We designed a particularly simple, compact and robust microscope for phase and fluorescent imaging. The phase-contrast image is reconstructed from a single, approximately 100 µm defocused image with an algorithm based on a constrained optimization of Fresnel diffraction model. Fluorescence image is recorded in-focus. No mechanical movement of neither sample nor objective or any other part of the system is needed to change between the phase-contrast and fluorescence modality. The change of focus between phase (out-of-focus) and fluorescence (in-focus) imaging is achieved with chromatic aberration specifically enhanced by the optical design of our system. Our microscope is sufficiently compact (10x10x10 cm^3) to fit into a standard biological incubator. The simple and robust design reduces the vibration and the drift of the sample. The absence of motorized components makes the system robust and resistant to the humid conditions inside the biological incubator. These aspects greatly facilitate the long-time observation of cell cultures.
We can observe a thousand of cells in parallel in a single field of view (1mm^2) with resolution down to 2 µm. We show FUCCI marked HeLa cell culture observed over three days directly in the incubator. FUCCI (fluorescence ubiquitination cell-cycle indicator), is a genetically encoded, two-colour (red and green), indicator of the progression through the cell cycle: the cells in G1 phase show red fluorescence nuclei while the cells in S, G2 and M phase display green fluorescence within the nuclei.
We use phase images for segmentation and tracking of the individual cells which allows us to determine the level of fluorescence in each cell in the green and red fluorescence channel. We compare the obtained statistics with the data from flow cytometer acquired at the end of the observation. We show that we can produce a statistically relevant time-resolved measurement of a cell population while keeping access to the individual cells.
We designed a simple, compact and robust microscope for phase and fluorescent imaging. No mechanical movement of neither sample nor objective or any other part of the system is needed to change between the phase-contrast and fluorescence modality. We can observe a thousand cells in parallel in a single field of view with resolution down to 2 µm. We demonstrate the system on a FUCCI marked HeLa cell culture observed over several days directly in the standard incubator. We compare the obtained statistics to the flow cytometer data and show that we can produce a statistically relevant time-resolved measurement
Lung tissue motion arising from breathing and heart beating has been described as the largest annoyance of in vivo
imaging. Consequently, infected lung tissue has never been imaged in vivo thus far, and little is known concerning the
kinetics of the mucosal immune system at the cellular level. We have developed an optimized post-processing strategy to
overcome tissue motion, based upon two-photon and second harmonic generation (SHG) microscopy.
In contrast to previously published data, we have freed the lung parenchyma from any strain and depression in order to
maintain the lungs under optimal physiological parameters. Excitation beams swept the sample throughout normal
breathing and heart movements, allowing the collection of many images. Given that tissue motion is unpredictably, it
was essential to sort images of interest. This step was enhanced by using SHG signal from collagen as a reference for
sampling and realignment phases. A normalized cross-correlation criterion was used between a manually chosen
reference image and rigid transformations of all others. Using CX3CR1+/gfp mice this process allowed the collection of
high resolution images of pulmonary dendritic cells (DCs) interacting with Bacillus anthracis spores, a Gram-positive
bacteria responsible for anthrax disease. We imaged lung tissue for up to one hour, without interrupting normal lung
physiology. Interestingly, our data revealed unexpected interactions between DCs and macrophages, two specialized
phagocytes. These contacts may participate in a better coordinate immune response. Our results not only demonstrate the
phagocytizing task of lung DCs but also infer a cooperative role of alveolar macrophages and DCs.
Lung efficiency as gas exchanger organ is based on the delicate balance of its associated mucosal immune system
between inflammation and sterility. In this study, we developed a dynamic imaging protocol using confocal and twophoton
excitation fluorescence (2PEF) on freshly harvested infected lungs. This modus operandi allowed the collection
of important information about CX3CR1+ pulmonary cells. This major immune cell subset turned out to be distributed in
an anisotropic way in the lungs: subpleural, parenchymal and bronchial CX3CR1+ cells have then been described. The
way parenchymal CX3CR1+ cells react against LPS activation has been considered using Matlab software,
demonstrating a dramatic increase of average cell speed. Then, interactions between Bacillus anthracis spores and
CX3CR1+ dendritic cells have been investigated, providing not only evidences of CX3CR1+ cells involvement in
pathogen uptake but also details about the capture mechanisms.
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