Compact portable multiphoton microscopy reveals histopathological hallmarks of unprocessed lung tumor tissue in real time

Abstract

During lung cancer operations a rapid and reliable assessment of tumor tissue can reduce operation time and potentially improve patient outcomes. We show that third harmonic generation (THG), second harmonic generation (SHG) and two-photon excited autofluorescence (2PEF) microscopy reveals relevant, histopathological information within seconds in fresh unprocessed human lung samples. We used a compact, portable microscope and recorded images within 1 to 3 seconds using a power of 5 mW. The generated THG/SHG/2PEF images of tumorous and nontumorous tissues are compared with the corresponding standard histology images, to identify alveolar structures and histopathological hallmarks. Cellular structures (tumor cells, macrophages and lymphocytes) (THG), collagen (SHG) and elastin (2PEF) are differentiated and allowed for rapid identification of carcinoid with solid growth pattern, minimally enlarged monomorphic cell nuclei with salt-and-pepper chromatin pattern, and adenocarcinoma with lipidic

Quenched coumarin derivatives as fluorescence lifetime phantoms for NADH and FAD

“…To further exploit this valuable information for medical diagnostics (eg, in the form of FLIM endoscopy), new instrumentation (eg, by integration of FLIM capability into devices similar to multiphoton This manuscript is part of the inaugural thesis of Christian Freymüller to be submitted to the Medical Faculty of LMU Munich. systems described in References [2,3]) and investigation protocols need to be devised. For the development, testing or comparison of medical diagnostic systems, it is usually necessary to evaluate a system by a defined calibration protocol/procedure.…”

Non-Invasive Skin Imaging Assessment of Human Stress During Head-Down Bed Rest Using a Portable Handheld Two-Photon Microscope

“…Since the two-photon microscopy imaging technology based on SHG and TPEF was first used in biomedicine, it has received extensive attention for its unique advantages. This technology is mainly used in fibrosis diagnosis and the grading of tissues and organs ( Sun et al, 2008 ), discrimination of tumors ( van Huizen et al, 2020 ), and morphological observation of the living tissue cell matrix in vivo or in vitro ( Zoumi et al, 2002 ) . The endogenous fluorophores of the skin allow label-free visualization of the cellular and subcellular features of various skin layers ( Yew et al, 2014 ).…”

Pulse-Picking Multimodal Nonlinear Optical Microscopy

“…The commonly used nonlinear optical processes for imaging include multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and coherent Raman scattering (CRS). MPEF is sensitive to probing intrinsic fluorophores such as nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide (FAD). , SHG is selective to noncentrosymmetric compositions such as collagen and elastin. , CRS processes, including both coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), harness molecular vibrational transitions excited by ultrafast laser pulses for chemical analysis. − These modalities can be integrated into a single laser-scanning microscope for multimodal imaging. − In general, MPEF and SHG use femtosecond (fs) laser pulses for signal excitation. − CRS, however, prefers picosecond (ps) laser pulses to achieve high spectral resolution. , Hyperspectral CRS microscopy applies either two narrow-band ps laser pulses, combined narrow and broadband laser pulses, or spectrally chirped broadband laser pulses. − The different requirement makes hyperspectral CRS less compatible with MPEF and SHG. It is necessary to develop effective ways to better integrate these imaging modalities without increasing laser phototoxicity.…”