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.

Frequency-encoded two-photon excited fluorescence microscopy.

Two-photon excited fluorescence (2PEF) microscopy is the most popular non-linear imaging method of biomedical samples. State-of-the art 2PEF microscopes use multiple detectors and spectral filter sets to discriminate different fluorophores based on their distinct emission behavior (emission discrimination). One drawback of 2PEF is that fluorescence photons outside the filter transmission range are inherently lost, thereby reducing the imaging efficiency and speed. Furthermore, emission discrimination of different fluorophores may fail if their emission profiles are too similar. Here, we present an alternative 2PEF method that discriminates fluorophores based on their excitation spectra (excitation discrimination). For excitation we use two lasers of different wavelengths (ω1, ω2) resulting in excitation energies at 2ω1, 2ω2, and the mixing energy ω1+ω2. Both lasers are frequency encoded (FE) by an intensity modulation at distinct frequencies while all 2PEF emission is collected on a single detector. The signal is fed into a lock-in-amplifier and demodulated at various frequencies simultaneously. A customized nonnegative matrix factorization (NNMF) then generates fluorescence images that are free of cross talk. Combining FE-2PEF with multiple detectors has the potential to enable the simultaneous imaging of an unprecedented number of fluorophores.

Laser scanning dark-field coherent anti-Stokes Raman scattering (DF-CARS): a numerical study.

We present and model a dark-field illumination scheme for coherent anti-Stokes Raman scattering (DF-CARS) that highlights the interfaces of an object with chemical sensitivity. The proposed DF-CARS scheme uses dedicated arrangements of the pump kp1, Stokes kS and probe kp2 beams' k-wave-vectors to address the sample's interfaces along the x, y or z axis. The arrangements of the incident k-wave-vectors are derived from the Ewald sphere representation of the outgoing anti-Stokes radiation and the effective CARS excitation wave-vector keff = kp1 + kp2 - kS under the intention to avoid probing the object frequency K(0,0,0), i.e., the contribution of a homogeneous sample (dark-field configuration). We suggest a possible experimental realization using simple masks placed in the back pupil of the excitation microscope objective lens. Applying a full vectorial model, the proposed experimental implementation is numerically investigated on grounds of the Debye-Wolff integral and dynadic Green function to confirm the predicted chemical interface contrast.

3D-coherent anti-Stokes Raman scattering Fourier ptychography tomography (CARS-FPT).

Fourier ptychography tomography (FPT) is a novel computational technique for coherent imaging in which the sample is numerically reconstructed from images acquired under various illumination directions. FPT is able to provide three-dimensional (3D) reconstructions of the complex sample permittivity with an increased resolution compared to standard microscopy. In this work, FPT is applied to coherent anti-Stokes Raman scattering (CARS) imaging. We show on synthetic data that complex third-order susceptibilities can be reconstructed in 3D from a limited number of widefield CARS images. In addition, we observe that the non-linear interaction increases significantly the potential of CARS-FPT compared to linear FPT in terms of resolution. In particular, with a careful choice of the pump and Stokes beam directions, CARS-FPT is able to provide optical sectioning even in transmission configuration.

Simultaneous stimulated Raman gain and loss detection (SRGAL).

The fidelity of stimulated Raman scattering (SRS) microscopy images is impaired by artifacts such as thermal lensing, cross-phase modulation and multi-photon absorption. These artifacts affect differently the stimulated Raman loss (SRL) and stimulated Raman gain (SRG) channels making SRL and SRG image comparisons attractive to identify and correct SRS image artifacts. To provide answer to the question: "Can I trust my SRS images?", we designed a novel, but straightforward SRS scheme that enables the dectection of the stimulated Raman gain and loss (SRGAL) simultaneously at the same pixel level. As an advantage over the conventional SRS imaging scheme, SRGAL doubles the SRS signal by acquiring both SRL as well as SRG and allows for the identification of SRS artifacts and their reduction via a balanced summation of the SRL and SRG images.

Background-suppressed SRS fingerprint imaging with a fully integrated system using a single optical parametric oscillator.

Stimulated Raman Scattering (SRS) imaging can be hampered by non-resonant parasitic signals that lead to imaging artifacts and eventually overwhelm the Raman signal of interest. Stimulated Raman gain opposite loss detection (SRGOLD) is a three-beam excitation scheme capable of suppressing this nonlinear background while enhancing the resonant Raman signal. We present here a compact electro-optical system for SRGOLD excitation which conveniently exploits the idler beam generated by an optical parametric oscillator (OPO). We demonstrate its successful application for background suppressed SRS imaging in the fingerprint region. This system constitutes a simple and valuable add-on for standard coherent Raman laser sources since it enables flexible excitation and background suppression in SRS imaging.

Coherent anti-Stokes Raman Fourier ptychography.

We present a theoretical and numerical study of coherent anti-Stokes Raman scattering Fourier ptychography microscopy (CARS-FPM), a scheme that has not been considered so far in the previously reported CARS wide-field imaging schemes. In this approach, the distribution of the Raman scatterer density of the sample is reconstructed numerically from CARS images obtained under various angles of incidences of the pump or Stokes beam. Our inversion procedure is based on an accurate vectorial model linking the CARS image to the sample and yields both the real and imaginary parts of the susceptibility, the latter giving access to the Raman information, with an improved resolution.

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.

Dual-focus coherent anti-Stokes Raman scattering microscopy using a compact two-beam fiber laser source.

We have developed a dual-focus coherent anti-Stokes Raman scattering (CARS) microscope based on a dual output, compact fiber laser source. The underlying concepts of time-multiplexed, two-beam scanning and demultiplexed detection that we already employed for second-harmonic generation are here naturally extended for CARS microscopy. The layout of a robust, all-fiber laser source was reconfigured to provide two outputs, each containing the two colors necessary for the CARS process. The utilization of the design for simultaneously imaging two laterally or axially separated fields of view and, thus, inherently speeding up the image acquisition process, is demonstrated on human artery tissue samples.

Pseudo-HE images derived from CARS/TPEF/SHG multimodal imaging in combination with Raman-spectroscopy as a pathological screening tool.

BACKGROUND: Due to the steadily increasing number of cancer patients worldwide the early diagnosis and treatment of cancer is a major field of research. The diagnosis of cancer is mostly performed by an experienced pathologist via the visual inspection of histo-pathological stained tissue sections. To save valuable time, low quality cryosections are frequently analyzed with diagnostic accuracies that are below those of high quality embedded tissue sections. Thus, alternative means have to be found that enable for fast and accurate diagnosis as the basis of following clinical decision making. METHODS: In this contribution we will show that the combination of the three label-free non-linear imaging modalities CARS (coherent anti-Stokes Raman-scattering), TPEF (two-photon excited autofluorescence) and SHG (second harmonic generation) yields information that can be translated into computational hematoxylin and eosin (HE) images by multivariate statistics. Thereby, a computational HE stain is generated resulting in pseudo-HE overview images that allow for identification of suspicious regions. The latter are analyzed further by Raman-spectroscopy retrieving the tissue's molecular fingerprint. RESULTS: The results suggest that the combination of non-linear multimodal imaging and Raman-spectroscopy possesses the potential as a precise and fast tool in routine histopathology. CONCLUSIONS: As the key advantage, both optical methods are non-invasive enabling for further pathological investigations of the same tissue section, e.g. a direct comparison with the current pathological gold-standard.

Hepatic Vitamin A Content Investigation Using Coherent Anti-Stokes Raman Scattering Microscopy.

Standard techniques for examining the distribution of vitamin A in liver either require staining or lead to rapid photobleaching of the molecule. A potentially better alternative approach is to use coherent anti-Stokes Raman scattering (CARS) microscopy; a fast, label-free, non-disruptive imaging method that provides contrast based on molecular vibrations. This contribution evaluates the viability of CARS microscopy for imaging vitamin A within thick hepatic tissue under physiological conditions by tuning into its characteristic vibrational band in the fingerprint region. Additional information about the morphology and architecture of the tissue was acquired using second harmonic generation (SHG) and multi-photon excited fluorescence (MPEF) to help mapping the intra-lobular positions of the vitamin A droplets. We demonstrate the capability of our multimodal imaging framework to selectively image lipid-soluble vitamin A droplets deep in bulk liver tissue with a high contrast while co-registering a complementary morphological background that clearly visualizes hepatic lobules. The results obtained envisage the good prospect of the technique for in vivo studies assessing vitamin A distribution heterogeneity and how it is affected by the progression of hepatic diseases.

Vibrational phase imaging in wide-field CARS for nonresonant background suppression.

Coherent Anti-Stokes Raman Scattering (CARS) microscopy is a valuable tool for label-free imaging of biological samples. As a major drawback quantification based on CARS images is compromised by the appearance of a nonresonant background. In this paper we propose and demonstrate a wide-field CARS vibrational phase imaging scheme that allows for nonresonant background suppression. Several CARS images at a few consecutive planes perpendicular to the propagation direction were recorded to reconstruct a phase map utilizing the iteration phase retrieval method. Experimental results verify that the CARS background is efficiently suppressed by the phase imaging approach, as compared to traditional CARS imaging without background correction. The proposed background correction method is robust against environmental disturbance, since the experimental implementation of the suggested detection scheme requires no reference beam.

Fiber-based dual-focus time-demultiplexed second harmonic generation microscopy.

We present a dual-focus second harmonic generation (SHG) microscopy approach based on stable, compact, and inexpensive fiber technology. One-tenth of the fiber laser output is coupled into a 100 m (≙500 ns) long single-mode fiber and further amplified to achieve two separately guided beams with time-alternating pulse trains. SHG detection is performed sequentially, generating two individual images in one scan. Thus, the configuration allows for imaging of distinct areas within the field of view at twice the repetition rate of the fiber laser but is readily extended to a multiple of the repetition rate with tens of foci.

In vivo organoid growth monitoring by stimulated Raman histology.

Patient-derived tumor organoids have emerged as a crucial tool for assessing the efficacy of chemotherapy and conducting preclinical drug screenings. However, the conventional histological investigation of these organoids necessitates their devitalization through fixation and slicing, limiting their utility to a single-time analysis. Here, we use stimulated Raman histology (SRH) to demonstrate non-destructive, label-free virtual staining of 3D organoids, while preserving their viability and growth. This novel approach provides contrast similar to conventional staining methods, allowing for the continuous monitoring of organoids over time. Our results demonstrate that SRH transforms organoids from one-time use products into repeatable models, facilitating the efficient selection of effective drug combinations. This advancement holds promise for personalized cancer treatment, allowing for the dynamic assessment and optimization of chemotherapy treatments in patient-specific contexts.

Automated seeding-based nuclei segmentation in nonlinear optical microscopy.

Nonlinear optical (NLO) microscopy based, e.g., on coherent anti-Stokes Raman scattering (CARS) or two-photon-excited fluorescence (TPEF) is a fast label-free imaging technique, with a great potential for biomedical applications. However, NLO microscopy as a diagnostic tool is still in its infancy; there is a lack of robust and durable nuclei segmentation methods capable of accurate image processing in cases of variable image contrast, nuclear density, and type of investigated tissue. Nonetheless, such algorithms specifically adapted to NLO microscopy present one prerequisite for the technology to be routinely used, e.g., in pathology or intraoperatively for surgical guidance. In this paper, we compare the applicability of different seeding and boundary detection methods to NLO microscopic images in order to develop an optimal seeding-based approach capable of accurate segmentation of both TPEF and CARS images. Among different methods, the Laplacian of Gaussian filter showed the best accuracy for the seeding of the image, while a modified seeded watershed segmentation was the most accurate in the task of boundary detection. The resulting combination of these methods followed by the verification of the detected nuclei performs high average sensitivity and specificity when applied to various types of NLO microscopy images.

Coherent Stokes Raman scattering microscopy (CSRS).

We report the first implementation of laser scanning coherent Stokes Raman scattering (CSRS) microscopy. To overcome the major challenge in CSRS imaging, we show how to suppress the fluorescence background by narrow bandpass filter and a lock-in based demodulation. Near background free CSRS imaging of polymer beads, human skin, onion cells, avocado flesh and the wing disc of a drosphila larva are presented. Finally, we explain and demonstrate numerically that CSRS solves a major obstacle of other coherent Raman techniques by sending a significant part (up to 100%) of the CSRS photons into the backward direction under tight focusing conditions. We believe that this discovery will pave the way for numerous technological advances, e.g., in epi-detected coherent Raman multi-focus imaging, real-time laser scanning based spectroscopy or efficient endoscopy.

Live Stimulated Raman Histology for the Near-Instant Assessment of Central Nervous System Samples.

Central nervous system tumors encompass many heterogeneous neoplasms with different outcomes and treatment strategies. The current classification of these tumors is based on molecular parameters in addition to histopathology to define tumor entities. This genomic characterization of tumors is also becoming increasingly essential for physicians to identify targeted therapy options. The deployment of such genomic profiling relies on an efficient surgical sampling. To perform an appropriate tumor resection and a correct sampling of the tumor, the neurosurgeon may request an intraoperative pathological consultation. Stimulated Raman histology (SRH), an emerging nondestructive imaging technology, can address this challenge. SRH allows for a rapid and label-free microscopic examination of unprocessed tissues samples in near-perfect concordance with standard histology. In this study we showed that SRH enabled the near-instant microscopic examination of various central nervous system samples without any tissue processing such as labeling, freezing nor sectioning. Since SRH imaging is a nondestructive approach, we demonstrated that the tissue could be readily recovered after SRH imaging and reintroduced into the conventional pathology workflow including immunohistochemistry and genomic profiling to establish a definitive diagnosis.

Shot-noise limited tunable dual-vibrational frequency stimulated Raman scattering microscopy.

We present a shot-noise limited SRS implementation providing a >200 mW per excitation wavelength that is optimized for addressing two molecular vibrations simultaneously. As the key to producing a 3 ps laser of different colors out of a single fs-laser (15 nm FWHM), we use ultra-steep angle-tunable optical filters to extract 2 narrow-band Stokes laser beams (1-2 nm & 1-2 ps), which are separated by 100 cm-1. The center part of the fs-laser is frequency doubled to pump an optical parametric oscillator (OPO). The temporal width of the OPO's output (1 ps) is matched to the Stokes beams and can be tuned from 650-980 nm to address simultaneously two Raman shifts separated by 100 cm-1 that are located between 500 cm-1 and 5000 cm-1. We demonstrate background-free SRS imaging of C-D labeled biological samples (bacteria and Drosophila). Furthermore, high quality virtual stimulated Raman histology imaging of a brain adenocarcinoma is shown for pixel dwell times of 16 µs.

Spatial frequency modulated imaging in coherent anti-Stokes Raman microscopy.

For sparse samples or in the presence of ambient light, the signal-to-noise ratio (SNR) performance of single-point-scanning coherent anti-Stokes Raman scattering (CARS) images is not optimized. As an improvement, we propose replacing the conventional CARS focus-point illumination with a periodically structured focus line while continuing to collect the transmitted CARS intensity on a single detector. The object information along the illuminated line is obtained by numerically processing the CARS signal recorded for various periods of the structured focus line. We demonstrate experimentally the feasibility of this spatial frequency modulated imaging (SPIFI) in CARS (SPIFI-CARS) and SHG (SPIFI-SHG) and identify situations where its SNR is better than that of the single-point-scanning approach.