We present a method of high resolution, non-invasive, in vivo vascular imaging obtained using watersoluble and bright SWIR-emitting gold nanoclusters presenting an anisotropic surface charge combined with SWIR detection and Monte Carlo processing of the images. We applied this approach to quantify vessel complexity in mice presenting vascular disorders.
Quantitative phase imaging (QPI) allows the monitoring of adherent cell cultures continuously over long time periods and it delivers an image of the cell with pixel intensities corresponding to the optical path difference (OPD). These images can be processed to quantify several cellular features. In particular, cell OPD measurements allows the estimation of the cell dry mass, an important metric to study cell mass and growth kinetics.
If the ability of QPI to provide phase-contrast images of cells is taken for granted, the accuracy and the precision of QPI cell OPD measurements can still be questioned. Indeed, the reported QPI cell measurements have not yet been assessed with any reference method (e.g. microfluidic resonators). And there is a lack of independent experimental comparison and validation which can hinder the acceptance of QPI in the realms of live-cell mass profiling.
With the aim of filling this gap, here we compare three different methods: digital holographic microscopy, quadriwave lateral sheering interferometry and lens-free microscopy (not yet established as a QPI technique). The experimental design is based on the inter-modality comparisons of OPD measurements performed over several tens of cells. To ensure consistency, we performed OPD measurements on a fixed cell culture the same day on the same location. Importantly, the statistical analysis of these measurements allowed us to estimate the precision of QPI cell OPD measurements without any reference material. In addition, we have evaluated the influence of the post-processing steps (baseline subtraction, cell segmentation) on the precision of QPI cell measurements.
We present our implementation of lens-free video microscopy setup for the monitoring of adherent cell cultures. We use a multi-wavelength LED illumination together with a dedicated holographic reconstruction algorithm that allows for an efficient removal of twin images from the reconstructed phase image for densities up to those of confluent cell cultures (>500 cells/mm2). We thereby demonstrate that lens-free video microscopy, with a large field of view (~30 mm2) can enable us to capture the images of thousands of cells simultaneously and directly inside the incubator. It is then possible to trace and quantify single cells along several cell cycles. We thus prove that lens-free microscopy is a quantitative phase imaging technique enabling estimation of several metrics at the single cell level as a function of time, for example the area, dry mass, maximum thickness, major axis length and aspect ratio of each cell. Combined with cell tracking, it is then possible to extract important parameters such as the initial cell dry mass (just after cell division), the final cell dry mass (just before cell division), the average cell growth rate, and the cell cycle duration. As an example, we discuss the monitoring of a HeLa cell cultures which provided us with a data-set featuring more than 10 000 cell cycle tracks and more than 2x106 cell morphological measurements in a single time-lapse.
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