Pulse-Picking Multimodal Nonlinear Optical Microscopy

Abstract

Nonlinear optical microscopy techniques can map chemical compositions in biological samples in a label-free manner. Commonly used nonlinear optical processes for imaging include multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and coherent Raman scattering (CRS). Femtosecond lasers are typically used for MPEF and SHG due to the requirement of high peak power for excitation, while picosecond lasers are preferred for CRS due to the need for high spectral resolution. Therefore, it is challenging to integrate CRS with MPEF and SHG for chemical imaging. We develop a pulsepicking strategy based on an acousto-optic modulator that can program the duty cycle of the laser pulse train, significantly increasing the pulse peak power at low input average power. This approach offers strong enhancement of nonlinear optical signals and makes hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy compatible with MPEF and SHG for multimodal imaging at low laser average power. The pulse-picking method also enables the evaluation and comparison of phototoxicity of laser pulses at different average and peak power levels. The photo-perturbations to biological samples are evaluated using cellular dynamics and sample morphological changes, allowing the selection of optimal laser power for the best sensitivity and minimal phototoxicity.

Coherent Raman scattering microscopy of lipid droplets in cells and tissues

“…This method has been used for hyperspectral CARS and SRS microscopy. 70,71 To demonstrate hyperspectral SRS imaging, we acquired images from MIA PaCa2 cells using spectral focusing similar to previous reports. 72 Figure 3A displays an SRS image integrated from 2800 to 3050 cm À1 , whereas Figure 3B-E shows the maps of LD, ER, protein, and nuclei images, respectively.…”

“…LD dynamics quantified by CARS microscopy are applied to evaluate the phototoxicity of laser pulses at different average and peak power ranges. 71 This approach allows to find the optimal imaging condition for live cells using nonlinear optical modalities. Aside from quantification of LD dynamics, a recent technology termed real-time precision opto-control has demonstrated the potential of controlling LD dynamics at subcellular compositions when applied with photoswitchable tubulin inhibitors.…”

“…Many research groups have developed methods to assess phototoxicity induced by laser pulses with different energy and pulse width. 71,96,[102][103][104] Cell morphological change, 96 membrane blebbing, 102 RNA sequencing, 104 cell proliferation, 104 reactive oxygen species, 104 and intracellular dynamics 71 have been used to measure the phototoxicity of laser pulses to samples. The sensitivity of these methods for evaluating phototoxicity has not been compared.…”

“…Selecting the best laser repetition rate and pulse energy or using the pulse picking method allows to find the optimal average and peak power range for minimal phototoxicity and the best image contrast. 71,102…”

Full-Spectrum CARS Microscopy of Cells and Tissues with Ultrashort White-Light Continuum Pulses

“…Furthermore, the generated WLC shows long-term stability and does not suffer from wavelength-dependent intensity fluctuations that may affect the shape of the CARS spectrum. The use of high pulse energies also results in stronger CARS signals, improving the signal quality and acquisition speed of the setup …”