dye laser for excitation from 337.1 nm through the near infrared, a quartz fiber-optic probe for remote light delivery and collection, and amplified detectors for rapid spectral and temporal detection from 350 to 800 nm. The spectral resolution of the FLS was determined to be 3 nm, which is sufficient for accurately detecting the broad spectral bands associated with biological fluorophores. The FLS was able to detect ${\text{5×10}}^{\text{−7}}$ M fluorescein dye concentrations with spectral signal-to-noise ratios (SNRs) of 29. Time-resolved detection with the FLS had a dynamic range of approximately three decades with a SNR of 200. Using fluorescence lifetime standards, the FLS was determined to be capable of accurately resolving fluorophore lifetimes from hundreds of picoseconds to tens of nanoseconds in duration, with an ultimate temporal resolution of 360 ps.