Achieving magnification smaller than 1 in lensless microscopy by illumination with a convergent wavefront.

Lensless or lensfree microscopy is now available commercially. With these new microscopes, it is possible to record images in contact mode with a magnification of 1 or in holographic mode with a magnification larger than 1. In this Letter, we present an original setup that allows us to record the image of an object whose surface is larger than that of the image sensor without placing an optical component between the object and the image sensor.

A novel heterotrifunctional peptide-based cross-linking reagent for facile access to bioconjugates. Applications to peptide fluorescent labelling and immobilisation.

A convenient, versatile and straightforward synthesis of a novel heterotrifunctional peptide-based linker molecule is described. This generic bio-labelling reagent contains an amine-reactive N-hydroxysuccinimidyl carbamate moiety, an aldehyde/ketone-reactive aminooxy group and a thiol group with a propensity to form urea, oxime and thioether linkages respectively. The full chemical orthogonality between the free aminooxy and thiol functionalities was demonstrated through the preparation of a fluorescent reagent suitable for the selective staining of a carboxaldehyde-modified surface by means of oxime ligation. The absence of reactivity of these two functions toward the nucleophile-sensitive active carbamate was obtained by using temporary aminooxy- and thiol-protecting groups removable under mild conditions. The utility of the linker molecule to cross-link three different molecular partners has been illustrated by the preparation of fluorescent tripod-functionalised surfaces which may be useful in developing new peptide microarrays and related immunosensors.

Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model.

During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.

Time-lapse contact microscopy of cell cultures based on non-coherent illumination.

Video microscopy offers outstanding capabilities to investigate the dynamics of biological and pathological mechanisms in optimal culture conditions. Contact imaging is one of the simplest imaging architectures to digitally record images of cells due to the absence of any objective between the sample and the image sensor. However, in the framework of in-line holography, other optical components, e.g., an optical filter or a pinhole, are placed underneath the light source in order to illuminate the cells with a coherent or quasi-coherent incident light. In this study, we demonstrate that contact imaging with an incident light of both limited temporal and spatial coherences can be achieved with sufficiently high quality for most applications in cell biology, including monitoring of cell sedimentation, rolling, adhesion, spreading, proliferation, motility, death and detachment. Patterns of cells were recorded at various distances between 0 and 1000 µm from the pixel array of the image sensors. Cells in suspension, just deposited or at mitosis focalise light into photonic nanojets which can be visualised by contact imaging. Light refraction by cells significantly varies during the adhesion process, the cell cycle and among the cell population in connection with every modification in the tridimensional morphology of a cell.

A new generation of scanners for DNA chips.

Today, most of the DNA chips are used with fluorescent markers. Associated with fluorescence confocal scanners, this technology achieves remarkable performances in terms of sensitivity and accuracy. The main technical issues related to these scanners have already been reviewed. However, these scanners are costly, especially when high density chips are used. In this case, a mechanical precision of 1 microm or less is required to achieve the measurement precision required. This cost level prevents the spread of this technology in the diagnostic market. We will present a new concept for scanners with equivalent or superior performances, with a cost cut of 5-10. This concept is inspired from the field of optical disk and reader. Basically, an optical format is added to the chip, before DNA deposition. This format contains tracks which are superimposed to the DNA features. These tracks define the path that an optical head of a CD player must follow in order to scan the surface of the DNA chip. Such a head is a very cheap component, and has a precision of less than 100 nm thanks to real-time focus and tracking. These functions are fulfilled by electromagnetic actuators mounted on the support of the frontal lens. We show here that it is possible to use such a head to build a fluorescence confocal scanner with equivalent or even better performances than conventional scanners.

Photobiomodulation inside the brain: a novel method of applying near-infrared light intracranially and its impact on dopaminergic cell survival in MPTP-treated mice.

OBJECT: Previous experimental studies have documented the neuroprotection of damaged or diseased cells after applying, from outside the brain, near-infrared light (NIr) to the brain by using external light-emitting diodes (LEDs) or laser devices. In the present study, the authors describe an effective and reliable surgical method of applying to the brain, from inside the brain, NIr to the brain. They developed a novel internal surgical device that delivers the NIr to brain regions very close to target damaged or diseased cells. They suggest that this device will be useful in applying NIr within the large human brain, particularly if the target cells have a very deep location. METHODS: An optical fiber linked to an LED or laser device was surgically implanted into the lateral ventricle of BALB/c mice or Sprague-Dawley rats. The authors explored the feasibility of the internal device, measured the NIr signal through living tissue, looked for evidence of toxicity at doses higher than those required for neuroprotection, and confirmed the neuroprotective effect of NIr on dopaminergic cells in the substantia nigra pars compacta (SNc) in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson disease in mice. RESULTS: The device was stable in freely moving animals, and the NIr filled the cranial cavity. Measurements showed that the NIr intensity declined as distance from the source increased across the brain (65% per mm) but was detectable up to 10 mm away. At neuroprotective (0.16 mW) and much higher (67 mW) intensities, the NIr caused no observable behavioral deficits, nor was there evidence of tissue necrosis at the fiber tip, where radiation was most intense. Finally, the intracranially delivered NIr protected SNc cells against MPTP insult; there were consistently more dopaminergic cells in MPTP-treated mice irradiated with NIr than in those that were not irradiated. CONCLUSIONS: In summary, the authors showed that NIr can be applied intracranially, does not have toxic side effects, and is neuroprotective.

Ultra trace detection of explosives in air: development of a portable fluorescent detector.

This paper describes a system for the detection of nitroaromatic explosives consisting of a portable detector based on a specific fluorescent material. The developed sensor was able to perform an ultra trace detection of explosives, such as trinitrotoluene (TNT) or its derivate 2,4-dinitrotoluene (DNT), in ambient air or on objects tainted with explosives. In the presence of nitroaromatic vapors, the fluorescence of the material was found to decrease due to the adsorption of nitroaromatic molecules on its specific adsorption sites. The sensor exhibited a large sensitivity to TNT or DNT at their vapor pressures (respectively 6 and 148 ppbv) and the detection threshold was evaluated on a laboratory test setup and was found to be 0.75 ppbv for TNT. Moreover, the detector demonstrated no loss of performance in the presence of humidity or interfering compounds. All the tests led to the conclusion that the sensor fulfills the main requirements for the identification of suspect luggage, forensic analyses or battlefields clearing.

Solid-phase immobilized tripod for fluorescent renewable immunoassay. A concept for continuous monitoring of an immunoassay including a regeneration of the solid phase.

A new concept of immunoassay based on the use of a trifunctional reagent (tripod) and fluorescence resonance energy transfer (FRET) phenomenon is described. This procedure involves differential steps: (1) the tripod bearing (i) a fluorophore, (ii) a molecule structurally close to the target, and (iii) a linker reacts with the solid phase; (2) the solid phase is further activated with an anti-target antibody labeled with a quencher molecule, generating the decrease of the fluorophore emission via FRET; (3) FRET being distance dependent, the presence of the target by competing with the tripod for binding the quencher-labeled antibody leads to a rise of the fluorescence signal; (4) the solid phase is reactivated simply, by adding the quencher-labeled antibody. This method was evaluated in microtiter plates using the susbtance P as model while fluorescein and TAMRA were used as donor and acceptor, respectively. Results clearly illustrated the interest of the method, by allowing (i) a simple regeneration procedure, without requiring any drastic treatment, (ii) a direct fluorescence measurement onto the solid support, leading to a localized and cumulative signal, (iii) an increase of the signal when detecting the target, unlike classical competitive immunoassays, and (iv) a real-time monitoring of the competition and regeneration steps.