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 mm<sup>2</sup>) 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 mm<sup>2</sup> 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 cm<sup>2</sup> 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 mm<sup>2</sup>) and in final point (10 cm<sup>2</sup>) to assess the combination of these two complementary modalities. In the future, we aim at combining directly super wide field of view acquisitions (>10 cm<sup>2</sup>) with real time ability inside the incubator.
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 mm<sup>2</sup>) 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.
We demonstrated that the use of thin wetting film focusing allows detection of single micrometer-size objects with 24 mm<sup>2</sup> lensfree imaging. In order to refine the technique and push the detection limit down to the nanometer scale, a deep insight in the imaging mechanisms is necessary. We constructed a model based on wetting film microfluidics and Fresnel diffraction of light. This model properly fits the intensity measurements acquired on micro-particles with our lensfree imaging setup. When the particle diameter is 1 µm, a microlens is formed by a liquid surface deformation of about 100 nm in height over few microns radial distance. The measured point spread function of the light deflected by such microlens presents a constant beam intensity over a long range, between 50 µm and 250 µm from the object plane. This is very similar to what is obtained by illuminating an axicon with a Gaussian beam, i.e. the central beam propagates for several Rayleigh ranges without appreciable divergences. In the lensfree imaging setup, the detector plane is far apart from the object (≈500 µm). Thus, it is a true advantage to form axicon lens that can propagate strong intensity beams up to the detector plane. Most important, our model predicts that the detection of smaller objetcs needs thinner films. These results are important for further detecting viruses with lensfree imaging techniques.
This article [J. Biomed. Opt.. 17, , 106014 (2012)] was originally published online on 10 October 2012 with an error on page 5, column 2, line 5. The unit of measure "88 mW/cm2" should have read "8 mW/cm2."
This article was corrected online on 11 October 2012. The article appears correctly in print.
Over the last few years, near-infrared (NIR) fluorescence imaging has witnessed rapid growth and is already used in clinical trials for various procedures. However, most clinically compatible imaging systems are optimized for large, open-surgery procedures. Such systems cannot be employed during head and neck oncologic surgeries because the system is not able to image inside deep cavities or allow the surgeon access to certain tumors due to the large footprint of the system. We describe a miniaturized, low-cost, NIR fluorescence system optimized for clinical use during oral oncologic surgeries. The system, termed FluoSTIC, employs a miniature, high-quality, consumer-grade lipstick camera for collecting fluorescence light and a novel custom circular optical fiber array for illumination that combines both white light and NIR excitation. FluoSTIC maintains fluorescence imaging quality similar to that of current large-size imaging systems and is 22 mm in diameter and 200 mm in height and weighs less than 200 g.
In the context of continuous wave fluorescence-enhanced diffuse optical tomography, we show that the reconstructed
fluorescence depends on the local diffusion coefficient and demonstrate that the a priori knowledge of specific optical
parameters may lead to the reconstruction of absolute quantification of the fluorophore distribution. In this context, we
point out the potentiality of a bimodal instrument coupling functional and morphological information to provide
knowledge of the distribution of optical parameters of internal organs. We show some quantitative results on simulated
and experimental data on phantoms and conclude suggesting the use of optical parameters atlases to achieve an absolute
quantification of fluorophore distribution in real contexts.
Due to low light scattering, bacteria are difficult to detect using lensless imaging systems. In
order to detect individual bacteria, we report a method based on a thin wetting film imaging
that produces a micro-lens effect on top of each bacterium when the sample dries up. The
imaging using a high-end CMOS sensor is combined with an in-line holographic
reconstruction to improve positive detection rate up to 95% with micron-sized beads at high
density of ~10<sup>3</sup> objects/mm<sup>2</sup>. The system allows detecting from single bacterium to densely
packed objects (10<sup>3</sup> bacteria/μl) within 10μl sample. As an example, E.coli, Bacillus subtilis
and <i>Bacillus thuringiensis</i>, has been successfully detected with strong signal to noise ratio across a 24mm<sup>2</sup> field of view.
Lensless imaging has recently attracted a lot of attention as a compact, easy-to-use method to image or detect biological
objects like cells, but failed at detecting micron size objects like bacteria that often do not scatter enough light. In order
to detect single bacterium, we have developed a method based on a thin wetting film that produces a micro-lens effect.
Compared with previously reported results, a large improvement in signal to noise ratio is obtained due to the presence
of a micro-lens on top of each bacterium. In these conditions, standard CMOS sensors are able to detect single
bacterium, e.g. <i>E.coli, Bacillus subtilis and Bacillus thuringiensis</i>, with a large signal to noise ratio. This paper presents our sensor optimization to enhance the SNR; improve the detection of sub-micron objects; and increase the imaging
FOV, from 4.3 mm<sup>2</sup> to 12 mm<sup>2</sup> to 24 mm<sup>2</sup>, which allows the detection of bacteria contained in 0.5μl to 4μl to 10μl, respectively.
An instrument dedicated to the co-registration of optical and X-ray measurements is presented: specific acquisition
protocol and reconstruction software have been developed for carrying out fluorescence diffuse optical tomography in a
cylindrical geometry consistent with XCT. Actual animal geometry provided by the X-ray tomography is used to give
animal boundaries to the diffuse optical tomography reconstruction algorithm. To evaluate performances of this new
optical imaging system, experiments have been conducted on phantoms, mice with fluorescent capillaries, and finally on
mice bearing tumors. The fluorescence reconstructions are shown to be geometrically consistent with X-ray ones. We
determined that the sensibility limit of the system to detect fluorescence signal over intrinsic ones is 2 pmol for lungs
area and 5 pmol for the abdomen area.
We present first results of a fluorescence optical diffusion tomography experiment coupled to a X-ray computed
tomography reconstruction. An instrument, dedicated to the co-registration of optical and X-ray measurements, has been
developed: specific acquisition protocol and reconstruction software have been developed for carrying out fluorescence
diffuse optical tomography in a cylindrical geometry consistent with X-ray tomography. Actual animal geometry
provided by the X-ray tomography is used to give animal boundaries to the diffuse optical tomography reconstruction
algorithm. Experiments have been conducted on sacrificed mice and fluorescence reconstructions have been evaluated
and are geometrically consistent with X-ray ones.
The lifetime of optical components submitted to high laser fluences is degraded under organic contaminant environment.
The molecular background of the Ligne d'Integration Laser (LIL), prototype of the future Laser Megajoule, might reduce
the laser damage threshold of exposed fused silica surfaces. This paper reports the interaction effects between pure
model contaminant deposits and a pulsed 1064 nm laser radiation on the coming out of mirror damage. The experimental
setup allowed us to condense nanolayers of model contaminants on optics, the deposit impacts were then investigated by
Laser Induced Damage Threshold (LIDT) tests in Rasterscan mode. In order to highlight physical processes emphasizing
the emergence of optics damage, we characterized the irradiated deposit using interferometric microscopy analysis and
spectrophotometric analysis. The challenge was to determine physical and phenomenological processes occurring during
the irradiation of a pure contaminant deposit with a 1064 nm pulsed laser and to study the impact of this model
contaminant on the LIDT of dielectric SiO<sub>2</sub>/HfO<sub>2</sub> mirrors.
Surface incandescence properties of proton implanted fused silica have been researched with a focused CO<sub>2</sub>
laser. We have discovered that in the initial stage of incandescence a thermoluminescent peak appears. We call it
blackbody thermoluminescence. In our silica samples, with a 100 micron spatial resolution, the blackbody
thermoluminescence mapping reveals surface and sub surfaces defects made by the polishing process. We show how
laser damage and laser conditioning are the same two facets of this blackbody thermoluminescence occurrence.
Laser damage at 3ω, 351 nm, of fused silica optical components is a major concern for LMJ maintenance.
Indeed, even a low density of damage sites is unacceptable due to the exponential growth of surface damage with a series
of laser shots. A technique is now used to prevent the growth of initiated damage sites : this mitigation technique consists
in a local melting and evaporation of silica by CO<sub>2</sub> laser irradiation on the damage site. Even if the growth is stopped in
most cases, we showed previously that some of the mitigated sites re-initiate on their peripheral area, where most of redeposited
debris are located. To further increase the efficiency of mitigation technique, the treatment was improved by
varying the spatial profile of the CO<sub>2</sub> laser beam. We present here the new set-up and the results obtained in terms of
laser damage resistance: about 98% of the mitigated sites sustained 200 shots of a 10 J/cm<sup>2</sup> 3ω YAG laser without