Optimization of wide-field second-harmonic generation microscopy for fast imaging of large sample areas in biological tissues

Abstract

Second-harmonic generation (SHG) microscopy is a label-free imaging method that can be used to visualize the detailed arrangement of collagen structures in biological tissues. Here, we sought to optimize the speed of microscopic SHG image acquisition of macroscopic fixed tissue sample areas by employing the wide-field imaging with a high power and medium, 1 MHz pulse repetition frequency laser in combination with a mechanical sample scanning. Unlike in the conventional laser-scanning microscopy, the optimum of the wide-field acquisition entails an interplay between the size of the illuminated area and the intensity of the generated signal. We delineate quantitative procedures to set the image parameters for the maximum speed of the tiled image acquisition, and also describe the possible optimization of the laser parameters for further enhancement of the speed of acquisition.

Photoluminescence Dynamics of MAPI Perovskite Films Induced by Lateral Electric Field

“…EFISH Microscopy: SHG imaging was performed on a custom-built wide-field nonlinear microscopy setup based on a modular microscope from Applied Scientific Instruments (ASI), as described previously. [26] In brief, a sample area of about 300 Â 300 μm 2 was illuminated with FemtoLux3 laser (Ekspla) with 1030 nm wavelength, 262 fs pulse duration, 100 kHz pulse repetition frequency, and 100 mW of average power at the sample. The resulting signal was detected in the forward direction with Neo 5.5 s CMOS (Andor) with 10Â magnification.…”

Quantitative and qualitative analysis of pulmonary arterial hypertension fibrosis using wide-field second harmonic generation microscopy

“…All imaging was performed on a custom-built wide-field non-linear microscopy setup based on a modular microscope (Applied Scientific Instruments, USA), as described previously 28 . In brief, a sample area of about 150 × 150 µm 2 was illuminated with FemtoLux3 laser (Ekspla, Lithuania) with 1030 nm wavelength, 262 fs pulse duration, 1 MHz pulse repetition frequency, and 1.5 W of average power at the sample.…”

Wide-Field Multicontrast Nonlinear Microscopy for Histopathology

“…For wide-field nonlinear microscopy, a high-power ultrafast laser is employed to provide enough pulse energy for a large area illumination. The illumination is achieved either by a high-power oscillator [34], an amplified laser system [35], or a fiber laser [36]. Wide-field illumination using high-powered lasers differently affects biological samples when compared to a raster-scanning with tightly focused laser beam.…”