Conventional multiphoton microscopy uses periodically pulsed sources as excitation and the sample is illuminated uniformly by the laser. While necessary for structural imaging, monitoring dynamic biological functions such as neuronal activity in the brain typically only requires imaging of the region of interest (ROI), e.g., the neurons. The adaptive excitation source enables imaging of the region of interest only. It reduces the requirement for the output power of the excitation source (by at least an order of magnitude) and simultaneously reduces the excitation power to the sample for obtaining the necessary information (e.g., neuronal activity). We demonstrate three-photon imaging of brain activity in awake transgenic mice (jRGECO1a), with highest speed (30 frames/s), large field-of-view (620x620 μm/512x512 pixels) and deep penetration (750 μm beneath the dura).
We demonstrate a robust, all-fiber, two-wavelength time-lens source for background-free coherent anti-Stokes Raman scattering (CARS) imaging. The time-lens source generates two picosecond pulse trains simultaneously: one at 1064 nm and the other tunable between 1040 nm and 1075 nm (~ 400 mW for each wavelength). When synchronized to a modelocked Ti:Sa laser, the two wavelengths are used to obtain on- and off-resonance CARS images. Real-time subtraction of the nonresonant background in the CARS image is achieved by the synchronization of the pixel clock and the time-lens source. Background-free CARS imaging of sebaceous glands in ex vivo mouse tissue is demonstrated.
Though single-color coherent Raman microscopy has been widely used for vibrational imaging of isolated Raman bands, it is still challenging to visualize molecules having overlapping Raman bands. We address this issue by developing a spectroscopic SRS microscope with a time-lens laser source synchronized to a femtosecond laser. The time-lens source provides 2-ps pulse at the wavelength of 1064 nm. A pulse shaper is installed for intra-pulse spectral scanning of the femtosecond laser output. By electronically modulating the time-lens source at MHz frequency, spectroscopic stimulated Raman loss (SRL) images were obtained on a laser-scanning microscope. Using this microscope, we have been able to detect 0.2% DMSO in aqueous solution. Spectroscopic SRL images of prostate cancer cells were obtained. Multivariate curve resolution analysis was further applied to decompose the SRL images into concentration maps of proteins and lipids. With high sensitivity and high spectral resolution, this method offers exciting potential in label-free imaging of live cells using fingerprint Raman bands.
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