Non-invasive detection of regulatory T cells with Raman spectroscopy.

Regulatory T cells (Tregs) are a type of lymphocyte that is key to maintaining immunological self-tolerance, with great potential for therapeutic applications. A long-standing challenge in the study of Tregs is that the only way they can be unambiguously identified is by using invasive intracellular markers. Practically, the purification of live Tregs is often compromised by other cell types since only surrogate surface markers can be used. We present here a non-invasive method based on Raman spectroscopy that can detect live unaltered Tregs by coupling optical detection with machine learning implemented with regularized logistic regression. We demonstrate the validity of this approach first on murine cells expressing a surface Foxp3 reporter, and then on peripheral blood human T cells. By including methods to account for sample purity, we could generate reliable models that can identify Tregs with an accuracy higher than 80%, which is already comparable with typical sorting purities achievable with standard methods that use proxy surface markers. We could also demonstrate that it is possible to reliably detect Tregs in fully independent donors that are not part of the model training, a key milestone for practical applications.

Compressed sensing laser scanning microscopy.

We present a measurement and reconstruction method for laser-scanning microscopy based on compressed sensing, which enables significantly higher frame rates and reduced photobleaching. The image reconstruction accuracy is ensured by including a model of the physical imaging process into the compressed sensing reconstruction procedure. We demonstrate its applicability to unmodified commercial confocal fluorescence microscopy systems and for Raman imaging, showing a potential data reduction of 10-15 times, which directly leads to improvements in acquisition speed, or reduction of photobleaching, without significant loss of spatial resolution. Furthermore, the reconstruction model is also robust to noise, and effective for low-light applications. This method has promising applications for all imaging modalities based on laser-scanning acquisition, including fluorescence, Raman, and nonlinear microscopy.

[Keloid scars of the external ear: a non solved problem]. / Cicatrices queloideas en pabellón auricular: un problema no resuelto.

The external ear is a location with high risk of keloid scar formation. Its incidence is growing since general use of piercings and performance of plastic surgery of the external ear. The external ear keloid can be a devasting process for adolescent population which is worried about their appearance. Our aim is to attract attention about the risk of keloid scars of the external ear, reviewing our experience. After dismissing radiotherapy, corticoid infiltration and surgical removal are the most used options, with a high recurrence risk. We have reviewed traumatic, surgical and piercing wounds of the external ear, with a subsequent keloid formation treated in our outpatient clinic, collecting data about wound etiology, treatment and results. During the last 10 years we have found 11 keloid scars, 2 of them improved with topical corticosteroid. Treatment has been surgical in 9 cases, 4 of them with skin graft: 5 recovered and 4 recurred; 2 of them were reoperated. 2 of them were treated with intralesional corticosteroid solely, one recovered and the other one had improved. Treatment management of keloid scars is complex and there isn't a procedure with superior results than the others. Risk of complication must be explained within adolescent population.

Femtosecond laser nano-ablation in fixed and non-fixed cultured cells.

To understand the onset and morphology of femtosecond laser submicron ablation in cells and to study physical evidence of intracellular laser irradiation, we used transmission electron microscopy (TEM). The use of partial fixation before laser irradiation provides for clear images of sub-micron intracellular laser ablation, and we observed clear evidence of bubble-type physical changes induced by femtosecond laser irradiation at pulse energies as low as 0.48 nJ in the nucleus and cytoplasm. By taking ultrathin sliced sections, we reconstructed the laser affected subcellular region, and found it to be comparable to the point spread function of the laser irradiation. Laser-induced bubbles were observed to be confined by the surrounding intracellular structure, and bubbles were only observed with the use of partial pre-fixation. Without partial pre-fixation, laser irradiation of the nucleus was found to produce observable aggregation of nanoscale electron dense material, while irradiation of cytosolic regions produced swollen mitochondria but residual local physical effects were not observed. This was attributed to the rapid collapse of bubbles and/or the diffusion of any observable physical effects from the irradiation site following the laser exposure.

A femtosecond laser pacemaker for heart muscle cells.

The intracellular effects of focused near-infrared femtosecond laser irradiation are shown to cause contraction in cultured neonatal rat cardiomyocytes. By periodic exposure to femtosecond laser pulse-trains, periodic contraction cycles in cardiomyocytes could be triggered, depleted, and synchronized with the laser periodicity. This was observed in isolated cells, and in small groups of cardiomyocytes with the laser acting as pacemaker for the entire group. A window for this effect was found to occur between 15 and 30 mW average power for an 80 fs, 82 MHz pulse train of 780 nm, using 8 ms exposures applied periodically at 1 to 2 Hz. At power levels below this power window, laser-induced cardiomyocyte contraction was not observed, while above this power window, cells typically responded by a high calcium elevation and contracted without subsequent relaxation. This laser-cell interaction allows the laser irradiation to act as a pacemaker, and can be used to trigger contraction in dormant cells as well as synchronize or destabilize contraction in spontaneously contracting cardiomyocytes. By increasing laser power above the window available for laser-cell synchronization, we also demonstrate the use of cardiomyocytes as optically-triggered actuators. To our knowledge, this is the first demonstration of remote optical control of cardiomyocytes without requiring exogenous photosensitive compounds.

Slow Ca(2+) wave stimulation using low repetition rate femtosecond pulsed irradiation.

We demonstrated stimulation of Ca(2+) in living cells by near-infrared laser pulses operated at sub-MHz repetition rates. HeLa cells were exposed to focused 780 nm femtosecond pulses, generated by a titanium-sapphire laser and adjusted by an electro-optical modulator. We found that the laser-induced Ca(2+) waves could be generated over three orders of magnitude in repetition rates, with required laser pulse energy varying by less than one order of magnitude. Ca(2+) wave speed and gradients were reduced with repetition rate, which allows the technique to be used to modulate the strength and speed of laser-induced effects. By lowering the repetition rate, we found that the laser-induced Ca(2+) release is partially mediated by reactive oxygen species (ROS). Inhibition of ROS was successful only at low repetition rates, with the implication that ROS scavengers may in general be depleted in experiments using high repetition rate laser irradiation.

Linear phase imaging using differential interference contrast microscopy.

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier-space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.

Using the Hilbert transform for 3D visualization of differential interference contrast microscope images.

Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.