Inside Cover: Polarimetric imaging of biological tissues based on the indices of polarimetric purity (J. Biophotonics 4/2018)

The cover shows the image enhancement of biological tissues provided by the Indices of Polarimetric Purity (IPPs). By measuring the Mueller matrix of a biological sample, using an imaging polarimeter, the IPPs are calculated. They are polarimetric indicators providing further synthetization of depolarizing samples and leading to enhanced image contrast for some biological structures. Once the IPPs are calculated, a pseudo‐colouring technique is applied for higher visualization. Further details can be found in the article by Albert Van Eeckhout et al. ( e201700189 )

     

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.     

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.

     

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.

     

easySTORM: a robust,lower‐cost approach to localisation and TIRF microscopy

TIRF and STORM microscopy are super‐resolving fluorescence imaging modalities for which current implementations on standard microscopes can present significant complexity and cost. We present a straightforward and low‐cost approach to implement STORM and TIRF taking advantage of multimode optical fibres and multimode diode lasers to provide the required excitation light. Combined with open source software and relatively simple protocols to prepare samples for STORM, including the use of Vectashield for non‐TIRF imaging, this approach enables TIRF and STORM imaging of cells labelled with appropriate dyes or expressing suitable fluorescent proteins to become widely accessible at low cost.

     

Raman spectroscopy as a diagnostic tool for monitoring acute nephritis

Both acute nephritis and chronic nephritis account for substantial morbidity and mortality worldwide, partly due to the lack of reliable tools for detecting disease early and monitoring its progression non‐invasively. In this work, Raman spectroscopy coupled with multivariate analysis are employed for the first time to study the accelerated progression of nephritis in anti‐GBM mouse model. Preliminary results show up to 98% discriminant accuracy for the severe and midly diseased and the healthy among two strains of mice with different susceptibility to acute glomerulonephritis. This technique has the potential for non‐invasive or minimally‐invasive early diagnosis, prognosis, and monitoring of renal disease progression.

     

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     

Polarimetric assessment of healthy and radiofrequency ablated porcine myocardial tissue

Radiofrequency (RF) ablation offers a potential treatment for cardiac arrhythmia, where properly titrated energy delivered at critical sites can destroy arrhythmogenic foci. The resulting ablation lesion typically consists of a core (coagulative necrosis) surrounded by a rim of mixed viable and non‐viable cells. The extent of the RF lesion is difficult to delineate with current imaging techniques. Here, we explore polarization signatures of ten ex‐vivo samples from untreated (n = 5) and RF ablated porcine hearts (n = 5), in backscattered geometry through Mueller matrix polarimetry. Significant differences (p < 0.01) in depolarization, Δ_T, were observed between the healthy, RF ablated and rim regions. Linear retardance, δ, was significantly lower in the core and rim regions compared to healthy regions (p < 0.05). The results demonstrate a novel application of polarimetry, namely the characterization of RF ablation extent in myocardium, including the visualization of the important lesion rim region.

White light photo (top) of porcine myocardium tissue with radiofrequency ablation lesion and corresponding depolarization map (bottom). Depolarization is useful for visualizing the lesion core and rim.     

Inside Back Cover: The conformation of bovine serum albumin adsorbed to the surface of single all‐dielectric nanoparticles following light‐induced heating (J. Biophotonics 7/2018)

Germanium vs Silicon: All‐dielectric nanoparticles provides the heat resistance for proteins under light‐induced heating. Further details can be found in the article by Andrei A. Krasilin et al. ( e201700322 )

     

Photodamage in deep tissue two‐photon optical biopsy of human skin

Photodamage, induced by femtosecond laser radiation, was studied in thick samples of human skin tissue (healthy skin and neoplastic lesions). Photobleaching, photoionization, and thermomechanical damage effects were characterized comparatively. The laser power dependence of the damage rates allowed to connect macroscopic effects to underlying molecular processes. Optical effects were correlated to histopathological changes. Tissue alterations were found only from thermomechanical cavitation and limited to superficial layers of the epidermis. From the depth‐dependencies of all damage thresholds a depth‐dependent power‐compensation scheme was defined allowing for damage‐free deep tissue optical biopsy.

Damage‐induced luminescence pattern for different excitation powers and a corresponding threshold analysis.     

Investigation of the differentiation of ex vivo nerve and fat tissues using laser‐induced breakdown spectroscopy (LIBS): Prospects for tissue‐specific laser surgery

In the present study, the elemental compositions of fat and nerve tissue during their plasma mediated laser ablation are studied in the context of tissue differentiation for laser surgery applications by using Laser‐Induced Breakdown Spectroscopy (LIBS). Tissue samples of porcine fat and nerve were prepared as ex vivo experimental objects. Plasma mediated laser ablation is performed using an Nd : YAG laser in open air and under normal stray light conditions. The performed measurements suggest that the two tissue types show a high similarity in terms of qualitative elemental composition while at the same time revealing a distinct difference in the concentration of the constituent elements. Different analysis approaches are evaluated and discussed to optimize the tissue‐differentiation performance of the LIBS approach.

Plasma mediated laser tissue ablation.     

Recent advances in optical fiber devices for microfluidics integration

This paper examines the recent emergence of miniaturized optical fiber based sensing and actuating devices that have been successfully integrated into fluidic microchannels that are part of microfluidic and lab‐on‐chip systems. Fluidic microsystems possess the advantages of reduced sample volumes, faster and more sensitive biological assays, multi‐sample and parallel analysis, and are seen as the de facto bioanalytical platform of the future. This paper considers the cases where the optical fiber is not merely used as a simple light guide delivering light across a microchannel, but where the fiber itself is engineered to create a new sensor or tool for use within the environment of the fluidic microchannel.

Detection and trapping of molecules can be achieved with optical fibers directly located within the fluidic microchannel.     

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.

     

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.     

Human IgG detection in serum on polymer based Mach‐Zehnder interferometric biosensors

We report a new method for detecting human IgG (hIgG) in serum on integrated‐optical Mach‐Zehnder interferometer biosensors realized in a high index contrast polymer material system. In the linear range of the sensor (5–200 nM) we observed excellent signal recoveries (95–110%) in buffer and serum samples, which indicate the absence of matrix effects. Signal enhancement was reached by using secondary anti‐human IgG antibodies, which bind to immobilized target IgGs and allow detecting concentrations down to 100 pM. This polymer based optical sensor is fully compatible with cost‐efficient mass production technologies, which makes it an attractive alternative to inorganic optical sensors.

Graphical abstract of the hIgG measured on polymer based photonic sensors using a direct binding assay and a signal enhancement strategy with secondary antibodies.     

Assessment of ALA‐induced PpIX production in porcine skin pretreated with microneedles

Photodynamic therapy (PDT) is used for skin treatments of premalignant and cancer lesions and recognized as a non‐invasive technique that combines tissue photosensitization and subsequent exposure to light to induce cell death. However, it is limited to the treatment of superficial lesions, mainly due to the low cream penetration. Therefore, the improvement of transdermal distribution of aminolevulinic acid (ALA) is needed. In this study, the kinetics and homogeneity of production of ALA‐induced PpIX after the skin pre‐treatment with microneedles rollers of 0.5, 1.0 and 1.5 mm length were investigated. An improvement in homogeneity and production of PpIX was shown in a porcine model.

Widefield fluorescence imaging three hours after the topical application of ALA‐cream in the combined treatment with microeedles rollers.     

Real‐time in vivo cancer diagnosis using raman spectroscopy

Raman spectroscopy has becoming a practical tool for rapid in vivo tissue diagnosis. This paper provides an overview on the latest development of real‐time in vivo Raman systems for cancer detection. Instrumentation, data handling, as well as oncology applications of Raman techniques were covered. Optic fiber probes designs for Raman spectroscopy were discussed. Spectral data pre‐processing, feature extraction, and classification between normal/benign and malignant tissues were surveyed. Applications of Raman techniques for clinical diagnosis for different types of cancers, including skin cancer, lung cancer, stomach cancer, oesophageal cancer, colorectal cancer, cervical cancer, and breast cancer, were summarized.

Schematic of a real‐time Raman spectrometer for skin cancer detection. Without correction, the image captured on CCD camera for a straight entrance slit has a curvature. By arranging the optic fiber array in reverse orientation, the curvature could be effectively corrected.     

Toward the measurement of multiple fluorescence lifetimes in flow cytometry: maximizing multi‐harmonic content from cells and microspheres

Flow cytometry is a powerful means for in vitro cellular analyses where multi‐fluorescence and multi‐angle light scattering can indicate unique biochemical or morphological features of single cells. Yet, to date, flow cytometry systems have lacked the ability to capture complex fluorescence dynamics due to the transient nature of flowing cells. In this contribution we introduce a simple approach for measuring multiple fluorescence lifetimes from a single cytometric event. We leverage square wave modulation, Fourier analysis, and high frequency digitization and show the ability to resolve more than one fluorescence lifetime from fluorescently‐labelled cells and microspheres.

Illustration of a flow cytometer capable of capturing multiple fluorescence lifetime measurements; creating potential for multi‐parametric, time‐resolved signals to be captured for every color channel.     

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.     

Back Cover: Non‐invasive optical method for real‐time assessment of intracorneal riboflavin concentration and efficacy of corneal cross‐linking (J. Biophotonics 7/2018)

We disclose a theranostic device for performing image‐guided riboflavin/UV‐A corneal cross‐linking. The device determines treatment efficacy by real time monitoring of riboflavin concentration in the corneal stroma. The study shows efficacy of the device in eye bank human donor tissues. Further details can be found in the article by Giuseppe Lombardo et al. ( e201800028 )