Research is continuously developing imaging methods to better understand the structure and function of biological systems. In this paper, we describe our work to develop lens-free microscopy as a novel mean to observe and quantify cells in 2D and 3D cell culture conditions.
At first, we developed a lens-free video microscope based on multiple wavelength acquisitions to perform time-lapse 2D imaging of dense cell culture inside the incubator. We demonstrated that novel phase retrieval techniques enable imaging thin cell samples with high concentration (~15000 cells over a large field of view of 29.4 mm2). The experimental data can next be further analyzed with existing cell profiling and tracking algorithms. As an example, we showed that a 7 days acquisition of a culture of HeLa cells leads to a dataset featuring 2.106 cell point measurements and 104 cell cycle tracks.
Recently, we extended our work to the video-microscopy of 3D organoids culture. We showed the capability of lens-free microscopy to perform 3D+time acquisitions of 3D organoids culture. To our knowledge, our technique is the only one able to reconstruct very large volumes of 3D cell culture (~5 mm3) by phase contrast imaging. This new mean of microscopy allowed us to observe a broad range of phenomena present in 3D environments, e.g. self-organizations, displacement of large clusters, merging and interconnection over long distances (>1 mm). In addition, this 3D microscope can capture the interactions of single cells and organoids with their 3D environment, e.g. traction forces generated by large cell aggregates over long distances, up to 1.5 mm.
Overall, lens-free microscopy techniques favor ease of use and label-free experimentations as well as time-lapse acquisitions of large datasets. Importantly, we consider that these lens-free microscopy technique can thus expand the repertoire of phenomena that can be studied within 2D and 3D organoids cultures.
The cytology of the cerebrospinal fluid is traditionally performed by an operator (physician, biologist) by means of a conventional light microscope. The operator visually counts the leukocytes (white blood cells) present in a sample of cerebrospinal fluid (10 μl). It is a tedious job and the result is operator-dependent. Here in order to circumvent the limitations of manual counting, we approach the question of numeration of erythrocytes and leukocytes for the cytological diagnosis of meningitis by means of lens-free microscopy. In a first step, a prospective counts of leukocytes was performed by five different operators using conventional optical microscopy. The visual counting yielded an overall 16.7% misclassification of 72 cerebrospinal fluid specimens in meningitis/non-meningitis categories using a 10 leukocyte/μL cut-off. In a second step, the lens-free microscopy algorithm was adapted step-by-step for counting cerebrospinal fluid cells and discriminating leukocytes from erythrocytes. The optimization of the automatic lens-free counting was based on the prospective analysis of 215 cerebrospinal fluid specimens. The optimized algorithm yielded a 100% sensitivity and a 86% specificity compared to confirmed diagnostics. In a third step, a blind lens-free microscopic analysis of 116 cerebrospinal fluid specimens, including six cases of microbiology confirmed infectious meningitis, yielded a 100% sensitivity and a 79% specificity. Adapted lens-free microscopy is thus emerging as an operator-independent technique for the rapid numeration of leukocytes and erythrocytes in cerebrospinal fluid. In particular, this technique is well suited to the rapid diagnosis of meningitis at point-of-care laboratories.
Pathologist examination of tissue slides provides insightful information about a patient’s disease. Traditional analysis of tissue slides is performed under a binocular microscope, which requires staining of the sample and delays the examination. We present a simple cost-effective lensfree imaging method to record 2–4μm resolution wide-field (10 mm2 to 6 cm2) images of unstained tissue slides. The sample processing time is reduced as there is no need for staining. A wide field of view (10 mm2) lensfree hologram is recorded in a single shot and the image is reconstructed in 2s providing a very fast acquisition chain. The acquisition is multispectral, i.e. multiple holograms are recorded simultaneously at three different wavelengths, and a dedicated holographic reconstruction algorithm is used to retrieve both amplitude and phase. Whole tissue slides imaging is obtained by recording 130 holograms with X-Y translation stages and by computing the mosaic of a 25 x 25 mm2 reconstructed image. The reconstructed phase provides a phase-contrast-like image of the unstained specimen, revealing structures of healthy and diseased tissue. Slides from various organs can be reconstructed, e.g. lung, colon, ganglion, etc. To our knowledge, our method is the first technique that enables fast wide-field lensfree imaging of such unlabeled dense samples. This technique is much cheaper and compact than a conventional phase contrast microscope and could be made portable. In sum, we present a new methodology that could quickly provide useful information when a rapid diagnosis is needed, such as tumor margin identification on frozen section biopsies during surgery.
In order to extend the analysis of the datasets produced by lensfree video microscopy we have implemented a cell tracking algorithm to combine and correlate cell motility to the previously devised metrics to quantify e.g. cell adhesion and spreading, cell division, and cell death. In this paper we present the assessment of these new methodology on experiments involving three different cell lines, namely 3T3 fibroblast cells, primary HUVEC cells and macrophage THP1 cells. We demonstrate that the good spatial resolution and the fast frame rate obtained with of our lensfree video microscope allows standard cell tracking algorithm to be computed. The results is the possibility to analyze thousands of cells successfully tracked over tens of hours. The results is the possibility to compare different cell cultures in terms of e.g. cell motility and cell confinement ration. Ultimately we managed to measure the doubling time at single cell level over a large number of N=235 cells tracked over two days.
We developed a new imaging tool that can help pathologists in recording wide-field images of tissue slides. We present a simple cost-effective lens-free imaging method to record 2-4μm resolution wide-field (10 mm2 - 6 cm2) images of stained and unstained tissue slides. To our knowledge, our method is the first technique that enables fast (less than 5 minutes) wide-field lens-free imaging of such dense samples. Multiple holograms are recorded with different wavelength illumination, and a multispectral algorithm is used to retrieve both amplitude and phase. Our method can be used to retrieve images of stained tissue slides. For such absorbing object, the useful information is included in the modulus of the reconstructed complex field. Our method can also be applied to retrieve images of unstained tissue slides, where the useful information is in the retrieved phase. This technique is much cheaper and compact than a conventional microscope and could be made portable. Moreover, it enables wide field unstained tissue slides imaging, which could quickly provide useful information, for example on frozen section biopsies, when a rapid diagnosis is needed during surgery.