Common-path configuration in total internal reflection digital holography microscopy.

Total Internal Reflection Digital Holographic Microscopy (TIRDHM) is recognized to be a powerful tool for retrieving quantitative phase images of cell-substrate interfaces, adhesions, and tissue structures close to the prism surface. In this Letter, we develop an improved TIRDHM system, taking advantage of a refractive index mismatch between the prism and the sample substrate, to allow phase-shifting DH with just a single-beam interferometric configuration. Instead of the traditional off-axis method, phase-shift method is used to retrieve amplitude and phase images in coherent light and TIR modality. Essentially, the substrate-prism interface acts like a beam splitter generating a reference beam, where the phase-shift dependence on the incident angle is exploited in this common-path configuration. With the aim to demonstrate the technique's validity, some experiments are performed to establish the advantage of this compact and simple configuration, in which the reference arm in the setup is avoided.

Femtosecond digital lensless holographic microscopy to image biological samples.

The use of femtosecond laser radiation in digital lensless holographic microscopy (DLHM) to image biological samples is presented. A mode-locked Ti:Sa laser that emits ultrashort pulses of 12 fs intensity FWHM, with 800 nm mean wavelength, at 75 MHz repetition rate is used as a light source. For comparison purposes, the light from a light-emitting diode is also used. A section of the head of a drosophila melanogaster fly is studied with both light sources. The experimental results show very different effects of the pinhole size on the spatial resolution with DLHM. Unaware phenomena on the field of the DLHM are analyzed.

Generation of programmable 3D optical vortex structures through devil's vortex-lens arrays.

Different spatial distributions of optical vortices have been generated and characterized by implementing arrays of devil's vortex lenses in a reconfigurable spatial light modulator. A simple design procedure assigns the preferred position and topological charge value to each vortex in the structure, tuning the desired angular momentum. Distributions with charges and momenta of the opposite sign have been experimentally demonstrated. The angular velocity exhibited by the phase distribution around the focal plane has been visualized, showing an excellent agreement with the simulations. The practical limits of the method, with interest for applications involving particle transfer and manipulation, have been evaluated.

Single-exposure super-resolved interferometric microscopy by red-green-blue multiplexing.

We present single-exposure super-resolved interferometric microscopy (SESRIM) as a novel approach capable of providing one-dimensional (1-D) super-resolution (SR) imaging in holographic microscopy using a single illumination shot. The single-exposure SR working principle is achieved by combining angular and wavelength multiplexing incoming from a set of tilted beams with different wavelengths where each wavelength is tuned with the red-green-blue (RGB) channels of a color CCD. Thus, the information included in each color channel is retrieved by holographic recording using a single-color CCD capture and by analyzing the RGB channels. Finally, 1-D SR imaging is obtained after the digital postprocessing stage yielding the generation of a synthetic aperture. Experimental results are reported validating the proposed SESRIM approach while an extension of the proposed approach to the two-dimensional case is considered.

Resolution improvement by single-exposure superresolved interferometric microscopy with a monochrome sensor.

Single-exposure superresolved interferometric microscopy (SESRIM) by RGB multiplexing has recently been proposed as a way to achieve one-dimensional superresolved imaging in digital holographic microscopy by a single-color CCD snapshot [Opt. Lett. 36, 885 (2011)]. Here we provide the mathematical basis for the operating principle of SESRIM, while we also present a different experimental configuration where the color CCD camera is replaced by a monochrome (B&W) CCD camera. To maintain the single-exposure working principle, the object field of view (FOV) is restricted and the holographic recording is based on image-plane wavelength-dispersion spatial multiplexing to separately record the three bandpass images. Moreover, a two-dimensional extension is presented by considering two options: time multiplexing and selective angular multiplexing. And as an additional implementation, the FOV restriction is eliminated by varying the angle between the three reference beams in the interferometric recording. Experimental results are reported for all of the above-mentioned cases.

Comparison of Methods for Estimating Retinal Shape: Peripheral Refraction vs. Optical Coherence Tomography.

Retinal shape presents a clinical parameter of interest for myopia, and has commonly been inferred indirectly from peripheral refraction (PRX) profiles. Distortion-corrected optical coherence tomography (OCT) scans offer a new and direct possibility for retinal shape estimation. The current study compared retinal curvatures derived from OCT scans vs. PRX measurements in three refractive profiles (0° and 90° meridians, plus spherical equivalent) for 25 participants via Bland-Altman analysis. The radial differences between both procedures were correlated to axial length using Pearson correlation. In general, PRX- and OCT-based retinal radii showed low correlation (all intraclass correlation coefficients < 0.21). PRX found flatter retinal curvatures compared to OCT, with the highest absolute agreement found with the 90° meridian (mean difference +0.08 mm) and lowest in the 0° meridian (mean difference +0.89 mm). Moreover, a negative relation between axial length and the agreement of both methods was detected especially in the 90° meridian (R = -0.38, p = 0.06). PRX measurements tend to underestimate the retinal radius with increasing myopia when compared to OCT measurements. Therefore, future conclusions from PRX on retinal shape should be made cautiously. Rather, faster and more clinically feasible OCT imaging should be performed for this purpose.

In vitro cytotoxicity evaluation of cadmium by label-free holographic microscopy.

Among all environmental pollutants, the toxic heavy metal cadmium is considered as a human carcinogen. Cadmium may induce cell death by apoptosis in various cell types, although the underlying mechanisms are still unclear. In this paper we show how a label-free digital holography (DH)-based technique is able to quantify the evolution of key biophysical parameters of cells during the exposure to cadmium for the first time. Murine embryonic fibroblasts NIH 3T3 are chosen here as cellular model for studying the cadmium effects. The results demonstrate that DH is able to retrieve the temporal evolution of different key parameters such as cell volume, projected area, cell thickness and dry mass, thus providing a full quantitative characterization of the cell physical behaviour during cadmium exposure. Our results show that the label-free character of the technique would allow biologists to perform systematic and reliable studies on cell death process induced by cadmium and we believe that more in general this can be easily extended to others heavy metals, thus avoiding the time-consuming, expensive and invasive label-based procedures used nowadays in the field. In fact, pollution by heavy metals is severe issue that needs rapid and reliable methods to be settled.

Investigating fibroblast cells under "safe" and "injurious" blue-light exposure by holographic microscopy.

The exposure to visible light has been shown to exert various biological effects, such as erythema and retinal degeneration. However, the phototoxicity mechanisms in living cells are still not well understood. Here we report a study on the temporal evolution of cell morphology and volume during blue light exposure. Blue laser irradiation is switched during the operation of a digital holography (DH) microscope between what we call here "safe" and "injurious" exposure (SE & IE). The results reveal a behaviour that is typical of necrotic cells, with early swelling and successive leakage of the intracellular liquids when the laser is set in the "injurious" operation. In the phototoxicity investigation reported here the light dose modulation is performed through the very same laser light source adopted for monitoring the cell's behaviour by digital holographic microscope. We believe the approach may open the route to a deep investigation of light-cell interactions, with information about death pathways and threshold conditions between healthy and damaged cells when subjected to light-exposure. 3D Morphology and quantitative phase information from late stage of necrosis cell death.

Reversible Holographic Patterns on Azopolymers for Guiding Cell Adhesion and Orientation.

Topography of material surfaces is known to influence cell behavior at different levels: from adhesion up to differentiation. Different micro- and nanopatterning techniques have been employed to create patterned surfaces to investigate various aspects of cell behavior, most notably cellular mechanotransduction. Nevertheless, conventional techniques, once implemented on a specific substrate, fail in allowing dynamic changes of the topographic features. Here we investigated the response of NIH-3T3 cells to reversible topographic signals encoded on light-responsive azopolymer films. Switchable patterns were fabricated by means of a well-established holographic setup. Surface relief gratings were realized with Lloyd's mirror system and erased with circularly polarized or incoherent light. Cell cytoskeleton organization and focal adhesion assembly proved to be very sensitive to the underlying topographic signal. Thereafter, pattern reversibility was tested in air and wet environment by using temperature or light as a trigger. Additionally, pattern modification was dynamically performed on substrates with living cells. This study paves the way toward an in situ and real-time investigation of the material-cytoskeleton crosstalk caused by the intrinsic properties of azopolymers.