Clemens Roider

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.

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.

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.

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.

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.

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.

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.

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

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.

3D image scanning microscopy with engineered excitation and detection: publisher’s note

This publisher’s note corrects an award ID in the funding section of Optica4, 1373 (2017)OPTIC82334-253610.1364/OPTICA.4.001373.