The autofluorescence of biological tissue can be exploited for the detection and diagnosis of disease but, to date, its complex nature and relatively weak signal levels have impeded its widespread application in biology and medicine. We present here a portable instrument designed for the in situ simultaneous measurement of autofluorescence emission spectra and temporal decay profiles, permitting the analysis of complex fluorescence signals. This hyperspectral fluorescence lifetime probe utilizes two ultrafast lasers operating at 355 and $440\mathrm{nm}$ that can excite autofluorescence from many different biomolecules present in skin tissue including keratin, collagen, nicotinamide adenine dinucleotide (phosphate), and flavins. The instrument incorporates an optical fiber probe to provide sample illumination and fluorescence collection over a millimeter-sized area. We present a description of the system, including spectral and temporal characterizations, and report the preliminary application of this instrument to a study of recently resected $(<2\mathrm{h})$ ex vivo skin lesions, illustrating its potential for skin cancer detection and diagnosis.

We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor.

We present a time-gated, optically sectioned, hyperspectral fluorescence lifetime imaging (FLIM) microscope incorporating a tunable supercontinuum excitation source extending into the UV. The system is capable of resolving the excitation spectrum, emission spectrum, and fluorescence decays in an optically sectioned image.

(3-aminopropyl)triethoxysilane (APTES) is a commonly used organosilane on surface functionalization of silicon oxide surfaces. However, its deposition process from solution-phase usually involves the use of toluene, which has often been identified as crucial for the formation of an aminopropylsilane monolayer. Toluene is ranked as a problematic solvent in the guide developed by a group referred to as the solvent sub-team of CHEM21. In this work, we propose a facile synthetic route for functionalizing a silicon substrate with APTES via solution-phase approach using only solvents that are classified as recommended. The influence of the APTES concentration, reaction times and different post-deposition conditions using acetic acid and methanol were studied in order to evaluate the quality and thickness of the organosilane layers.•The method uses ethanol as APTES solvent for functionalizing silicon dioxide surfaces and only uses solvents classified as recommended.•The method uses a solution phase approach, does not require complicated equipment and can be prepared at room temperature.

This review provides a detailed picture of the innovative efforts to combine atomic force microscopy and different super-resolution microscopy techniques to elucidate biological questions.