Tual Monfort

Differential phase contrast (DPC) microscopy is widely used for label-free structural imaging of transparent specimens, yet whether phase-gradient signals can provide endogenous functional contrast remains unknown. Here we report dynamic reflection differential phase contrast (D-RDPC), a label-free imaging modality that reveals intracellular activity through temporal fluctuations of directional phase-gradient contrast. Using living Müller glial cells cultured on highly reflective substrates, we show that asymmetric illumination generates a directional dynamic signal that exhibits three characteristic signatures of phase-gradient imaging: contrast reversal upon illumination inversion, systematic enhancement with increasing illumination asymmetry, and recovery of spatial localization through directional Hilbert-transform reconstruction. These observations support the interpretation that the measured contrast originates from temporal fluctuations of a reflection differential phase contrast signal rather than from conventional dynamic interferometric scattering alone. D-RDPC reveals intracellular activity that remains poorly visible with conventional dynamic full-field optical coherence tomography and enables functional imaging at reflective biointerfaces where strong specular reflections typically limit performance. More broadly, our results establish temporal fluctuation analysis as a route to extending DPC microscopy from a structural imaging modality toward functional imaging.

Time-domain dynamic full-field optical coherence tomography (D-FFOCT) is a powerful label-free imaging modality that enables functional visualization of cellular activity in living tissues with subcellular resolution. However, its sensitivity remains a major limitation for imaging highly scattering three-dimensional (3D) biological models such as retinal organoids, where incoherent background and inefficient optical flux distribution reduce dynamic contrast and limit imaging depth. In this work, we introduce a ratio-free optical configuration for time-domain D-FFOCT that enables continuous tuning of the sample-to-reference field ratio while minimizing photon losses and suppressing parasitic reflections. This polarization-based architecture allows optimal redistribution of optical flux according to sample scattering conditions and improves sensitivity under both power-limited and dose-limited conditions. Compared with conventional non-polarizing beam splitter configurations, the proposed approach provides a √ 2-fold (3 dB) sensitivity improvement through optical optimization alone. In addition, we investigate for the first time the use of partial field illumination (PFI) in time-domain D-FFOCT to reduce incoherent background arising from multiple scattering. In retinal organoids imaged at 120 µm depth, PFI yields up to a 14.5-fold (23.2 dB) increase in dynamic signal sensitivity, while preserving functional contrast. When combined, ratio-free detection and PFI provide a cumulative sensitivity improvement of 20.5-fold (26.2 dB). These gains enable improved visualization of photoreceptor precursor organization, rosette structures, and Müller glial cell dynamics in both 3D retinal organoids and 2D cell cultures. This work establishes a practical framework for sensitivity optimization in D-FFOCT and expands its potential for functional imaging, disease modeling, and live-cell monitoring in complex biological systems.

Time domain Dynamic full-field optical coherence tomography (D-FFOCT) is a powerful label-free imaging modality that enables functional visualization of cellular activity in living tissues with subcellular resolution. However, its sensitivity remains a major limitation for imaging highly scattering three-dimensional (3D) biological models such as retinal organoids, where incoherent background and inefficient optical flux distribution reduce dynamic contrast and limit imaging depth. In this work, we introduce a ratio-free optical configuration for time-domain D-FFOCT that enables continuous tuning of the sample-to-reference field ratio while minimizing photon losses and suppressing parasitic reflections. This polarization-based architecture allows optimal redistribution of optical flux according to sample scattering conditions and improves sensitivity under both power-limited and dose-limited conditions. Compared with conventional non-polarizing beam splitter configurations, the proposed approach provides a -fold (3 dB) sensitivity improvement through optical optimization alone. In addition, we investigate for the first time the use of partial field illumination (PFI) in time-domain D-FFOCT to reduce incoherent background arising from multiple scattering. In retinal organoids imaged at 120 µm depth, PFI yields up to a 14.5-fold (23.2 dB) increase in dynamic signal sensitivity, while preserving functional contrast. When combined, ratio-free detection and PFI provide a cumulative sensitivity improvement of 20.5-fold (26.2 dB). These gains enable improved visualization of photoreceptor precursor organization, rosette structures, and Müller glial cell dynamics in both 3D retinal organoids and 2D cell cultures. This work establishes a practical framework for sensitivity optimization in D-FFOCT and expands its potential for functional imaging, disease modelling, and live-cell monitoring in complex biological systems. Abstract Figure:

Dynamic optical coherence tomography (DOCT) enhances conventional OCT by providing specific information related to flow dynamics, cell motility, and organelle metabolic activity. These biological phenomena can be detected with varying sensitivity depending on the OCT architecture parameters, including wavelength, numerical aperture, and implementation method (time domain or Fourier domain). Despite its potential, the field lacks standardization as various research groups have independently developed algorithms for specific applications. In this paper, we compare four widely used DOCT algorithms, each employing a distinct analytical approach: power spectral density moment analysis, frequency band visualization, logarithmic intensity variation evaluation, and motility-based analysis. These algorithms were originally optimized for different OCT technologies (full-field OCT, microscopic OCT, swept-source OCT, and spectral domain OCT), which vary in temporal and spatial resolution as well as susceptibility to motion artifacts. To conduct a fair evaluation, we perform comprehensive cross-wise comparisons using datasets acquired from each of these setups. Our findings reveal that each method exhibits unique advantages in specific imaging environments, thereby providing valuable guidance for algorithm selection based on particular application requirements.

Dynamic full-field optical coherence tomography (DFFOCT) has recently emerged as an invaluable label-free microscopy technique, owing to its sensitivity to cell activity, as well as speed and sectioning ability. However, the quality of DFFOCT images is often degraded due to phase noise and fringe artifacts. In this work, we present a new implementation, to the best of our knowledge, named rolling-phase (RP) DFFOCT, in which the reference arm is slowly scanned over magnitudes exceeding 2π. We demonstrate mathematically and experimentally that it shows superior image quality while enabling to extract both static and dynamic contrast simultaneously. We showcase RP-DFFOCT on a macaque retinal explant and demonstrate its ability to better resolve subcellular structures, including intranuclear activity.

Dynamic full-field optical coherence tomography (DFFOCT) has recently emerged as an invaluable label-free microscopy technique, owing to its sensitivity to cell activity, as well as speed and sectioning ability. However, the quality of DFFOCT images are often degraded due to phase noise and fringe artifacts. In this work, we present a new implementation named Rolling-Phase (RP) DFFOCT, in which the reference arm is slowly scanned over magnitudes exceeding 2$π$. We demonstrate mathematically and experimentally that it shows superior image quality while enabling to extract both static and dynamic contrast simultaneously. We showcase RP DFFOCT on monkey retinal explant, and demonstrate its ability to better resolve subcellular structures, including intranuclear activity.

We present Rolling-Phase D-FFOCT, a novel detection scheme for Dynamic Full-Field Optical Coherence Tomography. This technique uses homogeneous phase measurements. It improves signal-to-noise ratio, reduces speckle, and uncovers new sub-cellular structures compared to conventional methods.

Dynamic full-field optical coherence tomography (D-FFOCT) has recently emerged as a label-free imaging tool, capable of resolving cell types and organelles within 3D live samples, whilst monitoring their activity at tens of milliseconds resolution. Here, a D-FFOCT module design is presented which can be coupled to a commercial microscope with a stage top incubator, allowing non-invasive label-free longitudinal imaging over periods of minutes to weeks on the same sample. Long term volumetric imaging on human induced pluripotent stem cell-derived retinal organoids is demonstrated, highlighting tissue and cell organization processes such as rosette formation and mitosis as well as cell shape and motility. Imaging on retinal explants highlights single 3D cone and rod structures. An optimal workflow for data acquisition, postprocessing and saving is demonstrated, resulting in a time gain factor of 10 compared to prior state of the art. Finally, a method to increase D-FFOCT signal-to-noise ratio is demonstrated, allowing rapid organoid screening.

Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm⁻¹, is demonstrated.