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.

Fast compressive Raman micro-spectroscopy to image and classify microplastics from natural marine environment.

The fast and reliable detection of micron-sized plastic particles from the natural marine environment is an important topic that is mostly addressed using spontaneous Raman spectroscopy. Due to the long (>tens of ms) integration time required to record a viable Raman signal, measurements are limited to a single point per microplastic particle or require very long acquisition times (up to tens of hours). In this work, we develop, validate, and demonstrate a compressive Raman technology using binary spectral filters and single-pixel detection that can image and classify six types of marine microplastic particles over an area of 1 mm2 with a pixel dwell time down to 1.75 ms/pixel and a spatial resolution of 1 µm. This is x10-100 faster than reported in previous studies.

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.

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.

Coupling optimized bending-insensitive multi-core fibers for lensless endoscopy.

We report a bending-insensitive multi-core fiber (MCF) for lensless endoscopy imaging with modified fiber geometry that enables optimal light coupling in and out of the individual cores. In a previously reported bending insensitive MCF (twisted MCF), the cores are twisted along the length of the MCF allowing for the development of flexible thin imaging endoscopes with potential applications in dynamic and freely moving experiments. However, for such twisted MCFs the cores are seen to have an optimum coupling angle which is proportional to their radial distance from the center of the MCF. This brings coupling complexity and potentially degrades the endoscope imaging capabilities. In this study, we demonstrate that by introducing a small section (1 cm) at two ends of the MCF, where all the cores are straight and parallel to the optical axis one can rectify the above coupling and output light issues of the twisted MCF, enabling the development of bend-insensitive lensless endoscopes.

Fast interrogation wavelength tuning for all-optical photoacoustic imaging.

Optical detection of ultrasound for photoacoustic imaging provides a large bandwidth and high sensitivity at high acoustic frequencies. Therefore, higher spatial resolutions can be achieved using Fabry-Pérot cavity sensors than conventional piezoelectric detection. However, fabrication constraints during the deposition of the sensing polymer layer require precise control of the interrogation beam wavelength to provide optimal sensitivity. This is commonly achieved by employing slowly tunable narrowband lasers as interrogation sources, hence limiting the acquisition speed. We propose instead to use a broadband source and a fast-tunable acousto-optic filter to adjust the interrogation wavelength at each pixel within a few microseconds. We demonstrate the validity of this approach by performing photoacoustic imaging with a highly inhomogeneous Fabry-Pérot sensor.

Sub-diffraction computational imaging via a flexible multicore-multimode fiber.

An ultra-thin multimode fiber is an ideal platform for minimally invasive microscopy with the advantages of a high density of modes, high spatial resolution, and a compact size. In practical applications, the probe needs to be long and flexible, which unfortunately destroys the imaging capabilities of a multimode fiber. In this work, we propose and experimentally demonstrate sub-diffraction imaging through a flexible probe based on a unique multicore-multimode fiber. A multicore part consists of 120 Fermat's spiral distributed single-mode cores. Each of the cores offers stable light delivery to the multimode part, which provides optimal structured light illumination for sub-diffraction imaging. As a result, perturbation-resilient fast sub-diffraction fiber imaging by computational compressive sensing is demonstrated.

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.

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.

Fast Compressive Raman Imaging of Polymorph Molecules and Excipients in Pharmaceutical Tablets.

We implement a near-infrared (NIR) version of compressive Raman imaging that incorporates a digital micromirror device (DMD) and a single-pixel detector for fast chemometric analysis and microscopic imaging. The NIR compressive Raman system is successfully used to detect and image active pharmaceutical ingredients exhibiting polymorphism within compact pharmaceutical tablets. We report the chemical imaging of a mixture of two clopidogrel polymorphs and three excipients in solid tablets with a pixel dwell time of 2.5 ms (0.5 ms per species). These results open the road to fast pharmaceutical tablet quality control imaging using compressive Raman technology.

3D nanoparticle superlocalization with a thin diffuser.

We report on the use of a thin diffuser placed in the close vicinity of a camera sensor as a simple and effective way to superlocalize plasmonic nanoparticles in 3D. This method is based on holographic reconstruction via quantitative phase and intensity measurements of a light field after its interaction with nanoparticles. We experimentally demonstrate that this thin diffuser can be used as a simple add-on to a standard bright-field microscope to allow the localization of 100 nm gold nanoparticles at video rate with nanometer precision (1.3 nm laterally and 6.3 nm longitudinally). We exemplify the approach by revealing the dynamic Brownian trajectory of a gold nanoparticle trapped in various pockets within an agarose gel. The proposed method provides a simple but highly performant way to track nanoparticles in 3D.

Nearly degenerate two-color impulsive coherent Raman hyperspectral imaging.

Impulsive stimulated Raman scattering (ISRS) is a robust technique for studying low frequency (<300 cm-1) Raman vibrational modes, but ISRS has faced difficulty in translation to an imaging modality. A primary challenge is the separation of the pump and probe pulses. Here we introduce and demonstrate a simple strategy for ISRS spectroscopy and hyperspectral imaging that uses complementary steep edge spectral filters to separate the probe beam detection from the pump and enables simple ISRS microscopy with a single-color ultrafast laser source. ISRS spectra are obtained that span from the fingerprint region down to <50 cm-1 vibrational modes. Hyperspectral imaging and polarization-dependent Raman spectra are also demonstrated.

Miniature 120-beam coherent combiner with 3D-printed optics for multicore fiber-based endoscopy.

In this Letter, we report a high-efficiency, miniaturized, ultra-fast coherent beam, combined with 3D-printed micro-optics directly on the tip of a multicore fiber bundle. The highly compact device footprint (180 µm in diameter) facilitates its incorporation into a minimally invasive ultra-thin nonlinear endoscope to perform two-photon imaging.

An adaptive microscope for the imaging of biological surfaces.

Scanning fluorescence microscopes are now able to image large biological samples at high spatial and temporal resolution. This comes at the expense of an increased light dose which is detrimental to fluorophore stability and cell physiology. To highly reduce the light dose, we designed an adaptive scanning fluorescence microscope with a scanning scheme optimized for the unsupervised imaging of cell sheets, which underly the shape of many embryos and organs. The surface of the tissue is first delineated from the acquisition of a very small subset (~0.1%) of sample space, using a robust estimation strategy. Two alternative scanning strategies are then proposed to image the tissue with an improved photon budget, without loss in resolution. The first strategy consists in scanning only a thin shell around the estimated surface of interest, allowing high reduction of light dose when the tissue is curved. The second strategy applies when structures of interest lie at the cell periphery (e.g. adherens junctions). An iterative approach is then used to propagate scanning along cell contours. We demonstrate the benefit of our approach imaging live epithelia from Drosophila melanogaster. On the examples shown, both approaches yield more than a 20-fold reduction in light dose -and up to more than 80-fold- compared to a full scan of the volume. These smart-scanning strategies can be easily implemented on most scanning fluorescent imaging modality. The dramatic reduction in light exposure of the sample should allow prolonged imaging of the live processes under investigation.

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.

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.

Line-scan compressive Raman imaging with spatiospectral encoding.

We report a line-scanning imaging modality of compressive Raman technology with a single-pixel detector. The spatial information along the illumination line is encoded onto one axis of a digital micromirror device, while spectral coding masks are applied along the orthogonal direction. We demonstrate imaging and classification of three different chemical species.

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.