We use the time-lens concept to demonstrate a new scheme for synchronization of two pulsed light sources for biological
imaging. An all fiber, 1064 nm time-lens source is synchronized to a picosecond solid-state Ti: Sapphire mode-locked
laser by using the mode-locked laser pulses as the clock. We demonstrate the application of this synchronized source for
CARS and SRS imaging by imaging mouse tissues. Synchronized two wavelength pulsed source is a major technical
difficulty for CARS and SRS imaging. The time-lens source demonstrated here may provide an all-fiber, user friendly
alternative for future SRS imaging.
The development of methods that allow microscale studies of complex biomaterials based on their molecular composition is of great interest to a wide range of research fields. We show that stimulated Raman scattering (SRS) microscopy is an excellent analytical tool to study distributions of different biomolecules in multiphasic systems. SRS combines the label-free molecular specificity of vibrational spectroscopy with an enhanced sensitivity due to coherent excitation of molecular vibrations. Compared to previous imaging studies using coherent anti-Stokes Raman scattering microscopy, the main advantage of SRS microscopy is the absence of the unwanted nonresonant background, which translates into a superior sensitivity and undistorted vibrational spectra. We compare spectra of complex materials obtained with stimulated Raman scattering and spontaneous Raman scattering in the crowded fingerprint region. We find that, as expected, there is excellent correspondence and that the SRS spectra are free from interference from background fluorescence. In addition, we show high-resolution imaging of the distributions of selected biomolecules, such as lipids and proteins, in food products with SRS microscopy.
Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous
Raman microscopy provides specific vibrational signatures of chemical bonds, but is often
hindered by low sensitivity. Here we report a 3D multi-photon vibrational imaging
technique based on stimulated Raman scattering (SRS). The sensitivity of SRS is
significantly greater than that of spontaneous Raman scattering, and is further enhanced
by high-frequency (MHz) phase-sensitive detection. SRS microscopy has a major advantage
over previous coherent Raman techniques in that it offers
background-free and easily
interpretable chemical contrast. We show a variety of biomedical applications, such as
differentiating distributions of omega-3 fatty acids and saturated lipids in living cells,
imaging of brain and skin tissues based on intrinsic lipid contrast.
We report on the use of adaptive optics in coherent anti-Stokes Raman scattering microscopy (CARS) to improve the
image brightness and quality at increased optical penetration depths in biological material. The principle of the
technique is to shape the incoming wavefront in such a way that it counteracts the aberrations introduced by imperfect
optics and the varying refractive index of the sample. In recent years adaptive optics have been implemented in
multiphoton and confocal microscopy. CARS microscopy is proving to be a powerful tool for non-invasive and label-free
biomedical imaging with vibrational contrast. As the contrast mechanism is based on a 3rd order non-linear optical
process, it is highly susceptible to aberrations, thus CARS signals are commonly lost beyond the depth of ~100 μm in
tissue. We demonstrate the combination of adaptive optics and CARS microscopy for deep-tissue imaging using a
deformable membrane mirror. A random search optimization algorithm using the CARS intensity as the figure of merit
determined the correct mirror-shape in order to correct for the aberrations. We highlight two different methods of
implementation, using a look up table technique and by performing the optimizing in situ. We demonstrate a significant
increase in brightness and image quality in an agarose/polystyrene-bead sample and white chicken muscle, pushing the
penetration depth beyond 200 μm.