Abstract
Second-harmonic generation (SHG) is the most specific label-free indicator of collagen accumulation in widespread pathologies such as fibrosis, and SHG-based measurements hold important potential for biomedical analyses. However, efficient collagen SHG scoring in histological slides is hampered by the limited depth-of-field of usual nonlinear microscopes relying on focused Gaussian beam excitation. In this work we analyze theoretically and experimentally the use of Bessel beam excitation to address this issue. Focused Bessel beams can provide an axially extended excitation volume for nonlinear microscopy while preserving lateral resolution. We show that shaping the focal volume has consequences on signal level and scattering directionality in the case of coherent signals (such as SHG) which significantly differ from the case of incoherent signals (two-photon excited fluorescence, 2PEF). We demonstrate extended-depth SHG-2PEF imaging of fibrotic mouse kidney histological slides. Finally, we show that Bessel beam excitation combined with spatial filtering of the harmonic light in wave vector space can be used to probe collagen accumulation more efficiently than the usual Gaussian excitation scheme. These results open the way to SHG-based histological diagnoses.
Highlights
Second-harmonic generation (SHG) is the most specific label-free indicator of collagen accumulation in widespread pathologies such as fibrosis, and second harmonic generation (SHG)-based measurements hold important potential for biomedical analyses
We point out that the quadratic dependence will be lost in samples where collagen fibril polarity is heterogeneous at the submicron scale such as cartilage, as recently analyzed by interferometric SHG (I-SHG) studies[22]
Considering collagen imaging, we have shown that strong Bessel SHG signals are observed in dense collagen regions owing to coherence over the axially extended probe volume, despite the reduction in excitation intensity involved by beam shaping
Summary
Second-harmonic generation (SHG) is the most specific label-free indicator of collagen accumulation in widespread pathologies such as fibrosis, and SHG-based measurements hold important potential for biomedical analyses. Extracellular matrix remodeling may occur in tissues in response to various injuries, and in most cases, it exhibits abnormal accumulation of fibrillar collagen This process is called fibrosis and eventually leads to organ functional failure. We analyze more precisely the imaging properties of Bessel beams for SHG and two-photon-excited fluorescence (2PEF) microscopy in terms of signal level and emission directionality as a function of sample geometry. Overall, these results show how Bessel beam excitation can be used to probe collagen accumulation more rapidly and more reliably than the usual Gaussian excitation scheme
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