Finding the time for fluorescence. Its measurement and applications in life science

We summarise how developments in technology have brought fluorescence lifetime spectroscopy from being the preserve of the specialist to becoming a major tool for research across many science and engineering disciplines. We highlight the advantages which fluorescence lifetime measurements can bring, not only to underpin research, but also through application in helping to solve real-world problems. We illustrate this with recent examples in cancer and Alzheimer’s research, which are aimed at improving disease understanding, diagnosis and therapeutics

TCSPC camera for real time video rate FLIM acquisition based on CMOS technology

The use of fluorescence lifetime imaging microscopy (FLIM) is appealing for the study of biomolecular interactions. Traditionally FLIM has made use of scanning techniques to image a sample with a “single-pixel” detector, while widefield approaches have mainly related to intensity-based imaging. The latter is is advantageous to study mobile samples or kinetics, usually not achievable with FLIM. Rapid fluorescence lifetime imaging is especially important in the monitoring of biological samples, eg. because of cell movement. Recent advances in CMOS technology has led to the development of imaging sensors, based on arrays of pixels, with each pixel containing a single-photon avalanche photodiode (SPAD) and its associated timing electronics, based on a time to digital converter (TDC). This enables rapid (video rate) fluorescence lifetime determination based on the time-correlated single-photon counting technique (TCSPC) to be realised independently in each pixel. Here, we incorporate a 192 x 128 pixel image sensor1, implemented in STMicroelectronics 40nm CMOS technology, in a widefield epifluorescence microscope set-up. The sensor exhibits a 13% fill-factor and each 18.4 x 9.2 μm pixel contains a TDC with a resolution 30 fps) rate. This capability is demonstrated using standard samples and FUN-1 labelled yeast2

A 192×128 Time Correlated SPAD Image Sensor in 40-nm CMOS Technology

A 192 X 128 pixel single photon avalanche diode (SPAD) time-resolved single photon counting (TCSPC) image sensor is implemented in STMicroelectronics 40-nm CMOS technology. The 13% fill factor, 18.4\,\,\mu \text {m} \times 9.2\,\,\mu \text{m} pixel contains a 33-ps resolution, 135-ns full scale, 12-bit time-to-digital converter (TDC) with 0.9-LSB differential and 5.64-LSB integral nonlinearity (DNL/INL). The sensor achieves a mean 219-ps full-width half-maximum (FWHM) impulse response function (IRF) and is operable at up to 18.6 kframes/s through 64 parallelized serial outputs. Cylindrical microlenses with a concentration factor of 3.25 increase the fill factor to 42%. The median dark count rate (DCR) is 25 Hz at 1.5-V excess bias. A digital calibration scheme integrated into a column of the imager allows off-chip digital process, voltage, and temperature (PVT) compensation of every frame on the fly. Fluorescence lifetime imaging microscopy (FLIM) results are presented

A 192 x 128 time correlated single photon counting imager in 40nm CMOS technology

A 192 x 128 pixel single photon avalanche diode (SPAD) time-resolved single photon counting (TCPSC) image sensor is implemented in STMicroelectronics 40nm CMOS technology. The 13 % fill-factor, 18.4 x 9.2 mm pixel contains a 33 ps resolution, 135 ns full-scale, 12-bit time to digital converter (TDC) with 0.9 LSB differential and 8.7 LSB integral nonlinearity (DNL/INL). The sensor achieves a mean 219 ps fullwidth half maximum (FWHM) impulse response function (IRF) and a 5 mW core power consumption and is operable at up to 18.6 kfps. Cylindrical microlenses with a concentration factor of 3.15 increase the fill-factor to 41%. The median dark count rate (DCR) is 25 Hz at 1.5 V excess bias. Fluorescence lifetime imaging (FLIM) results are presented

Excitation of fluorescence decay using a 265 nm pulsed light-emitting diode: Evidence for aqueous phenylalanine rotamers

The authors describe the characteristics and application of a 265 nm AlGaN light-emitting diode (LED) operated at 1 MHz repetition rate, 1.2 ns pulse duration, 1.32 mu W average power, 2.3 mW peak power, and similar to 12 nm bandwidth. The LED enables the fluorescence decay of weakly emitting phenylalanine to be measured routinely, even in dilute solution. For pH of 6-9.2, the authors find evidence for a biexponential rather than monoexponential decay, providing direct evidence for the presence of phenylalanine rotamers with a photophysics closer to the other two fluorescent amino acids tryrosine and tryptophan than has previously been reported. (c) 2006 American Institute of Physics

Effect of ph on aqueous phenylalanine studied using a 265-nm pulsed light-emitting diode

Recently, we described the characteristics and application of a 265-nm AlGaN light-emitting diode (LED) operated at 1-MHz repetition rate, 1.2-ns pulse duration, 1.32-μW average power, 2.3-mW peak power, and approximately 12-nm bandwidth. The LED enables the fluorescence decay of weakly emitting phenylalanine to be measured routinely in the condensed phase, even in dilute solution. For a pH range of 1-11, we find evidence for a biexponential rather than a monoexponential decay, whereas at pH 13, only a monoexponential decay is present. These results provide direct evidence for the dominance of two phenylalanine rotamers in solution with a photophysics closer to the other two fluorescent amino acids, tyrosine and tryptophan, than has previously been reported. Although phenylalanine fluorescence is difficult to detect in most proteins because of its low quantum yield and resonance energy transfer from phenylalanine to tyrosine and tryptophan, the convenience of the 265-nm LED may well take protein photophysics in new directions, for example, by making use of this resonance energy transfer or by observing phenylalanine fluorescence directly in specific proteins where resonance energy transfer is inefficient