Polarization-controlled nonlinear computer-generated holography.

Dynamic phase-only beam shaping with a liquid crystal spatial light modulator is a powerful technique for tailoring the intensity profile or wave front of a beam. While shaping and controlling the light field is a highly researched topic, dynamic nonlinear beam shaping has hardly been explored so far. One potential reason is that generating the second harmonic is a degenerate process as it mixes two fields at the same frequency. To overcome this problem, we propose the use of type II phase matching as a control mechanism to distinguish between the two fields. Our experiments demonstrate that distributions of arbitrary intensity can be shaped in the frequency-converted field at the same quality as for linear beam shaping and with conversion efficiencies similar to without beam shaping. We envision this method as a milestone toward beam shaping beyond the physical limits of liquid crystal displays by facilitating dynamic phase-only beam shaping in the ultraviolet spectral range.

Uniform and efficient beam shaping for high-energy lasers.

Phase-only beam shaping with liquid crystal on silicon spatial light modulators (SLM) allows modulating the wavefront dynamically and generating arbitrary intensity patterns with high efficiency. Since this method cannot take control of all degrees of freedom, a speckle pattern appears and drastically impairs the outcome. There are several methods to overcome this issue including algorithms which directly control phase and amplitude, but they suffer from low efficiency. Methods using two SLMs yield excellent results but they are usually limited in the applicable energy due to damage to the SLM's backplane. We present a method which makes use of two SLMs and simultaneously gives way for high-energy laser applications. The algorithm and setup are designed to keep the fluence on the SLMs low by distributing the light over a large area. This provides stability against misalignment and facilitates experimental feasibility while keeping high efficiency.

Towards shifted position-diffuse reflectance imaging of anatomically correctly scaled human microvasculature.

Due to significant advantages, the trend in the field of medical technology is moving towards minimally or even non-invasive examination methods. In this respect, optical methods offer inherent benefits, as does diffuse reflectance imaging (DRI). The present study attempts to prove the suitability of DRI-when implemented alongside a suitable setup and data evaluation algorithm-to derive information from anatomically correctly scaled human capillaries (diameter: [Formula: see text], length: [Formula: see text]) by conducting extensive Monte-Carlo simulations and by verifying the findings through laboratory experiments. As a result, the method of shifted position-diffuse reflectance imaging (SP-DRI) is established by which average signal modulations of up to 5% could be generated with an illumination wavelength of [Formula: see text] and a core diameter of the illumination fiber of [Formula: see text]. No reference image is needed for this technique. The present study reveals that the diffuse reflectance data in combination with the SP-DRI normalization are suitable to localize human capillaries within turbid media.

Diffractive tunable lens for remote focusing in high-NA optical systems.

Remote focusing means to translate the focus position of an imaging system along the optical axis without moving the objective lens. The concept gains increasing importance as it allows for quick 3D focus steering in scanning microscopes, leaves the sample region unperturbed and is compatible with conjugated adaptive optics. Here we present a novel remote focusing approach that can be used in conjunction with high numerical aperture optics. Our method is based on a pair of diffractive elements, which jointly act as a tunable auxiliary lens. By changing the mutual rotation angle between the two elements, we demonstrate an axial translation of the focal spot produced by a NA = 0.95 air objective (corresponding to NA = 1.44 for an oil immersion lens) over more than 140 µm with largely maintained focus quality. We experimentally show that for the task of focus shifting, the wavefront produced by the high-NA design is superior to those produced by a parabolic lens design or a regular achromatic lens doublet.

Four-dimensional light shaping: manipulating ultrafast spatiotemporal foci in space and time.

The spectral dispersion of ultrashort pulses allows the simultaneous focusing of light in both space and time, which creates so-called spatiotemporal foci. Such space-time coupling may be combined with the existing holographic techniques to give a further dimension of control when generating focal light fields. In the present study, it is shown that a phase-only hologram placed in the pupil plane of an objective and illuminated by a spatially chirped ultrashort pulse can be used to generate three-dimensional arrays of spatio-temporally focused spots. By exploiting the pulse front tilt generated at focus when applying simultaneous spatial and temporal focusing (SSTF), it is possible to overlap neighboring foci in time to create a smooth intensity distribution. The resulting light field displays a high level of axial confinement, with experimental demonstrations given through two-photon microscopy and the non-linear laser fabrication of glass.

Assessing various Infrared (IR) microscopic imaging techniques for post-mortem interval evaluation of human skeletal remains.

Due to the influence of many environmental processes, a precise determination of the post-mortem interval (PMI) of skeletal remains is known to be very complicated. Although methods for the investigation of the PMI exist, there still remains much room for improvement. In this study the applicability of infrared (IR) microscopic imaging techniques such as reflection-, ATR- and Raman- microscopic imaging for the estimation of the PMI of human skeletal remains was tested. PMI specific features were identified and visualized by overlaying IR imaging data with morphological tissue structures obtained using light microscopy to differentiate between forensic and archaeological bone samples. ATR and reflection spectra revealed that a more prominent peak at 1042 cm-1 (an indicator for bone mineralization) was observable in archeological bone material when compared with forensic samples. Moreover, in the case of the archaeological bone material, a reduction in the levels of phospholipids, proteins, nucleic acid sugars, complex carbohydrates as well as amorphous or fully hydrated sugars was detectable at (reciprocal wavelengths/energies) between 3000 cm-1 to 2800 cm-1. Raman spectra illustrated a similar picture with less ν2PO43-at 450 cm-1 and ν4PO43- from 590 cm-1 to 584 cm-1, amide III at 1272 cm-1 and protein CH2 deformation at 1446 cm-1 in archeological bone material/samples/sources. A semi-quantitative determination of various distributions of biomolecules by chemi-maps of reflection- and ATR- methods revealed that there were less carbohydrates and complex carbohydrates as well as amorphous or fully hydrated sugars in archaeological samples compared with forensic bone samples. Raman- microscopic imaging data showed a reduction in B-type carbonate and protein α-helices after a PMI of 3 years. The calculated mineral content ratio and the organic to mineral ratio displayed that the mineral content ratio increases, while the organic to mineral ratio decreases with time. Cluster-analyses of data from Raman microscopic imaging reconstructed histo-anatomical features in comparison to the light microscopic image and finally, by application of principal component analyses (PCA), it was possible to see a clear distinction between forensic and archaeological bone samples. Hence, the spectral characterization of inorganic and organic compounds by the afore mentioned techniques, followed by analyses such as multivariate imaging analysis (MIAs) and principal component analyses (PCA), appear to be suitable for the post mortem interval (PMI) estimation of human skeletal remains.

High-resolution confocal Raman microscopy using pixel reassignment.

We present a practical modification of fiber-coupled confocal Raman scanning microscopes that is able to provide high confocal resolution in conjunction with high light collection efficiency. For this purpose, the single detection fiber is replaced by a hexagonal lenslet array in combination with a hexagonally packed round-to-linear multimode fiber bundle. A multiline detector is used to collect individual Raman spectra for each fiber. Data post-processing based on pixel reassignment allows one to improve the lateral resolution by up to 41% compared to a single fiber of equal light collection efficiency. We present results from an experimental implementation featuring seven collection fibers, yielding a resolution improvement of about 30%. We believe that our implementation represents an attractive upgrade for existing confocal Raman microscopes that employ multi-line detectors.

Deconvolution approach for 3D scanning microscopy with helical phase engineering.

RESCH (refocusing after scanning using helical phase engineering) microscopy is a scanning technique using engineered point spread functions which provides volumetric information. We present a strategy for processing the collected raw data with a multi-view maximum likelihood deconvolution algorithm, which inherently comprises the resolution gain of pixel-reassignment microscopy. The method, which we term MD-RESCH (for multi-view deconvolved RESCH), achieves in our current implementation a 20% resolution advantage along all three axes compared to RESCH and confocal microscopy. Along the axial direction, the resolution is comparable to that of image scanning microscopy. However, because the method inherently reconstructs a volume from a single 2D scan, a significantly higher optical sectioning becomes directly visible to the user, which would otherwise require collecting multiple 2D scans taken at a series of axial positions. Further, we introduce the use of a single-helical detection PSF to obtain an increased post-acquisition refocusing range. We present data from numerical simulations as well as experiments to confirm the validity of our approach.

Tilt-effect of holograms and images displayed on a spatial light modulator.

We show that a liquid crystal spatial light modulator (LCOS-SLM) can be used to display amplitude images, or phase holograms, which change in a pre-determined way when the display is tilted, i.e. observed under different angles. This is similar to the tilt-effect (also called "latent image effect") known from various security elements ("kinegrams") on credit cards or bank notes. The effect is achieved without any specialized optical components, simply by using the large phase shifting capability of a "thick" SLM, which extends over several multiples of 2π, in combination with the angular dependence of the phase shift. For hologram projection one can use the fact that the phase of a monochromatic wave is only defined modulo 2π. Thus one can design a phase pattern extending over several multiples of 2π, which transforms at different readout angles into different 2π-wrapped phase structures, due to the angular dependence of the modulo 2π operation. These different beams then project different holograms at the respective readout angles. In amplitude modulation mode (with inserted polarizer) the intensity of each SLM pixel oscillates over several periods when tuning its control voltage. Since the oscillation period depends on the readout angle, it is possible to find a certain control voltage which produces two (or more) selectable gray levels at a corresponding number of pre-determined readout angles. This is done with all SLM pixels individually, thus constructing different images for the selected angles. We experimentally demonstrate the reconstruction of multiple (Fourier- and Fresnel-) holograms, and of different amplitude images, by readout of static diffractive patterns in a variable angular range between 0° and 60°.

Lensless imaging through thin diffusive media.

Objects imaged through thin scattering media can be reconstructed with the knowledge of the complex transmission function of the diffuser. We demonstrate image reconstruction of static and dynamic objects with numerical phase conjugation in a lensless setup. Data is acquired by single shot intensity capture of an object coherently illuminated and obscured by an inhomogeneous medium, i.e. light diffracted at a specimen is scattered by a polycarbonate diffuser and the resulting speckle field is recorded. As a preparational step, which has to be performed only one time before imaging, the complex speckle field diffracted by the diffuser to the camera chip is measured interferometrically, which allows to reconstruct the transmission function of the diffuser. After insertion of the specimen, the speckle field in the camera plane changes, and the complex field of the sample can be reconstructed from the new intensity distribution. After initial interferometric measurement of the diffuser field, the method is robust with respect to a subsequent misalignment of the diffuser. The method can be extended to image objects placed between a pair of thin scattering plates. Since the object information is contained in a single speckle intensity pattern, it is possible to image dynamic processes at video rate.

Axial super-localisation using rotating point spread functions shaped by polarisation-dependent phase modulation.

We present an approach for point spread function (PSF) engineering that allows one to shape the optical wavefront independently in both polarisation directions, with two adjacent phase masks displayed on a single liquid-crystal spatial light modulator (LC-SLM). The set-up employs a polarising beam splitter and a geometric image rotator to rectify and process both polarisation directions detected by the camera. We shape a single-lobe ("corkscrew") PSF that rotates upon defocus for each polarisation channel and combine the two polarisation channels with a relative 180° phase-shift on the computer, merging them into a single PSF that exhibits two lobes whose orientation contains information about the axial position. A major advantage lies in the possibility to measure and eliminate the aberrations in the two polarisation channels independently. We demonstrate axial super-localisation of isotropically emitting fluorescent nanoparticles. Our implementation of the single-lobe PSFs follows the method proposed by Prasad [Opt. Lett.38, 585 (2013)], and thus is to the best of our knowledge the first experimental realisation of this suggestion. For comparison we also study an approach with a rotating double-helix PSFs (in only one polarisation channel) and ascertain the trade-off between localisation precision and axial working range.

Dispersion tuning with a varifocal diffractive-refractive hybrid lens.

We present a hybrid diffractive-refractive optical lens doublet consisting of a varifocal Moiré Fresnel lens and a polymer lens of tunable refractive power. The wide range of focal tunability of each lens and the opposite dispersive characteristics of the diffractive and the refractive element are exploited to obtain an optical system where both the Abbe number and the refractive power can be changed separately. We investigate the performance of the proposed hybrid lens at zero overall refractive power by tuning the Abbe number of a complementary standard lens while maintaining a constant overall focal length for the central wavelength. As an application example, the hybrid lens is used to tune to an optimal operating regime for quantitative phase microscopy based on a two-color transport of intensity (TIE) approach which utilizes chromatic aberrations rather than intensity recordings at several planes to reconstruct the optical path length of a phase object.

A new tool to ensure the fluorescent dye labeling stability of nanocarriers: a real challenge for fluorescence imaging.

Numerous studies on nanocarriers use fluorescent dye labeling to investigate their biodistribution or cellular trafficking. However, when the fluorescence dye is not grafted to the nanocarrier, the question of the stability of the labeling arises. How can it be validated that the fluorescence observed during an experiment corresponds to the nanocarriers, and not to the free dye released from the nanocarriers? Studying the integrity of the labeling is challenging. Therefore, an innovative approach to confirm the labeling stability was developed, based on the transfer of a fluorescent dye from its hosting nanocarrier to a lipophilic compartment. Lipid nanocapsules (LNC) and triglyceride oil were used as models. The protocol involved mixing of LNC suspension and oil, and then separation by centrifugation. The quality of the separation was controlled by light scattering, using the derived count rate tool. Dye transfer from loaded LNCs to the lipophilic compartment or from a lipophilic compartment containing dye to non-loaded LNC was investigated by varying the nature of the dye and the oil, the oil volume and the LNC dilution. Tensiometry was used to define the dye location in the nanocarrier. Results showed that when dyes such as Nile Red and Coumarin-6 are located in oily core, the transfer occurred in a partition-dependent manner. In contrast, when the dye was entrapped in the surfactant shell of LNCs such as lipophilic indocarbocyanines (i.e. DiO, DiI and DiD), no transfer was observed. Dye diffusion was also observed in cell culture, with Nile Red inside lipid bodies of HEI-OC1 cells, without uptake of LNCs. In contrast, DiO-loaded LNCs had to be internalized to observe fluorescence inside the cells, providing a further confirmation of the absence of transfer in this case, and the stability of fluorescence labeling of the LNCs.

Enhancing diffractive multi-plane microscopy using colored illumination.

We present a method to increase the number of simultaneously imaged focal planes in diffractive multi-plane imaging. We exploit the chromatic properties of diffraction by using multicolor LED illumination and demonstrate time-synchronous imaging of up to 21 focal planes.We discuss the possibilities and limits given by the use of a liquid crystal spatial light modulator to display the diffractive patterns. The method is suitable for wide-field transmission and reflection microscopy.

Wide-field vibrational phase imaging in an extremely folded box-CARS scattering geometry.

We present a method that allows one to measure the real and imaginary parts of the third-order susceptibility in a wide-field coherent anti-Stokes Raman scattering setup using a quadriwave lateral shearing interferometer. This permits the retrieval of the undistorted Raman spectrum and the removal of a nonresonant signal from the surrounding solvent, which otherwise may overwhelm weak resonances.

Contrast enhancement in widefield CARS microscopy by tailored phase matching using a spatial light modulator.

We introduce a widefield CARS microscope implementation that uses a spatial light modulator to obtain extremely precise control over the pump/probe-beam incidence geometry, which provides the possibility to enhance the image contrast at specific target resonances by fine-tuning the incidence angles. We show how this technique can be used to optimize the image contrast between objects of different size and to practically eliminate the undesired signal from the solvent that embeds small target specimens. Changing the numerical aperture of the illumination from 1.27 to 1.24 improved the ratio of the signals of 500 nm polystyrene beads and the agarose solvent by about 20 dB.