We have developed a new shot noise limited light source for Coherent Raman microscopy and other nonlinear microscopy modalities such as SHG and multiphoton fluorescence. Compared to the market leading SRS light source it achieves 100x faster tuning for random wavelength access including power control and dispersion compensation. Further switching from 80 to 40MHz repetition rate with subharmonic modulation frequency of 20MHz reduces the pixel dwell time in theory by a factor of 8 for the same signal to noise ratio. We will present a comparison to the prevailing SRS light source along with fingerprint images of relevant biological samples.
We present a ready to use label free coherent Raman microscope (CARS/SRS) to perform instantaneous virtual histology and chemical imaging for scientific applications. The system is based on a picosecond laser source that can address two vibrational wavenumbers simultaneously across the entire vibrational spectrum (400cm-1 – 4000cm-1). The laser source is coupled to a ready to use beam scanning microscope enabling SRS, CARS and multiphoton microscopy. Targeting the CH2 and CH3 chemical bonds, we demonstrate instantaneous stimulated Raman histology in the brain and the GI. This new, ready to use, SRS/CARS and multiphoton microscope enables SRS detection with exquisite sensitivity (shot noise limited) and ratio-metric measurement by targeting simultaneously two vibrational frequencies that can be tuned anywhere across the vibrational spectrum.
We revisit wide field CARS imaging using speckle field illuminations. In the proposed scheme we break the CARS coherence using fast varying pump speckle illuminations while keeping static the Stokes speckle. Acquiring a large number of Stokes images enables dynamic speckle illumination (DSI) and random illumination microscopy (RIM). We show that the first one enables quasi-confocal axial sectioning (1/z) while the second one, through post-processing, can retrieve a superior image contrast, noise level and spatial resolution as an important step towards robust nonlinear super-resolution CARS microscopy.
Diagnostic genomic profiling constitutes one of the major challenges to cure brain tumors. The deployment of such analyses depends on the quality of the surgical specimen sent for histopathological examination and further molecular studies. The aim of our study was to assess the potential added value of Stimulated Raman Histology (SRH) for the assessment of freshly excised central nervous system samples. We showed that SRH enabled a near-instant microscopic examination of various central nervous system samples without any tissue processing such as labelling, freezing nor sectioning. Following SRH imaging, we demonstrated that the samples could be readily recovered and reintroduced into a conventional pathology workflow including immunohistochemistry and genomic profiling to establish a definitive diagnosis.
We present a shot noise limited, three-color SRS implementation to address two molecular vibrations simultaneously. The system allows fast, high quality stimulated Raman histology as well as background-free SRS imaging. It is ready to be tested in hospitals for its viability and image quality in comparison to classical rapid histology.
It is based on a mode-locked fs-laser from which 2 narrow-band Stokes laser beams are extracted and subsequently modulated at 13 and 20MHz. The center part of the fs-laser is frequency doubled to pump a picosecond optical parametric oscillator, which can be tuned from 500 to 5000 cm−1.
Raman microscopy is a powerful mode of label free nonlinear optical microscopy that is hampered by weak cross-sections, leading to slow imaging. We will discuss two advances in coherent Raman microscopy: 1) high speed coherent anti-Stokes Raman scattering microscopy imaging based on spatial frequency modulation imagining (SPIFI) where a structured line focus is used to image is used to image specimens with a single pixel detector. 2) Doppler Raman microscopy that exploits the extremely low timing jitter of modelocked lasers for ultrasensitive Raman spectroscopy and microscopy.
Raman microscopy has been widely developed for label free nonlinear optical microscopy of biological systems. Imaging speed in these systems is hampered by low Raman scattering cross sections and the requirement of scanning a focused laser beam through the sample in conventional Raman microscopy. The serial acquisition that is necessary in point scanning microscopy slows image acquisition and limits the dwell time are each image pixel. Here, we discuss two new imaging methods that are based on spatial frequency modulation imagining (SPIFI) [1-2], where a structured line focus is used to image is used to image specimens by collecting light on a single pixel detector. We discuss the use of SPIFI to improve the imaging speed of Spontaneous Raman scattering and coherent anti-Stokes Raman scattering microscopy. A detailed noise analysis highlighting the advantages and disadvantages of SPIFI as compared to conventional point scan imaging is presented.
Conventional CARS microscopy requires scanning a point focus through the specimen limits imaging speed. We present a spatial frequency projection imaging (SPIFI) method for CARS microscopy to spatially multiplex CARS microscopy. A spinning disk modulator is used to rapidly modulate the Stokes field with a rapidly swept spatially periodic transmission grating. SPIFI-CARS images are obtained by Fourier transforming the single pixel signal. Images of CARS and second harmonic generation from histological slices will be presented. The physics of image formation and the impact of multiplexing on SNR will be discussed. Prospects for scaling to high speed CARS imaging will be discussed.
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