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Significance: In multiphoton microscopy, two-photon excited fluorescence (TPEF) spectra carry valuable information on morphological and functional biological features. For measuring these biomarkers, separation of different parts of the fluorescence spectrum into channels is typically achieved by the use of optical band pass filters. However, spectra from different biomarkers can be unknown or overlapping, creating a crosstalk in between the channels. Previously, establishing these channels relied on prior knowledge or heuristic testing., Aim: The presented method aims to provide spectral bands with optimal separation between groups of specimens expressing different biomarkers., Approach: We have developed a system capable of resolving TPEF with high spectral resolution for the characterization of biomarkers. In addition, an algorithm is created to simulate and optimize optical band pass filters for fluorescence detection channels. To demonstrate the potential improvements in cell and tissue classification using these optimized channels, we recorded spectrally resolved images of cancerous (HT29) and normal epithelial colon cells (FHC), cultivated in 2D layers and in 3D to form spheroids. To provide an example of an application, we relate the results with the widely used redox ratio., Results: We show that in the case of two detection channels, our system and algorithm enable the selection of optimized band pass filters without the need of knowing involved fluorophores. An improvement of 31,5% in separating different 2D cell cultures is achieved, compared to using established spectral bands that assume NAD(P)H and FAD as main contributors of autofluorescence. The compromise is a reduced SNR in the images., Conclusions: We show that the presented method has the ability to improve imaging contrast and can be used to tailor a given label-free optical imaging system using optical band pass filters targeting a specific biomarker or application.

We have investigated the physical, biochemical, and cellular properties of an autologous leukocyte and platelet-rich fibrin patch. This was generated in an automated device from a sample of a patient's blood at the point of care. Using microscopy, cell counting, enzyme-linked immunosorbent assay, antibody arrays, and cell culture assays, we show that the patch is a three-layered membrane comprising a fibrin sheet, a layer of platelets, and a layer of leukocytes. Mean recovery of platelets from the donated blood was 98% (±95% CI 0.8%). Mean levels of platelet-derived growth factor AB, human transforming growth factor beta 1, and vascular endothelial growth factor extracted from the patch were determined as 127 ng (±95% CI 20), 92 ng (±95% CI 17), and 1.35 ng (±95% CI 0.37), respectively. We showed a continued release of PDGF-AB over several days, the rate of which was increased by the addition of chronic wound fluid. By comparison with traditional platelet-rich plasma, differences in immune components were found. The relevance of these findings was assessed by showing a mitogenic and migratory effect on cultured human dermal fibroblasts. Further, we showed that fibrocytes, a cell type important for acute wound healing, could be grown from the patch. The relevance of these findings in relation to the use of the patch for treating recalcitrant wounds is discussed. [ABSTRACT FROM AUTHOR]