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 μm x 9.2 μm 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.

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.