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