We have developed a video-rate stimulated Raman scattering (SRS) microscope with frame-by-frame wavenumber tunability. The system uses a 76-MHz picosecond Ti:sapphire laser and a subharmonically synchronized, 38-MHz Yb fiber laser. The Yb fiber laser pulses are spectrally sliced by a fast wavelength-tunable filter, which consists of a galvanometer scanner, a 4-f optical system and a reflective grating. The spectral resolution of the filter is ~ 3 cm-1. The wavenumber was scanned from 2800 to 3100 cm-1 with an arbitrary waveform synchronized to the frame trigger. For imaging, we introduced a 8-kHz resonant scanner and a galvanometer scanner. We were able to acquire SRS images of 500 x 480 pixels at a frame rate of 30.8 frames/s. Then these images were processed by principal component analysis followed by a modified algorithm of independent component analysis. This algorithm allows blind separation of constituents with overlapping Raman bands from SRS spectral images. The independent component (IC) spectra give spectroscopic information, and IC images can be used to produce pseudo-color images. We demonstrate various label-free imaging modalities such as 2D spectral imaging of the rat liver, two-color 3D imaging of a vessel in the rat liver, and spectral imaging of several sections of intestinal villi in the mouse. Various structures in the tissues such as lipid droplets, cytoplasm, fibrous texture, nucleus, and water-rich region were successfully visualized.
Stimulated Raman scattering (SRS) microscopy can visualize molecular vibration with high
sensitivity and high contrast, allowing label-free imaging of biological samples. In order to specify
molecules, it is important to obtain Raman spectrum at each pixel. High-speed wavelength scanning
would allow such spectral imaging. Here, we demonstrate a tunable optical filter for spectral
imaging with SRS microscopy. In the filter, an incident beam is reflected by a galvanometer scanner,
and then imaged onto a Littrow grating by 4-f relay lenses. The diffracted beam is reflected back to
the galvanometer scanner, and then launched into a fiber collimator. The transmission wavelength of
this filter can be tuned because the Littrow angle is dependent on the angle of the galvanometer
scanner. This configuration allows high spectral resolution of ~0.3 nm and high-speed wavelength
scanning with a response time of a few milliseconds. Furthermore, the optical path length is kept
constant when the wavelength is scanned. This property is important because SRS microscopy uses
two-color laser pulses, which should coincide in time. In the experiment, broadband pulses from a
38-MHz ytterbium fiber laser is filtered out by the optical filter, and then amplified by Yb-doped
fiber amplifiers. The wavelength of the amplified pulses is tunable over ~24 cm-1 and the spectral
width of the pulses is < 3.3 cm-1. These pulses are synchronized with a 76-MHz train of 5-ps pulses
generated by a Ti:sapphire laser. By using these two-color pulses, SRS spectral imaging of polymer
beads is successfully accomplished.
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