Fibrillogenesis in collagen hydrogels accelerated by carboxylated microbeads.

Collagen type I is a material widely used for 3D cell culture and tissue engineering. Different architectures, such as gels, sponges, membranes, and nanofibers, can be fabricated with it. In collagen hydrogels, the formation of fibrils and fibers depends on various parameters, such as the source of collagen, pH, temperature, concentration, age, etc. In this work, we study the fibrillogenesis process in collagen type I hydrogels with different types of microbeads embedded, using optical techniques such as turbidity assay and confocal reflectance microscopy. We observe that microbeads embedded in the collagen matrix hydrogels modify the fibrillogenesis. Our results show that carboxylated fluorescent microbeads accelerate 3.6 times the gelation, while silica microbeads slow down the formation of collagen fibrils by a factor of 1.9, both compared to pure collagen hydrogels. Our observations suggest that carboxylate microbeads act as nucleation sites and the early collagen fibrils bind to the microbeads.

Infrared Laser Effects on Cell Projection Depend on Irradiation Intermittence and Cell Activity.

Highly focused near-infrared (NIR) lasers have been used to induce fibroblast and neuron protrusions in a technique called optical guidance. However, little is known about the biochemical and biophysical effects that the laser provokes in the cell and optimal protocols of stimulation have not yet been established. Using intermittent NIR laser radiation and multivariate time series representations of cell leading edge movement, we analyzed the direction and velocity of cell protrusions. We found that the orientation and advance of PC12 neuron phenotype cells and 3T3 fibroblasts protrusions remain after the laser is turned off, but the observed increase in velocity stops when radiation ceases. For an increase in the speed and distance of cell protrusions by NIR laser irradiation, the cell leading edge needs to be advancing prior to the stimulation, and NIR irradiation does not enable the cell to switch between retracting and advancing states. Using timelapse imaging of actin-GFP, we observed that NIR irradiation induces a faster recruitment of actin, promoting filament formation at the induced cell protrusions. These results provide fresh evidence to understand the phenomenon of the optical guidance of cell protrusions.

Focus variation due to near infrared laser in a confocal microscope.

Focus precision and stability is crucial in confocal microscopy not only for image sharpness but also to avoid radiometric fluctuations that can wrongly be interpreted as variations of the fluorescence intensity in the sample. Here we report a focus variation provoked by a continuous wave laser of 810-nm wavelength introduced along the optical path of an inverted confocal microscope with an oil immersion ×60 objective. When the laser is turned on or off, the focus position drifts toward lower or high values of the vertical coordinate z, respectively. The maximum drift observed was 2.25 ðœ‡m for a laser power of 40 mW at the sample and over a 600-s exposure time. The temporal evolution of the focus position is well fitted by exponential curves that mimic temperature variations due to a heat source. Our analysis strongly suggests that the focus drift is due to heating of the immersion oil.

Pathogen associated molecular pattern-decorated mesoporous silica-A colloidal model for studying bacterial-host cell interactions.

Tuberculosis is the top infectious disease worldwide and the development of a vaccine and diagnostic tools to control the disease is a priority that requires a better understanding of the factors involved in the pathogenesis of Mycobacterium tuberculosis, the infectious agent. It is known that bacterial cell surface components are released, interact with immune cell receptors, and may traffic toward host cell structures. Many of these compounds are lipids that have been associated with mycobacterial virulence. However, their hydrophobic nature has frequently hampered their biological study. In this work, silica particles were coated with functional lipids to obtain a colloidal bioinspired system based on nonhydrosoluble glycolipids. Mycobacterium tuberculosis phosphatidylinositol mannosides (PIMs), known to interact with receptors of innate immune cells, were purified from the M. tuberculosis H37Rv type strain, and used to prepare large unilamellar liposomes in combination with zwitterionic phosphatidyl choline. Then, bacillary-like Santa Barbara Amorphous-15 (SBA-15) silica particles were cationized and the vesicle fusion method was used to promote the attachment of anionic PIM-containing lipid bilayers. Thermogravimetric analysis, x-ray diffraction, N2 adsorption-desorption isotherm analysis, Fourier transform infrared spectroscopy, electron microscopy, and zeta potential analyses were used to characterize the materials obtained. The as-prepared PIM-containing colloids, named PIM@SBA-15, showed biocompatibility toward human fibroblasts and were found to colocalize with Toll-like receptor (TLR)2 upon their incubation with THP1-derived macrophages. Furthermore, the particles induced the formation of pseudopods and were internalized into phagocytic cells. In all, these data suggest the usefulness of PIM@SBA-15 particles to better comprehend the interactions between immune cells and PIMs.

Effects of near infrared focused laser on the fluorescence of labelled cell membrane.

Near infrared (NIR) laser light can have important reactions on live cells. For example, in a macroscopic scale, it is used therapeutically to reduce inflammation and in a single-cell scale, NIR lasers have been experimentally used to guide neuronal growth. However, little is known about how NIR lasers produce such behaviours on cells. In this paper we report effects of focussing a continuous wave 810-nm wavelength laser on in vivo 3T3 cells plasma membrane. Cell membranes were labelled with FM 4-64, a dye that fluoresces when associated to membrane lipids. Confocal microscopy was used to image cell membranes and perform fluorescence recovery after photobleaching (FRAP) experiments. We found that the NIR laser produces an increase of the fluorescence intensity at the location of laser spot. This intensity boost vanishes once the laser is turned off. The mean fluorescence increase, calculated over 75 independent measurements, equals 19%. The experiments reveal that the fluorescence rise is a growing function of the laser power. This dependence is well fitted with a square root function. The FRAP, when the NIR laser is acting on the cell, is twice as large as when the NIR laser is off, and the recovery time is 5 times longer. Based on the experimental evidence and a linear fluorescence model, it is shown that the NIR laser provokes a rise in the number of molecular associations dye-lipid. The results reported here may be a consequence of a combination of induced increments in membrane fluidity and exocytosis.

Optical concatenation of a large number of beads with a single-beam optical tweezer.

Optical tweezers consist of the spatial confinement of microscopic dielectric particles by the action of forces produced by the change in momentum of the photons of a highly focused laser beam that are deviated by the particle. In experiments that use a single laser beam, it is common to capture not only one but a few particles in the optical trap. However, to our knowledge, the formation of a long chain of beads optically confined with a single laser beam has never been reported. In this work, up to 73 silica spheres immersed in water are seen concatenated along the propagation direction of a 976-nm wavelength Gaussian laser of 300 mW of power. This long chain of beads is obtained when the laser is focused through an oil-immersion DIN microscope objective with 100× magnification and a numerical aperture of 1.25. When performing the same experiment using an infinity-corrected UplanFLN 100× objective with a numerical aperture of 1.3, the maximum number of concatenated beads is only 14. Our results suggest that the mechanisms responsible for the observed phenomena involve successive refocusing of the laser beam by each trapped sphere, optically induced dipole coupling (commonly referred to as optical binding), and aberrations generated by the DIN microscope objective.

Phase change in a diffracted wave: a Cornu spiral perspective.

A simple evaluation of the phase change in a diffracted wave, in terms of the Cornu spiral, is presented to complement the well-known intensity change, which is routinely obtained for this elegant graphical construction of the Fresnel integrals. This is, to the best of our knowledge, the first presentation of this evaluation. It is shown that the phase of a wave diffracted by a slit is equal to the slope of the line tangent to the Cornu spiral, shifted by π/4.

On the normalization of scintillation autocovariance for generalized SCIDAR.

The Generalized SCIDAR (Scintillation Detection and Ranging) technique consists in the computation of the mean autocorrelation of double-star scintillation images taken on a virtual plane located a few kilo-meters below the telescope pupil. This autocorrelation is normalized by the autocorrelation of the mean image. Johnston et al. in 2002 pointed out that this normalization leads to an inexact estimate of the optical-turbulence strength C(2)(N). Those authors restricted their analysis to turbulence at ground level. Here we generalize that study by calculating analytically the error induced by that normalization, for a turbulent layer at any altitude. An exact expression is given for any telescope-pupil shape and an approximate simple formula is provided for a full circular pupil. We show that the effect of the inexact normalization is to overestimate the C(2)(N) values. The error is larger for higher turbulent layers, smaller telescopes, longer distances of the analysis plane from the pupil, wider double-star separations, and larger differences of stellar magnitudes. Depending on the observational parameters and the turbulence altitude, the relative error can take values from zero up to a factor of 4, in which case the real C(2)(N) value is only 0.2 times the erroneous one. Our results can be applied to correct the C(2)(N) profiles that have been measured using the Generalized SCIDAR technique.