Multi-scale high-throughput cell culture monitoring by lensfree imaging S. Vinjimore Kesavan1, C.P. Allier1, F. Navarro2, M. Menneteau2, B. Chalmond3,4, J-M Dinten1 1 CEA DRT Leti DTBS/STD/LISA, 2CEA DRT Leti DTBS/SBSC/LBAM, 3Université de CergyPontoise, 4ENS-Cachan Research is continuously developing new imaging methods to better understand the structure and function of biological systems at the microscopic scale. Despite our ability to peer through the cells using increasingly powerful optical instruments, fundamental biology questions remain unanswered at larger scales. Hence we are developing lensfree imaging as an alternative method bringing new perspectives, i.e. our system (Fig. 1a) aims at, • Multiscale observation capability across three orders of magnitude, e.g. from mm2 to µm2 • Large field of view >20 mm2 • Live capture inside the incubator over several days (acquisition every 5 minutes) • Simplicity of use, small form factor (495cm3) and low cost (< 200 Euros) In this presentation, we demonstrate the potentialities of lensfree imaging for cell culture monitoring and describe the innovative technologies that underpin its feasibility. The technique is based on inline holography as invented by Gabor. Albeit the existence of the method since 1970, the recent development of digital sensors, popularized by their use in cameras, helped realize the full potential of this method in the recent years. When illuminated with coherent light, cells diffract the incident wave and produce interferences that can be recorded by a sensor (Fig. 1b). LED, pinhole, and CMOS sensor are the three basic components of our lensfree imaging system. The absence of bulky optical components makes the system simple and hence it could be placed inside the incubator to monitor cells in real time. Several systems could also be placed simultaneously to observe several conditions in parallel (Fig 1c). The field of the view (FOV) of the system is 24 mm², in other words, the system is capable of monitoring several thousands of cells at the same time. Hence it provides the ability to perform high throughput analyses (>5000 cells), e.g. cell tracking and cell density measurement. An automated reconstruction algorithm reconstructs the interference (holographic) patterns recorded over the entire field of view of the system (Fig 1d). When we digitally zoom into the reconstructed image (few mm2 FOV), cell-cell interaction, and cell division could be visualized in detail (Fig 1e). We could further zoom to obtain the morphology of the cells with a resolution close to 2 µm (Fig 1f). This allows the visualization of single cell motility with filopodial extensions and focal adhesion points. 3D reconstruction results from the calculation of phase with a precision of 1µm. Figure 1g shows, in 3D, a moving cell which later divides (marked by red arrow). Overall, the system offers a resolution close to 2µm over a field of view of 24mm² which makes it capable of monitoring cell culture at different scales, from very large field of view (>20mm2) down to the single cell. High through-put analysis of fundamental properties of cell populations could be performed without the necessity of markers, e.g. cell adhesion, cell division, cell migration and cell morphology.
Figure 1: Photograph of the lensfree imaging system developed at CEA-Leti, (b) Schematic diagram explaining the principle of lensfree imaging system, (c)Photograph of three lensfree imaging systems inside the incubator, (d) Reconstructed image showing cells in a field of view of 24 mm², (e) Trajectories followed by cells (reconstructed) during several hours of imaging inside the incubator, (f) Reconstructed image of a moving cell (filopodial extensions) and a dividing cell, (g)3D reconstruction montage of a moving cell which later divides (Red arrow). Cell line: NIH 3T3 fibroblasts.
Supplemental data: The petridish containing the cells is placed on the system which is placed inside the incubator. The images recorded by the system are sent to the laptop using a USB chord, which is inserted in to the incubator from the back. An example of a raw image (without reconstruction) obtained using our system is shown below.
Figure: 3T3 cells inside the petridish as imaged by the system. Scale bar: 1000 µm. Number of cells: Approximately 5000. As explained in the abstract, the raw image by itself renders several information regarding the cells (cell adhesion, cell proliferation, cell motility, etc). Moreover, any region of interest from the image or the entire image can be further reconstructed to obtain minute details with resolution close to 2 µm.
