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We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 +/- 1 ns). The approximately 1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional approximately 13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at approximately 440 nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore.

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Extracellular vesicles (EVs) have numerous potential applications in the field of healthcare and diagnostics, and research into their biological functions is rapidly increasing. Mainly because of their small size and heterogeneity, there are significant challenges associated with their analysis and despite overt evidence of the potential of EVs in clinical diagnostic practice, guidelines for analytical procedures have not yet been properly established. Here, we present an overview of the main methods for studying the properties of EVs based on the principles of fluorescence. Setting aside the isolation, purification and physicochemical characterization strategies which answer questions about the size, surface charge and stability of EVs (reviewed elsewhere), we focus on available optical tools that enable the direct analysis of phenotype and mechanisms of interaction with tissues. In brief, the topics on which we elaborate range from the most popular approaches such as nanoparticle tracking analysis and flow cytometry, to less commonly used techniques such as fluorescence depolarization and microarrays as well as emerging areas such as fast fluorescence lifetime imaging microscopy (FLIM). We highlight that understanding the strengths and limitations of each method is essential for choosing the most appropriate combination of analytical tools. Finally, future directions of this rapidly developing area of medical diagnostics are discussed.

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 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 kfps. 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. Fluorescence lifetime imaging microscopy (FLIM) results are presented.Index Terms-single photon avalanche diode, CMOS image sensor, fluorescence lifetime imaging microscopy. laser ranging.

Nanomedicine involves measurement and therapy at the level of 1-100 nm. Although the science is still in its infancy, it has major potential applications in diabetes. These include solving needs such as non-invasive glucose monitoring using implanted nanosensors, with key techniques being fluorescence resonance energy transfer (FRET) and fluorescence lifetime sensing, as well as new nano-encapsulation technologies for sensors such as layer-by-layer (LBL) films. The latter might also achieve better insulin delivery in diabetes by both improved islet encapsulation and oral insulin formulations. An 'artificial nanopancreas' could be an alternative closed-loop insulin delivery system. Other applications of nanomedicine include targeted molecular imaging in vivo (e.g. tissue complications) using quantum dots (QDs) or gold nanoparticles, and single-molecule detection for the study of molecular diversity in diabetes pathology.

The aggregation of silica particles during hydrogel polymerization has been observed in situ with angstrom resolution using the combined fluorescence anisotropy decay of solvated and bound dye. Primary particles of mean hydrodynamic radius approximately 1.5 nm are found to be present within 20 min of mixing sodium silicate solution and sulfuric acid. Clustering then occurs during siloxane polymerization to produce after approximately 30 h secondary particles with a mean radius up to approximately 4.5 nm at a growth rate that depends on silicate concentration and time to microgelation t(g). Subsequent condensation to approximately 4 nm radius occurs within 1 week as particle syneresis dominates.

Fluorescence of synthetic melanin in dimethyl sulfoxide has been excited by two-photon absorption at 800 nm, using 120 fs pulses with photon flux densities > or = 10(27) cm-2 s-1. The shortest main component of the three-exponential decay of fluorescence is 200 +/- 2 ps. The overall spectral shape is red-shifted with respect to the 400 nm excited fluorescence. Two-photon excited melanin fluorescence also has been measured from excised samples of healthy human skin tissue. Because of the selectivity of melanin excitation via resonant two-photon absorption, it is hypothesized that fluorescence excited in this way may yield information on malignant transformation.