Development and characterization of a two-photon fluorescence microscope for collagen tissue based on a nanosecond laser

The structural and microscopic examination of cartilage and tendon tissue is of central interest for the characterization of torn ligament healing or pathological tissue changes. Conventional microscopy or optical coherence tomography methods can only partially resolve tissue types and their structural orientation. Two or multiphoton microscopy and second harmonic generation provide information about the material composition or alignment of nanoscaled structural proteins. In most systems, femtosecond lasers are used, which leads to high system costs and requires special system components. The aim of this work is to investigate a narrowband nanosecond laser with a pulse energy several orders of magnitude higher, reducing the required number of laser pulses to be averaged per pixel. Second harmonic generation and two photon excited fluorescence can be used to non-invasively examine deeper tissue structures. High lateral resolution was achieved by scanning the sample. Simultaneous real-time visualization of collagenous and cellular structures was attained. Defined aligned collagenous fibers of a sheep tendon were investigated. The anisotropy of the collagenous structures could be demonstrated. It was possible to realize a two-dimensional imaging method with a maximum point density of 5080 PPI and a numerical aperture of 0.15. The method allows the simultaneous separate observation of collagen fibers through the use of second harmonic generation and cellular tissue using a narrow-band nanosecond laser for two photon excited fluorescence.