Simultaneous label-free autofluorescence-multiharmonic microscopy and beyond

Abstract

Without sophisticated data inversion algorithms, nonlinear optical microscopy can acquire images at subcellular resolution and relatively large depth, with plausible endogenous contrasts indicative of authentic biological and pathological states. Although independent contrasts have been derived by sequentially imaging the same sample plane or volume under different and often optimized excitation conditions, new laser source engineering with inputs from key biomolecules surprisingly enable real-time simultaneous acquisition of multiple endogenous molecular contrasts to segment a rich set of cellular and extracellular components. Since this development allows simple single-beam single-shot excitation and simultaneous multicontrast epidirected signal detection, the resulting platform avoids perturbative sample pretreatments such as fluorescent labeling, mechanical sectioning, scarce or interdependent contrast generation, constraints to the sample or imaging geometry, and intraimaging motion artifacts that have limited in vivo nonlinear optical molecular imaging.

Label-free imaging of human brain tissue at subcellular resolution for potential rapid intra-operative assessment of glioma surgery

“…NADH and FAD are often sequentially excited at 700-750 nm and 850-920 nm, respectively, in order to obtain cleaner (or less mixed) fluorescence signals. This sequential excitation at different wavelengths, however, would lead to a lower imaging frame rate, and more importantly, it would become more difficult in ensuring pixel-level registration between the NADH and FAD channels in the case of dynamic samples (e.g., when performing imaging in vivo ) 36 , 37 . Here we chose 780 nm laser to simultaneously excite and collect the fluorescence of NADH and FAD to avoid the above mentioned difficulties.…”

Weakly supervised identification of microscopic human breast cancer-related optical signatures from normal-appearing breast tissue

“…However, the H&E sample preparation process requires tissue fixation, sectioning, and staining, which may unavoidably cause tissue distortions and feature disappearance. Therefore, a label-free virtual histopathology technology is fulfilled by nonlinear optical techniques which enable the observation of the unperturbed biochemical microenvironment 3,26 . In this study, we leverage MIL for human breast cancer-related optical signature identification based on label-free virtual histopathology, where only ambiguous whole-image-level annotations are available.…”

Multiplex‐multiphoton microscopy and computational strategy for biomedical imaging

“…Indeed, the remote multiphoton excitation spectra of standard fluorophores are spread on large bandwidths covering about 300 nm in the NIR, sometimes without any overlapping. This property limits the dynamic imaging of samples across different species, which can only be alternatively imaged with a multishot excitation (Boppart, You, Li, Chen, & Tu, 2019). Then, a numerical merging of resulting images is mandatory for obtaining a multiplex image and artifacts due to misalignment during the recording can appear.…”

“…Then, a numerical merging of resulting images is mandatory for obtaining a multiplex image and artifacts due to misalignment during the recording can appear. This sequential procedure drastically increases imaging delay, a critical parameter in the field of dynamic multiplex imaging or for detecting phenomena at video rates (Abdeladim et al, 2019; Boppart et al, 2019; Kirkpatrick et al, 2012). The technological transition from standard multishot MPM toward a one‐shot strategy of excitation for multiplex imaging thus still requires the implantation of an alternative excitation solution for MPM.…”