Dual‐polarization Raman spectral imaging to extract overlapping molecular fingerprints of living cells

Raman spectral imaging is gaining more and more attention in biological studies because of its label‐free characteristic. However, the discrimination of overlapping chemical contrasts has been a major challenge. In this study, we introduce an optical method to simultaneously obtain two orthogonally polarized Raman images from a single scan of the sample. We demonstrate how this technique can improve the quality and quantity of the hyperspectral Raman dataset and how the technique is expected to further extend the horizons of Raman spectral imaging in biological studies by providing more detailed chemical information.

The dual‐polarization Raman images of a HeLa cell.     

Volumetric optoacoustic imaging feedback during endovenous laser therapy – an ex vivo investigation

Endovenous laser therapy (ELT) was introduced in clinical practice for treating incompetent veins about fifteen years ago. Despite the considerable clinical evidence collected so far, no rigorous guidelines are yet available regarding the optimal energy deposition protocols while incidence of recanalization, lack of vessel occlusion and collateral damage remains variable among patients. Online monitoring and feedback‐based control over the lesion progression may improve clinical outcomes. Yet the currently employed monitoring tools, such as Doppler ultrasound, often do not provide sufficient contrast as well as three‐dimensional imaging capacity for accurate lesion assessment during thermal treatments. Here we investigate on the utility of volumetric optoacoustic tomography for real‐time monitoring of the ELT procedures. Experiments performed in subcutaneous veins of an ox foot model revealed the accurate spatio‐temporal maps of the lesion progression and characteristics of the vessel wall. Optoacoustic images further correlated with the temperature elevation measured in the area adjacent to the coagulation spot and made it possible to track the position of the fiber tip during its pull back in real time and in all three dimensions. Overall, we showcase that volumetric optoacoustic tomography is a promising tool for providing online feedback during endovenous laser therapy.

     

Improved detectability of microcirculatory dynamics by laser speckle flowmetry

Mechanisms of renal autoregulation generate oscillations in arterial blood flow at several characteristic frequencies. Full‐field laser speckle flowmetry provides a real‐time imaging of superficial blood microcirculation. The possibility to detect changes in oscillatory dynamics is an important issue in biomedical applications. In this paper we show how laser power density affects quality of the recorded signal and improves detectability of temporal changes in microvascular perfusion.

     

Probe‐based confocal laser endomicroscopy (pCLE) – a new imaging technique for in situ localization of spermatozoa

In azoospermic patients, spermatozoa are routinely obtained by testicular sperm extraction (TESE). However, success rates of this technique are moderate, because the site of excision of testicular tissue is determined arbitrarily. Therefore the aim of this study was to establish probe‐based laser endomicroscopy (pCLE) a noval biomedical imaging technique, which provides the opportunity of non‐invasive, real‐time visualisation of tissue at histological resolution. Using pCLE we clearly visualized longitudinal and horizontal views of the tubuli seminiferi contorti and localized vital spermatozoa. Obtained images and real‐time videos were subsequently compared with confocal laser scanning microscopy (CLSM) of spermatozoa and tissues, respectively.

Comparative visualization of single native Confocal laser scanning microscopy (CLSM, left) and probe‐based laser endomicroscopy (pCLE, right) using Pro Flex^TM UltraMini O after staining with acriflavine.     

Wide‐field hyperspectral 3D imaging of functionalized gold nanoparticles targeting cancer cells by reflected light microscopy

We present a new hyperspectral reflected light microscopy system with a scanned broadband supercontinuum light source. This wide‐field and low phototoxic hyperspectral imaging system has been successful for performing spectral three‐dimensional (3D) localization and spectroscopic identification of CD44‐targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic‐based imaging, disease detection and treatment in complex biological environment. The presented system can be used for real‐time 3D NP tracking as spectral sensors, thus providing new avenues in the spatio‐temporal characterization and detection of bioanalytes.

3D image of the distribution of functionalized AuNPs attached to CD44‐expressing MDA‐MB‐231 human cancer cells.     

A new approach for optical assessment of directional anisotropy in turbid media

A study of polarized light transport in scattering media exhibiting directional anisotropy or linear birefringence is presented in this paper. Novel theoretical and experimental methodologies for the quantification of birefringent alignment based on out‐of‐plane polarized light transport are presented here. A polarized Monte Carlo model and a polarimetric imaging system were devised to predict and measure the impact of birefringence on an impinging linearly polarized light beam. Ex‐vivo experiments conducted on bovine tendon, a biological sample consisting of highly packed type I collagen fibers with birefringent property, showed good agreement with the analytical results.

Top view geometry of the in‐plane ( a ) and the out‐of‐plane ( b ) detection. Letter C indicates the location of the detection arm.     

Holographic imaging of unlabelled sperm cells for semen analysis: a review

Male reproductive health in both humans and animals is an important research field in biological study. In order to characterize the morphology, the motility and the concentration of the sperm cells, which are the most important parameters to feature them, digital holography demonstrated to be an attractive technique. Indeed, it is a label‐free, non‐invasive and high‐resolution method that enables the characterization of live specimen. The review is intended both for summarizing the state‐of‐art on the semen analysis and recent achievement obtained by means of digital holography and for exploring new possible applications of digital holography in this field.

Quantitative phase maps of living swimming spermatozoa.     

Hyperspectral imaging in medicine: image pre‐processing problems and solutions in Matlab

The paper presents problems and solutions related to hyperspectral image pre‐processing. New methods of preliminary image analysis are proposed. The paper shows problems occurring in Matlab when trying to analyse this type of images. Moreover, new methods are discussed which provide the source code in Matlab that can be used in practice without any licensing restrictions.

The proposed application and sample result of hyperspectral image analysis.     

Multimodal and multiplex spectral imaging of rat cornea ex vivo using a white‐light laser source

We applied our multimodal nonlinear spectral imaging microscope to the measurement of rat cornea. We successfully obtained multiple nonlinear signals of coherent anti‐Stokes Raman scattering (CARS), third‐order sum frequency generation (TSFG), and second harmonic generation (SHG). Depending on the nonlinear optical processes, the cornea tissue was visualized with different image contrast mechanism simultaneously. Due to white‐light laser excitation, multiplex CARS and TSFG spectra were obtained. Combined multimodal and spectral analysis clearly elucidated the layered structure of rat cornea with molecular structural information. This study indicates that our multimodal nonlinear spectral microscope is a promising bioimaging method for tissue study.

Multimodal nonlinear spectral images of rat cornea at corneal epithelium and corneal stroma in the in‐plane (XY) direction. With use of the combinational analysis of different nonlinear optical processes, detailed molecular structural information is available without staining or labelling.     

Optical hyperspectral imaging in microscopy and spectroscopy – a review of data acquisition

Rather than simply acting as a photographic camera capturing two‐dimensional (x, y) intensity images or a spectrometer acquiring spectra (λ), a hyperspectral imager measures entire three‐dimensional (x, y, λ) datacubes for multivariate analysis, providing structural, molecular, and functional information about biological cells or tissue with unprecedented detail. Such data also gives clinical insights for disease diagnosis and treatment. We summarize the principles underpinning this technology, highlight its practical implementation, and discuss its recent applications at microscopic to macroscopic scales.

Datacube acquisition strategies in hyperspectral imaging x, y, spatial coordinates; λ, wavelength.     

Fiber probe for nonlinear imaging applications

Over the past years it had been demonstrated that multimodal imaging combining the nonlinear modalities coherent anti‐Stokes Raman scattering (CARS), two‐photon excited auto‐fluorescence (TPEF) and second harmonic generation (SHG) show a great potential for tissue diagnosis and tumor identification. To extend the applicability of this multimodal imaging approach for in‐vivo tissue screening of difficult to access body regions the development of suitable fiber optic probes is required. Here we report about a novel CARS imaging fiber probe consisting of 10,000 coherent light guiding elements preserving the spatial relationship between the entrance and the output of the fiber. Therefore the scanning procedure can be shifted from the distal to the proximal end of the fiber probe and no moving parts or driving current are required to realize in‐vivo CARS endoscopy.

Back scattered CARS image of rabbit aorta with plaques (white) using a laser scanning microscope and an imaging fiber.     

Cell nuclei have lower refractive index and mass density than cytoplasm

Common perception regards the nucleus as a densely packed object with higher refractive index (RI) and mass density than the surrounding cytoplasm. Here, the volume of isolated nuclei is systematically varied by electrostatic and osmotic conditions as well as drug treatments that modify chromatin conformation. The refractive index and dry mass of isolated nuclei is derived from quantitative phase measurements using digital holographic microscopy (DHM). Surprisingly, the cell nucleus is found to have a lower RI and mass density than the cytoplasm in four different cell lines and throughout the cell cycle. This result has important implications for conceptualizing light tissue interactions as well as biological processes in cells.

     

Synergistic effects of atmospheric pressure plasma‐emitted components on DNA oligomers: a Raman spectroscopic study

Cold atmospheric‐pressure plasmas have become of increasing importance in sterilization processes especially with the growing prevalence of multi‐resistant bacteria. Albeit the potential for technological application is obvious, much less is known about the molecular mechanisms underlying bacterial inactivation. X‐jet technology separates plasma‐generated reactive particles and photons, thus allowing the investigation of their individual and joint effects on DNA. Raman spectroscopy shows that particles and photons cause different modifications in DNA single and double strands. The treatment with the combination of particles and photons does not only result in cumulative, but in synergistic effects. Profilometry confirms that etching is a minor contributor to the observed DNA damage in vitro.

Schematics of DNA oligomer treatment with cold atmospheric‐pressure plasma.     

Comparing Raman and fluorescence lifetime spectroscopy from human atherosclerotic lesions using a bimodal probe

Fluorescence lifetime imaging (FLIm) and Raman spectroscopy are two promising methods to support morphological intravascular imaging techniques with chemical contrast. Both approaches are complementary and may also be used in combination with OCT/IVUS to add chemical specificity to these morphologic intravascular imaging modalities. In this contribution, both modalities were simultaneously acquired from two human coronary specimens using a bimodal probe. A previously trained SVM model was used to interpret the fluorescence lifetime data; integrated band intensities displayed in RGB false color images were used to interpret the Raman data. Both modalities demonstrate unique strengths and weaknesses and these will be discussed in comparison to histologic analyses from the two coronary arteries imaged.

     

Label‐free and non‐invasive monitoring of porcine trophoblast derived cells: differentiation in serum and serum‐free media

Traditional approaches to characterize stem cell differentiation are time‐consuming, lengthy and invasive. Here, Raman microspectroscopy (RM) and atomic force microscopy (AFM) – both considered as non‐invasive techniques – are applied to detect the biochemical and biophysical properties of trophoblast derived stem‐like cells incubated up to 10 days under conditions designed to induce differentiation. Significant biochemical and biophysical differences between control cells and differentiated cells were observed. Quantitative real time PCR was also applied to analyze gene expression. The relationship between cell differentiation and associated cellular biochemical and biomechanical changes were discussed.

Monitoring trophoblast cells differentiation     

Automated quantitative cytological analysis using portable microfluidic microscopy

In this article, a portable microfluidic microscopy based approach for automated cytological investigations is presented. Inexpensive optical and electronic components have been used to construct a simple microfluidic microscopy system. In contrast to the conventional slide‐based methods, the presented method employs microfluidics to enable automated sample handling and image acquisition. The approach involves the use of simple in‐suspension staining and automated image acquisition to enable quantitative cytological analysis of samples. The applicability of the presented approach to research in cellular biology is shown by performing an automated cell viability assessment on a given population of yeast cells. Further, the relevance of the presented approach to clinical diagnosis and prognosis has been demonstrated by performing detection and differential assessment of malaria infection in a given sample.

     

Multi‐modal acousto‐optic/ultrasound imaging of ex vivo liver tumors at 790 nm using a Sn2P2S6 wavefront adaptive holographic setup

Biological tissues are very strong light‐scattering media. As a consequence, current medical imaging devices do not allow deep optical imaging unless invasive techniques are used. Acousto‐optic imaging is a light‐ultrasound coupling technique that takes advantage of the ballistic propagation of ultrasound in biological tissues to access optical contrast with a millimeter resolution. We have developed a photorefractive‐crystal‐based system that performs self‐adaptive wavefront holography and works within the optical therapeutic window. As it works at an appropriate wavelength range for biological tissues imaging, it was tested on ex vivo liver samples containing tumors as a pre‐clinical study. Optical contrast was obtained even if acoustical one was not significant.

Ultrasound image (left) and acousto‐optic image (right) of a liver biopsy with tumors. Acousto‐optic imaging exhibits tumors that are not detected through ultrasound.     

In vivo nonlinear optical imaging to monitor early microscopic changes in a murine cutaneous squamous cell carcinoma model

Early detection of cutaneous squamous cell carcinoma (cSCC) can enable timely therapeutic and preventive interventions for patients. In this study, in vivo nonlinear optical imaging (NLOI) based on two‐photon excitation fluorescence (TPEF) and second harmonic generation (SHG), was used to non‐invasively detect microscopic changes occurring in murine skin treated topically with 7,12‐dimethylbenz(a)anthracene (DMBA). The optical microscopic findings and the measured TPEF‐SHG index show that NLOI was able to clearly detect early cytostructural changes in DMBA treated skin that appeared clinically normal. This suggests that in vivo NLOI could be a non‐invasive tool to monitor early signs of cSCC.

In vivo axial NLOI scans of normal murine skin (upper left), murine skin with preclinical hyperplasia (upper right), early clinical murine skin lesion (lower left) and late or advanced murine skin lesion (lower right).     

Subcellular measurements of mechanical and chemical properties using dual Raman‐Brillouin microspectroscopy

Brillouin microspectroscopy is a powerful technique for noninvasive optical imaging. In particular, Brillouin microspectroscopy uniquely allows assessing a sample's mechanical properties with microscopic spatial resolution. Recent advances in background‐free Brillouin microspectroscopy make it possible to image scattering samples without substantial degradation of the data quality. However, measurements at the cellular‐ and subcellular‐level have never been performed to date due to the limited signal strength. In this report, by adopting our recently optimized VIPA‐based Brillouin spectrometer, we probed the microscopic viscoelasticity of individual red blood cells. These measurements were supplemented by chemically specific measurements using Raman microspectroscopy.