Development of a high-speed line-scanning fluorescence lifetime imaging microscope for biological imaging.

We report the development of a novel line-scanning microscope capable of acquiring high-speed time-correlated single-photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) imaging. The system consists of a laser-line focus, which is optically conjugated to a 1024 × 8 single-photon avalanche diode (SPAD)-based line-imaging complementary metal-oxide semiconductor (CMOS), with 23.78 µm pixel pitch at 49.31% fill factor. Incorporation of on-chip histogramming on the line-sensor enables acquisition rates 33 times faster than our previously reported bespoke high-speed FLIM platforms. We demonstrate the imaging capability of the high-speed FLIM platform in a number of biological applications.

Calcaneal Tendon Collagen Fiber Morphometry and Aging.

Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (~7 days) to elderly (~38 months) rabbits were monitored in intact (n tendons=24) and microtome sectioned (n tendons=11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Λ) started at very low values (A=2.0±0.6 µm, Λ=19±4 µm) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development.