Lens-Free microscopy aims at recovering an observed object such as cell cultures from its diffraction measurements. Diffraction acquisitions are processed with an inverse problem approach to recover optical path difference (OPD) images of the object. Phase unwrapping issue is solved here by using a convolutional neural network (CNN) trained on simulations. The procedure was applied successfully on a neuron cells culture video acquisition.
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.
KEYWORDS: Detection and tracking algorithms, Video microscopy, Video, Image segmentation, Microscopes, Signal to noise ratio, Holography, Cell death, Time metrology, Reconstruction algorithms
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.
In this paper, we discuss a new methodology based on lens-free imaging to perform wound healing assay with unprecedented statistics. Our video lens-free microscopy setup is a simple optical system featuring only a CMOS sensor and a semi coherent illumination system. Yet it is a powerful means for the real-time monitoring of cultivated cells. It presents several key advantages, e.g., integration into standard incubator, compatibility with standard cell culture protocol, simplicity and ease of use. It can perform the follow-up in a large field of view (25 mm2) of several crucial parameters during the culture of cells i.e. their motility, their proliferation rate or their death. Consequently the setup can gather large statistics both in space and time. But in the case of tissue growth experiments, the field of view of 25 mm2 remains not sufficient and results can be biased depending on the position of the device with respect to the recipient of the cell culture. Hence, to conduct exhaustive wound healing assay, here we propose to enlarge the field of view up to 10 cm2 through two different approaches. The first method consists in performing a scan of the cell culture by moving the source/sensor couple and then stitch the stack of images. The second is to make an acquisition by scanning with a line scan camera. The two approaches are compared in term of resolution, complexity and acquisition time. Next we have performed acquisitions of wound healing assay (keratinocytes HaCaT) both in real-time (25 mm2) and in final point (10 cm2) to assess the combination of these two complementary modalities. In the future, we aim at combining directly super wide field of view acquisitions (>10 cm2) with real time ability inside the incubator.
Quantification of cell proliferation and monitoring its kinetics are essential in fields of research such as developmental biology, oncology, etc. Although several proliferation assays exist, monitoring cell proliferation kinetics remains challenging. We present a novel cell proliferation assay based on real-time monitoring of cell culture inside a standard incubator using a lensfree video-microscope, combined with automated detection of single cell divisions over a population of several thousand cells. Since the method is based on direct visualization of dividing cells, it is label-free, continuous, and not sample destructive. Kinetics of cell proliferation can be monitored from a few hours to several days. We compare our method to a standard assay, the EdU proliferation assay, and as proof of principle, we demonstrate concentration-dependent and time-dependent effect of actinomycin D—a cell proliferation inhibitor.
Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell microscopy techniques, we are developing lensfree video microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree video microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm2) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree microscopy with 3D cell culture in the study of epithelial tissue morphogenesis. In summary, we demonstrate that lensfree video microscopy is a powerful tool to conduct cell assays in 2D and 3D culture experiments. The applications are in the realms of fundamental biology, tissue regeneration, drug development and toxicology studies.
A new contrast agent, LipImage™ 815, has been designed and compared to previously described indocyanine green (ICG)-loaded lipid nanoparticles (ICG-lipidots®). Both contrast agents display similar size (50-nm diameter), zeta potential, high IC50 in cellular studies, near-infrared absorption and emission wavelengths in the “imaging window,” long-term shelf colloidal and optical stabilities with high brightness (>10 6 L mol −1 cm −1 ) in ready-to-use storage conditions in aqueous buffer (4°C in dark), therefore being promising fluorescence contrast agents for in vivo imaging. However, while ICG-lipidots® display a relatively short plasma lifetime, LipImage™ 815 circulates in blood for longer times, allowing the efficient uptake of fluorescence signal in human prostate cancer cells implanted in mice. Prolonged tumor labeling is observed for more than 21 days.
Owing to its simplicity, lensless imaging system is adept at continuous monitoring of adherent cells inside the incubator.
The setup consists of a CMOS sensor with pixel pitch of 2.2 μm and field of view of 24 mm2, LED with a dominating
wavelength of 525 nm, along with a pinhole of 150 μm as the source of illumination. The in-line hologram obtained
from cells depends on the degree of cell-substrate adhesion. Drastic difference is observed between the holographic
patterns of floating and adherent cells. In addition, the well-established fact of reduction of cell-substrate contact during
cell division is observed with our system based on corresponding spontaneous transition in the holographic pattern. Here,
we demonstrate that by recognizing this specific holographic pattern, number of cells undergoing mitosis in a cell culture
with a population of approximately 5000 cells, can be estimated in real-time. The method is assessed on comparison with
Edu-based proliferation assay. The approach is straightforward and it eliminates the use of markers to estimate the
proliferation rate of a given cell culture. Unlike most proliferation assays, the cells are not harvested enabling continuous
monitoring of cell culture.
The use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker®705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC50 > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications.
Fabrice Navarro, Denise Bechet, Thomas Delmas, Pierre Couleaud, Céline Frochot, Marc Verhille, Ezatul Kamarulzaman, Régis Vanderesse, Patrick Boisseau, Isabelle Texier, Julien Gravier, Françoise Vinet, Muriel Barberi-Heyob, Anne Claude Couffin
PhotoDynamic Therapy (PDT) has been established as a potent and less invasive treatment for different kinds of
cancer. Among various attempts to enhance the therapeutics efficacy of PDT, the specific delivery of the PhotoSensitizer
(PS) in the tumor is expected to increase its clinical applications, since unwanted accumulation, especially in the skin,
impairs the patients' quality of life (prolonged cutaneous photosensitivity). The aim of this study was to engineer Lipid
Nanoparticles (LNP) with different sizes and various PS contents, using simple, solvent-free and easily scale up
manufacturing processes. Meso-tetra (hydroxyphenyl) chlorin (mTHPC) is one of the most potent photoactive
compounds for clinical use and it has been successfully applied in the treatment of various indications, such as the head
and neck, prostate and pancreatic cancers. Here, a derivative of mTHPC was efficiently incorporated into the lipid core
of LNP, leading to a large range of stable and reproducible mTHPC-loaded LNP with narrow size distribution. The
photophysical and photochemical properties of mTHPC-loaded LNP were studied by measuring absorbance and
fluorescence spectra, colloidal stability, particle size and zeta potential, as well as singlet oxygen luminescence. The
photocytotoxicity of three selected mTHPC-loaded LNP (25 nm, 45 nm and 95 nm of diameter, respectively) was
evaluated on MCF-7 cells, in comparison to free mTHPC, under irradiation at 652 nm with a range of light fluence from
1 to 5 J/cm2. All the physico-chemical, photophysical and biological measurements allow us to conclude that LNP is a
promising nano-drug delivery system for PDT.
Fluorescence imaging in diffusive media locates tumors tagged by injected fluorescent markers in NIR wave-lengths.
For deep embedded markers, natural autofluorescence of tissues comes to be a limiting factor to
tumor detection and accurate FDOT reconstructions. A spectroscopic approach coupled with Non-negative
Matrix Factorization source separation method is explored to discriminate fluorescence sources according to
their fluorescence spectra and remove unwanted autofluorescence. We successfully removed autofluorescence
from acquisitions on living mice with a single subcutaneous tumor or two capillary tubes inserted at different
depths.
Fluorescence imaging in diffusive media is an emerging imaging modality for medical applications that uses injected fluorescent markers that bind to specific targets, e.g., carcinoma. The region of interest is illuminated with near-IR light and the emitted back fluorescence is analyzed to localize the fluorescence sources. To investigate a thick medium, as the fluorescence signal decreases with the light travel distance, any disturbing signal, such as biological tissues intrinsic fluorescence (called autofluorescence) is a limiting factor. Several specific markers may also be simultaneously injected to bind to different molecules, and one may want to isolate each specific fluorescent signal from the others. To remove the unwanted fluorescence contributions or separate different specific markers, a spectroscopic approach is explored. The nonnegative matrix factorization (NMF) is the blind positive source separation method we chose. We run an original regularized NMF algorithm we developed on experimental data, and successfully obtain separated in vivo fluorescence spectra.
Fluorescence imaging is a valuable tool for the study of living systems. It can be used with good resolution from
the micro- to the macroscopic range. However, for macroscopic use in living animals or humans, fluorescent probes must
overcome several obstacles such as aqueous solubility, suitable circulating lifetime and clearance. Fluorescent probes
should also display high molar extinction coefficient and fluorescence quantum yield. In this article, we report the
encapsulation of five hydrophobic or amphiphilic fluorophores (DiO, DiI, DiD, DiR and ICG) with emission wavelength
ranging from 500 to 800 nm, in long-circulating Lipid NanoParticles (LNP). Loading of these commercially available
indocyanines in LNP is highly efficient (from 77 to 97 %), and fluorescence quantum yields range from 7 to 53%,
depending on the dye, in the standard formulation (50 nm diameter nanoparticles). Given the wide range of wavelengths
covered and the stability of particle dispersion in aqueous buffer, dye-loaded LNP should be a valuable tool for both in
vivo and in vitro fluorescence imaging.
Fluorescence imaging (FLI) allows the in vivo monitoring of biological events associated with disease and
represents a new promising tool for drug discovery. In particular, it speeds up the development and assessment of new
therapies in oncology, helps in diagnosis, and improves surgery by fluorescence-guided tumor resection. This technique
is highly sensitive, non-ionizing, easy to use and relatively inexpensive.
Nevertheless, the main limitation of FLI lies in the optical properties of biological tissues. Mainly because of
haemoglobin and water absorption, only near-infrared (NIR) light is adapted to image tissues in depth. Using a
contrasting agent absorbing and emitting in the NIR region is therefore necessary to improve the background signal ratio,
and thus the image contrast.
Among many commercially available NIR optical contrast agents, only indocyanine green (ICG), has been
approved by the United State Food and Drug Administration (FDA) for various medical applications. However, its
instability (photo-degradation, thermal-degradation and low aqueous solubility) limits its applications as a fluorescent
probe for imaging purposes. In order to improve the effectiveness of ICG, we engineered ICG-doped lipid nanoparticles
(LNP).
In this communication, we will report the design of these novel fluorescent nanoparticle probes. These low cost
nanocarriers have numerous advantages, including their high chemical stability and biocompatibility. The
characterization of the optical properties of the nanoparticles entrapping ICG will also be discussed. Finally, the
biodistribution in mice of ICG when delivered through nanoparticles in comparison to free ICG in solution is presented.
It demonstrates the efficient accumulation of ICG-doped nanoparticles in the tumor site.
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