Double-modulation stimulated Raman scattering: how to image up to 16-fold faster.

A stimulated Raman microscope is conventionally performed by modulating either the pump or Stokes beam and demodulating the other. Here, we propose a double modulation scheme that modulates both beams at fm and 2fm. Exploiting aliasing and reduction of the repetition rate, we show that the proposed double modulation scheme amplifies the signal amplitude by a factor of 1.5, 2, and 4 for different modulation frequencies and experimental realizations for the same average power at the sample. By deriving the noise power for different sources, we show that the double modulation scheme can perform stimulated Raman scattering (SRS) imaging with an up to 16-fold speed improvement as compared with single beam modulation.

High-speed chemical imaging of dynamic and histological samples with stimulated Raman micro-spectroscopy.

We report a shot noise limited high-speed stimulated Raman microscopy platform allowing to acquire molecular vibrational spectra over 200 cm-1 in 12 µs at a scan rate of 40kHz. Using spectral focusing together with optimized acousto-optics programmable dispersive filters, the designed low noise imaging platform performs chemical imaging of dynamical processes such as Mannitol crystal hydration and reaches a signal to noise ratio sufficient to perform label free histological imaging on frozen human colon tissue slides.

Low-aberration high-speed-compatible optical delay line.

We describe a simple approach to dispersion-free optical delay line design that provides very low aberration over an extended delay range. In this approach, we minimize aberrations by directing non-axial beam displacements along a line of symmetry built into the apparatus. We show improved performance and significant reduction of wavefront aberrations by comparing simulation and experimental results with a similar delay line that lacks this line of symmetry. The new design facilitates transform-limited recovery of spectral resolution in Fourier transform coherent anti-Stokes Raman scattering, and accordingly, we demonstrate 3.5cm-1 spectral resolution with a 10 ps delay scan range.

Sub-second hyper-spectral low-frequency vibrational imaging via impulsive Raman excitation.

Real-time vibrational microscopy has been recently demonstrated by various techniques, most of them utilizing the well-known schemes of coherent anti-stokes Raman scattering and stimulated Raman scattering. These techniques readily provide valuable chemical information mostly in the higher vibrational frequency regime (>400 cm-1). Addressing the low vibrational frequency regime (<200 cm-1) is challenging due to the usage of spectral filters that are required to isolate the signal from the Rayleigh scattered excitation field. In this Letter, we report on rapid, high-resolution, low-frequency (<130 cm-1) vibrational microscopy using impulsive coherent Raman excitation. By combining impulsive excitation with a fast acousto-optic delay line, we detect the Raman-induced optical Kerr lensing and spectral shift effects with a 25 µs pixel dwell time to produce shot-noise limited, low-frequency hyper-spectral images of various samples.

Dual-focus stimulated Raman scattering microscopy: a concept for multi-focus scaling.

High-speed imaging is of the utmost importance for video-rate live cell investigations or to study extended sample areas at sufficient spatial resolution within reasonable time scales. Improving the speed of single-focus stimulated Raman scattering (SRS) microscopy is ultimately restricted by the sample's damage threshold and the shot noise of the demodulated laser source. To overcome this limitation, we present a dual-focus SRS approach modulating the pump laser for each focus at a distinct frequency. The corresponding probe beams are detected each by a photodiode and demodulated individually by two separate lock-in units to avoid inter-focal cross-talk. Two laterally or axially displaced images as well as hyperspectral SRS images can be obtained simultaneously within the field of view of the objective lens. The modular implementation presented here can be extended to multiple foci by using multi-channel acousto-optics modulators in combination with multi-channel lock-in amplifiers.

Simultaneous dual-channel stimulated Raman scattering microscopy demultiplexed at distinct modulation frequencies.

To increase the information per pixel in stimulated Raman scattering (SRS) microscopy as well as to correct from artifacts, it is valuable to acquire images at two different Raman shifts. We present a three-color SRS approach acquiring two perfectly registered SRS images where both pump beams are modulated at distinct frequencies while demodulating the Stokes beam. Our implementation uses two optical parametric oscillators that can be tuned to an almost arbitrary energy difference of Raman shifts, allowing investigation of fingerprint resonances simultaneously to CH-stretch vibrations.

Pump-probe micro-spectroscopy by means of an ultra-fast acousto-optics delay line.

We demonstrate femtosecond pump-probe transient absorption spectroscopy using a programmable dispersive filter as an ultra-fast delay line. Combined with fast synchronous detection, this delay line allows for recording of 6 ps decay traces at 34 kHz. With such acquisition speed, we perform single point pump-probe spectroscopy on bulk samples in 80 µs and hyperspectral pump-probe imaging over a field of view of 100 µm in less than a second. The usability of the method is illustrated in a showcase experiment to image and discriminate between two pigments in a mixture.

Fast stimulated Raman and second harmonic generation imaging for intraoperative gastro-intestinal cancer detection.

Conventional haematoxylin, eosin and saffron (HES) histopathology, currently the 'gold-standard' for pathological diagnosis of cancer, requires extensive sample preparations that are achieved within time scales that are not compatible with intra-operative situations where quick decisions must be taken. Providing to pathologists a close to real-time technology revealing tissue structures at the cellular level with HES histologic quality would provide an invaluable tool for surgery guidance with evident clinical benefit. Here, we specifically develop a stimulated Raman imaging based framework that demonstrates gastro-intestinal (GI) cancer detection of unprocessed human surgical specimens. The generated stimulated Raman histology (SRH) images combine chemical and collagen information to mimic conventional HES histopathology staining. We report excellent agreements between SRH and HES images acquire on the same patients for healthy, pre-cancerous and cancerous colon and pancreas tissue sections. We also develop a novel fast SRH imaging modality that captures at the pixel level all the information necessary to provide instantaneous SRH images. These developments pave the way for instantaneous label free GI histology in an intra-operative context.