Temporal focusing (TF) is a key tool in wide-field two-photon excitation fluorescence microscopy for confining fluorescence excitation to a thin layer around the focal plane. TF is typically implemented with 100 femtosecond laser pulses and large microscope magnifications (>40). In this work, we demonstrate that performing TF with a random speckled illumination rather than a collimated beam significantly improves the optical sectioning for long pulses and small magnifications. We derive simple formulas of the optical sectioning as a function of speckle angular divergence, pulse bandwidth, and microscope parameters. Our approach paves the way for optically sectioned wide-field nonlinear imaging using >200 fs pulses and low magnifications enabling large fields of view.

Two-photon microscopy is a widely used technique for three-dimensional deep-tissue imaging, relying on pixel-by-pixel scanning of a tightly focused laser beam and fluorescence detection with a bucket detector such as a photomultiplier tube. Wide-field two-photon microscopy implementations using cameras have the potential to outperform conventional point-scanning approaches in terms of field of view, imaging speed, and reduced phototoxicity.However, wide-field twophoton methods have faced major limitations, including poor axial sectioning, restricted field of view, and the risk of damaging objective lenses due to the high laser powers required for wide-field nonlinear excitation. Here, we introduce a wide-field two-photon microscope that overcomes these limitations by combining temporal focusing with speckle illumination and galvo-based beam scanning. In our approach, temporally focused speckle illumination is confined to a 50 & micro;m elementary field of view that is rapidly scanned across the sample while fluorescence is recorded on a camera. The microscope achieves a 5 & micro;m axial resolution and operates at 10 frames per second over a 300 & times; 300 & micro;m2 field of view. We demonstrate multicolor two-photon imaging on a variety of samples, including fluorescent beads, cultured cells, Drosophila larval epidermis, and organoids. We show that speckle illumination not only protects the objective lens from damage but also provides superior axial sectioning in scattering media. Furthermore, it enables super-resolution operation in the random illumination microscopy (RIM) mode, in which temporally focused speckle images are acquired at successive translation steps of a static speckle pattern across a region of interest. By leveraging temporal focusing for efficient rejection of out-of-focus fluorescence, we demonstrate that temporally focused RIM in thick scattering samples yields higher contrast images than conventional two-photon RIM and achieves spatial resolution beyond the diffraction limit.

Biological and biomedical samples are routinely examined using focused two-photon (2P) fluorescence microscopy due to its intrinsic axial sectioning and reduced out-of-focus bleaching. However, 2P imaging often requires excitation intensities that can damage samples through ionization and radical formation. Additionally, the lateral resolution of 2P microscopy is lower compared to linear one-photon (1P) fluorescence microscopy. Widefield 2P microscopy, using cameras, holds promise for reducing photo-toxicity while maintaining high image acquisition rates. Widefield imaging trades the high power and short integration times of sequential single point scanning for the low power and extended integration times of parallel detection across millions of pixels. However, generating effective axial sectioning over arbitrarily large fields of view (FOVs) has remained a challenge. In this work, we introduce 2P Random Illumination Microscopy (2P-RIM), an easy-to-implement 2P widefield technique, that achieves low photo-damage, fast imaging, micrometric axial sectioning, and enhanced lateral resolution for arbitrarily large FOVs. By using widefield speckled illuminations in conjunction with an image standard deviation matching algorithm, 2P-RIM demonstrated multicolor imaging over FOVs greater than 200 um, lateral resolution 220 nm, axial sectioning 2 um, and peak excitation powers about 10 times lower than those used in focused laser scanning microscopy.

Biological and biomedical samples are routinely examined using focused two-photon (2P) fluorescence microscopy due to its intrinsic axial sectioning and reduced out-of-focus bleaching. However, 2P imaging often requires excitation intensities that can damage samples through ionization and radical formation. In addition, the lateral resolution of 2P microscopy is lower compared to linear one-photon (1P) fluorescence microscopy. Wide-field 2P microscopy, using cameras, holds promise for reducing phototoxicity while maintaining high image acquisition rates. Wide-field imaging trades the high power and short integration times of sequential single point scanning for the low power and extended integration times of parallel detection across millions of pixels. However, generating effective axial sectioning over arbitrarily large fields of view (FOVs) has remained a challenge. In this work, we introduce 2P random illumination microscopy (2P-RIM), an easy-to-implement 2P wide-field technique that achieves low photo-damage imaging, micrometric axial sectioning, and enhanced lateral resolution for arbitrarily large FOVs. By using wide-field speckled illuminations in conjunction with an image standard deviation matching algorithm, 2P-RIM demonstrated multicolor imaging over FOVs greater than 200 μm, lateral resolution of 220 nm, axial sectioning of 2 μm, and peak excitation powers about 10 times lower than those used in focused laser scanning microscopy.

Stimulated Raman scattering (SRS) microscopy is a nonlinear imaging technique that visualizes chemical composition by detecting molecular-vibrational bonds. We present a shot-noise limited, tunable SRS Microscopy scheme optimized for fast virtual histology. (c) 2025 The Author(s)

We propose a temporal focusing scheme with galvo scanners to perform two-photon fluorescence and CARS microscopy of biological samples over an unprecedently large field of view (>300x300 mu m(2)) at >10 frames/s with 3.8 mu m optical sectioning ability. (c) 2025 The Author(s)

We present a widefield two-photon fluorescence technique for biological imaging. Using a random illumination microscopy scheme, we achieve 2 mu m axial resolution and a 1.7x improvement in lateral resolution over a field of view of 250 mu m. (c) 2025 The Author(s)

Stimulated Raman Scattering (SRS) microscopy was developed for the label-free detection of molecular groups, addressing the speed limitations of spontaneous Raman microscopy. Standard SRS microscopy typically operates with laser sources at an 80 MHz repetition rate and a color-tuning speed of approximately 0.1 Hz to target different molecular groups. Here, we present a novel laser system that overcomes these speed limitations, achieving an order-of-magnitude improvement in both color-tuning and imaging speed. Our system features a reduced repetition rate of 40 MHz, enabling SRS imaging that is ten times faster than standard systems while maintaining the same average power at the sample. This is achieved through increased pulse energy and laser modulation at half the repetition rate. Furthermore, the system provides nearly ten times faster color-tuning across an extended range (660-1010 nm) by employing angle-tuning of nonlinear crystals instead of temperature-tuning. The improved performance is demonstrated in direct comparison with a standard SRS laser system, showcasing the potential for significantly enhanced imaging capabilities. ### Competing Interest Statement AE: Abberior Instruments GmbH (I), GS, PT, SP & IR: APE Angewandte Physik & Elektronik GmbH (E).

Wide-field two-photon microscopy using a collimated excitation is plagued by poor optical sectioning. In this work, we show analytically that exciting the sample with randomly varying speckled illuminations could significantly improve the optical sectioning ability while keeping the same large Field of View.

Coherent non-linear wide-field imaging reduces phototoxicity compared to focused approaches and holds the potential for the highest possible image acquisition rates, but misses optical sectioning. Here, we reintroduce optical sectioning using fast changing speckle illuminations.

In the last decades, nonlinear optical microscopy techniques, such as two-Photon Excited Fluorescence (2PEF), Sum Frequency Generation (SFG), and Hyperspectral Coherent Anti-Stokes Raman Scattering (H-CARS) microscopy, emerged as powerful tools to image biological samples in a label-free, fast, and non-destructive way. Traditionally, these techniques are performed with single point-scanning acquisition schemes featuring a limited field of view and often leading to sample damage when high laser power is used to increase the acquisition speed. Wide-field illumination and camera-based detection schemes offer a solution to these issues, enabling fast imaging over a large field view. However, current laser sources used for nonlinear microscopy operating at several MHz repetition rates are not able to induce nonlinear effects over areas larger than 100 × 100 µm2. Here, we present a powerful 200 kHz repetition rate laser source based on an Ytterbium fiber laser pumping two picosecond optical parametric amplifiers tunable in the 700–900 nm spectral range and rapidly tunable (up to 100 KHz) within a 20 nm sub-range. We exemplify the possibilities of this laser system to perform rapid CARS spectroscopy (2 ms/spectrum) and nonlinear wide-field imaging, up to 3.3 frames/s for 2PEF and SFG and 0.3 hypercubes/s for H-CARS, over a field of view >300 × 300 µm2.

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.

We describe the basic principles of super-resolved Random Illumination Microscopy (RIM) and present different applications in fluorescence and non-linear imaging.

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.