FFOTT is a non-invasive, non-destructive method of imaging that was found promising for a broad range of applications. We applied FFOTT to compare intracellular dynamic signals, a proxy for cellular metabolic activity. We investigated the metabolic changes associated with the transition from and towards polar night in the polar diatom Fragilariopsis cylindrus , grown under continuous illumination or kept in darkness for six weeks. Our results revealed a tenfold signal decrease in darkness and a rapid signal recovery upon re-illumination. Photosynthetic performance was assessed in parallel. Biovolume determinations allowed the computation of the metabolic rates of F. cylindrus grown under both light and dark conditions, which were compared to the optical signal variations.

We developed a label-free optical microscopy method to study movements of different frequencies and amplitudes within a cell. We use optical transmission tomography (OTT) that operates in transmission, and we record the changes of signal values of all the pixels of movies taken for a few seconds (dynamic signal). This signal is a metabolic signal in algae as it decreased in the presence of photosystem II inhibitors or when samples were illuminated at wavelengths where the photoreceptors are poorly operative. We used as model organism Chlamydomonas for which mutants are available. We used a mutant deleted of the chloroplastic gene encoding the large subunit of the Rubisco, ΔrbcL. This mutant is unable to fix atmospheric CO2 and is devoid of pyrenoid. We compared the dynamic signal between wild-type strain and ΔrbcL mutant of Chlamydomonas grown in dark condition and found it to be 5 to 10 times higher. This mutant overproduced starch, and we tempted to associate the metabolic signal to the cost in ATPeq consumption for building starch. The method is easy to implement and could be very valuable for studies of phytoplankton in situ or virus-infected cells.

Membrane vesicles (MVs) are produced by cells from all domains of life and could be involved in the horizontal gene transfer (HGT). They were found in some environments as seawater and rivers but technological improvements must be required to found out their roles in the ecosystems. In this work, we developed a method using flow cytometry (FC) after lipid staining dye FM4-64 and compared the results to those obtained with Interferometric Microscopy (IM). The abundance of nanoparticles in different lakes determined by the two methods were in agreement. Moreover, nanoparticles with a high refraction index were particularly abundant suggesting that MVs could be associated with several virus particles or to very dense compounds such as iron oxides. ### Competing Interest Statement The authors have declared no competing interest.

DNA methylation loss at transposable elements (TEs) can affect neighboring genes and be epigenetically inherited in plants, yet the determinants and importance of this additional system of inheritance are unknown. In this work, we demonstrate in Arabidopsis thaliana that transgenerational stability of experimentally induced hypomethylation at TE loci is constrained by small RNAs derived from related copies. Using data from more than 700 strains collected worldwide, we uncover similar and recurrent hypomethylation at hundreds of these TE loci, often near genes. Most natural epivariants that we tested can be inherited without DNA sequence changes and are therefore bona fide epialleles, although genetic factors modulate their recurrence or persistence. Epiallelic variants often cause gene expression changes and may be targets of selection, revealing their contribution to heritable phenotypic variation in nature.

Last year we introduced a new Full Field Optical Transmission Tomography (FFOTT) technique that we applied to CELL studies. This interferometric technique is based on the use of the Gouy phase shift that takes place close to a microscope objective focus. We will now show results obtained in biological tissues using both a static mode (morphology) and dynamic one (metabolic contrast). In particular we have been able with our 400 $ microscope to section in tissues. We will discuss the importance of the spatial coherence of the illumination and compare the results with those obtained with Full Field OCT.

Live-cell imaging generally requires pretreatment with fluorophores to either monitor cellular functions or the dynamics of intracellular processes and structures. We have recently introduced full-field optical coherence tomography for the label-free live-cell imaging of fungi with potential clinical applications for the diagnosis of invasive fungal mold infections. While both the spatial resolution and technical set up of this technology are more likely designed for the histopathological analysis of tissue biopsies, there is to our knowledge no previous work reporting the use of a light interference-based optical technique for direct mycological examination and monitoring of intracellular processes. We describe the first application of dynamic full-field optical transmission tomography (D-FF-OTT) to achieve both high-resolution and live-cell imaging of fungi. First, D-FF-OTT allowed for the precise examination and identification of several elementary structures within a selection of fungal species commonly known to be responsible for invasive fungal infections such as Candida albicans, Aspergillus fumigatus, or Rhizopus arrhizus. Furthermore, D-FF-OTT revealed the intracellular trafficking of organelles and vesicles related to metabolic processes of living fungi, thus opening new perspectives in fast fungal infection diagnostics.

Our past contribution was to introduce a Gouy phase Full Field Optical Transmission technique applied to detect and characterise nanoparticles such as virus, vesicles and nano plastics in terms of size and refractive index. More recently we have adapted this interferometric approach to achieve optical tomography in cells and tissues. We will show improvements in sensitivity and resolution obtained with 20 nm virus as well as with biological tissues using both a static mode (morphology) and dynamic one (metabolic contrast). We discuss the importance of the illumination spatial coherence and compare the results with those obtained with Full Field OCT.

Transgenerational epigenetic inheritance (TEI) mediated by transposable elements (TEs) is well established in plants. However, the molecular determinants and functional impact of this additional system of inheritance are poorly characterized. Through comprehensive analyses of severe DNA methylation loss over TEs in an experimental population and in >700 natural strains of Arabidopsis thaliana, we uncovered hundreds of TE epivariants in nature, most of which can be inherited independently of DNA sequence changes and thus are true epialleles. Furthermore, natural epivariants are enriched near genes and we uncovered a novel role for the Paf1 complex as a major facilitator of their prevalence. Conversely, related TE copies limit epiallelic variation, by producing the small RNAs that guide DNA methylation in trans. Crucially, natural epivariants mainly affect stress-responsive genes, making them unique targets for selection. Our findings therefore demonstrate that TEs are important mediators of transgenerational epigenetic variation in nature, with singular properties for plant adaptation. ### Competing Interest Statement The authors have declared no competing interest.

At Langevin Institute, ESPCI Paris, we have developed a label-free approach to detect and track viruses in aquatic solutions using an interferometric microscopy approach working in transmission. By improving the experimental setup design, we have increased the sensitivity to detect viruses as small as 20nm in diameter, AAV viruses, which are of paramount importance for gene therapy. Furthermore, based on the variation of the interferometric signal along the axial position, we introduce a new way to perform single-shot volumetric imaging which enables high-precision 3D tracking.

La tomographie par cohérence optique ou OCT a révolutionné le diagnostic oculaire. Nous avons proposé une variante de l’OCT, dite « plein champ » (FFOCT) dont les principales applications se sont situées en cancérologie ex et in vivo. Nous souhaitons ici introduire une nouvelle méthode de tomographie plein champ par transmission (FFOTT) et comparer les avantages et inconvénients des deux méthodes.

We have recently (BOE July 2022) proposed an interferometric approach called full-field transmission tomography (FFOTT) based on the use of the Gouy phase shift that manifests at the focus of microscope objectives. Forward scattering of cellular structures larger than 100 nm being much greater than backscattering (used in OCT) performances constraints of the imaging system are strongly relaxed and setups using cheap microscopes and a smartphones become possible. Note that good quality 100X, NA+1.25 objectives are available for less than $100. We show cells and tissues images through their morphological or metabolic contrasts modified by a chemical or biological environments.

Viruses have a profound influence on all forms of life, motivating the development of rapid and minimally invasive methods for virus detection. In this study, we present a novel methodology that enables quantitative measurement of the interaction between individual biotic nanoparticles and antibodies in solution. Our approach employs a label-free, full-field common-path interferometric technique to detect and track biotic nanoparticles and their interactions with antibodies. It is based on the interferometric detection of light scattered by viruses in aqueous samples for the detection of individual viruses.We employ single-particle tracking analysis to characterize the size and properties of the detected nanoparticles, and to monitor the changes in their diffusive mobility resulting from interactions. To validate the sensitivity of our detection approach, we distinguish between particles having identical diffusion coefficients but different scattering signals, using DNA-loaded and DNA-devoid capsids of the Escherichia coli T5 virus phage.In addition, we have been able to monitor, in real time, the interaction between the bacteriophage T5 and purified antibodies targeting its major capsid protein pb8, as well as between the phage SPP1 and nonpurified anti-SPP1 antibodies present in rabbit serum. Interestingly, these virus-antibody interactions are observed within minutes. Finally, by estimating the number of viral particles interacting with antibodies at different concentrations, we successfully quantify the dissociation constant Kd of the virus-antibody reaction using single-particle tracking analysis.

ABSTRACTThe dynamic movement of cell organelles is an important and poorly understood component of cellular organisation and metabolism. In this work we present a non-invasive non-destructive method (Dynamic Cell Imaging, DCI) based on light scattering and interferometry to monitor dynamic events within photosynthetic cells using the diatom Phaeodactylum tricornutum as a model system. For this monitoring we acquire few seconds movies of the signals that are related to the motion of dynamic structures within the cell (denoted scatterers), followed by a statistical analysis of each pixel time series. Illuminating P.tricornutum with LEDs of different wavelengths associated to short pulsed or continuous-wave modes of illumination revealed that dynamic movements depend on chloroplast activity, in agreement with the reduction in the number of pixels with dynamic behaviour after addition of photosystemII inhibitors. We studied P. tricornutum under two environmentally relevant stresses, iron and phosphate deficiency. The major dynamic sites were located within lipid droplets and chloroplast envelope membranes. By comparing standard deviation and cumulative sum analysis of the time series, we showed that within the droplets two types of scatterer movement could be observed: random motions (Brownian type) but also anomalous movements corresponding to a drift which may relate to molecular fluxes within a cell. The method appears valuable for studying the effects of various environments on a large variety of microalgae in the laboratory as well as in natural aquatic environments.HIGHLIGHTsLight scattering an alternative to fluorescence to rapidly evidence dynamic processes.Lipid droplets the major metabolic active sites under stressA non-destructive visualisation method for laboratory microalgae and aquatic samples..SIGNIFICANCE STATEMENTLight scattering could be an alternative to fluorescence techniques to study dynamic processes within photosynthetic cells. We used a method combining light scattering and interferometry to analyse movements of intracellular scatterers in the marine diatom Phaedactylum tricornutum under two environmentally relevant stresses, iron and phosphate deficiency. Lipid droplets were the major active sites under stress. The method which is rapid and non destructive can be broadly expanded to study other microalgae and their stress responses, in the laboratory and in aquatic environments.

This work compares two tomographic imaging technologies, time-domain fullfield optical coherence tomography (FFOCT) working in reflection and optical transmission tomography (OTT), using a new optical setup that combines both. We show that, due to forward-scattering properties, the axial sectioning and contrast in OTT can be optimized by tuning illumination. The influence of sample scattering and thickness are discussed. We illustrate the comparison of the two methods in static (morphology) and dynamic (metabolic contrast) regimes using cell cultures, tissues and entire organisms emphasizing the advantages of both approaches.

There is an increasing need for label free methods that could reveal intracellular structures and dynamics. In this context, we develop a new optical tomography method working in transmission - full-field optical transmission tomography (FF-OTT). The method can measure the forward scattering signals and reveals the time-dependent metabolic signals in living cells. FF-OTT is a common path interferometer taking advantage of the Gouy phase shift - a π phase shift that the light wave experiences around the focus. By modulating the position of the focus one can alter the phase of the scattered light. Demodulation of images with different phases rejects the background and enhances the light from the depth-of-field, thus producing an optical section. We test FF-OTT by imaging single-cell diatoms and ex vivo biological samples. In fresh samples, we show that the intracellular motions create visible intensity fluctuations in FF-OTT so that the method is able to reveal a metabolic dynamic contrast. FF-OTT was found to be an efficient label free technique that can be readily implemented thanks to a robust common-path speckle-free interferometer design using an incoherent light source.

Full Field Optical Transmission Tomography (FFOTT) relies on forward scattering using the Gouy’s phase shift modulation that is achieved close to the focus of a microscope objective. FFOTT principle is different than Full Field OCT (FFOCT) that relies on backscattering of light, This new type of endogenous cell imaging technique offers structural and metabolic contrasts and is particularly well suited for imaging cell culture on glass slide or Petri dishes avoiding fringes that mask cells in FFOCT as well as biological structures such as biofilms. The sectioning ability is close to confocal microscopy but no contrast agent is required.

Interferometric microscopy techniques have been recently developed to detect small particles at the nanoscale without the need for specific labeling. Here, we introduce a highly sensitive, label-free approach on a common path interferometric set-up working on a transmission that amplifies weak scattering signals coming from small particles. Using single-particle tracking analysis, we can track and differentiate viruses and other biotic particles in aquatic environment. Furthermore, we have developed a fast assay based on antibody recognition of targeted virus in solution. We associate changes in diffusion and in the interferometric signal of the detected particles with the immune reaction between antibodies and surface proteins of the virus.