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.     

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 )

     

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.

     

Light distribution and thermal effects in the rat brain under optogenetic stimulation

Optical brain stimulation gained a lot of attention in neuroscience due to its superior cell‐type specificity. In the design of illumination strategies, predicting the light propagation in a specific tissue is essential and requires knowledge of the optical properties of that tissue. We present the estimated absorption and reduced scattering in rodent brain tissue using non‐destructive contact spatially resolved spectroscopy (cSRS). The obtained absorption and scattering in the cortex, hippocampus and striatum are similar, but lower than in the thalamus, leading to a less deep but broader light penetration profile in the thalamus. Next, the light distribution was investigated for different stimulation protocols relevant for fiber‐optic based optogenetic experiments, using Monte Carlo simulation. A protocol specific analysis is proposed to evaluate the potential of thermally induced side effects.

     

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.     

Observation of an improved healing process in superficial skin wounds after irradiation with a blue‐LED haemostatic device

The healing process of superficial skin wounds treated with a blue‐LED haemostatic device is studied. Four mechanical abrasions are produced on the back of 10 Sprague Dawley rats: two are treated with the blue‐LED device, while the other two are left to naturally recover. Visual observations, non‐linear microscopic imaging, as well as histology and immunofluorescence analyses are performed 8 days after the treatment, demonstrating no adverse reactions neither thermal damages in both abraded areas and surrounding tissue. A faster healing process and a better‐recovered skin morphology are observed: the treated wounds show a reduced inflammatory response and a higher collagen content.

Blue LED induced photothermal effect on superficial abrasions.     

Protein,enzyme and carbohydrate quantification using smartphone through colorimetric digitization technique

In this paper the utilization of smartphone as a detection platform for colorimetric quantification of biological macromolecules has been demonstrated. Using V‐channel of HSV color space, the quantification of BSA protein, catalase enzyme and carbohydrate (using D‐glucose) have been successfully investigated. A custom designed android application has been developed for estimating the total concentration of biological macromolecules. The results have been compared with that of a standard spectrophotometer which is generally used for colorimetric quantification in laboratory settings by measuring its absorbance at a specific wavelength. The results obtained with the designed sensor is found to be similar when compared with the spectrophotometer data. The designed sensor is low cost, robust and we envision that it could promote diverse fields of bio‐analytical investigations.

Schematic illustration of the smartphone sensing mechanism for colorimetric analysis of biomolecular samples.     

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.

     

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.     

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.

     

In situ non‐destructive measurement of biofilm thickness and topology in an interferometric optical microscope

Biofilms are ubiquitous and impact the environment, human health, dental hygiene, and a wide range of industrial processes. Biofilms are difficult to characterize when fully hydrated, especially in a non‐destructive manner, because of their soft structure and water‐like bulk properties. Herein a method of measuring and monitoring the thickness and topology of live biofilms of using white light interferometry is described. Using this technique, surface morphology, surface roughness, and biofilm thickness were measured over time without while the biofilm continued to grow. The thickness and surface topology of a P. putida biofilm were monitored growing from initial colonization to a mature biofilm. Measured thickness followed expected trends for bacterial growth. Surface roughness also increased over time and was a leading indicator of biofilm growth.

     

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.     

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.     

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.

     

Cell perforation mediated by plasmonic bubbles generated by a single near infrared femtosecond laser pulse

We report on transient membrane perforation of living cancer cells using plasmonic gold nanoparticles (AuNPs) enhanced single near infrared (NIR) femtosecond (fs) laser pulse. Under optimized laser energy fluence, single pulse treatment (τ = 45 fs, λ = 800 nm) resulted in 77% cell perforation efficiency and 90% cell viability. Using dark field and ultrafast imaging, we demonstrated that the generation of submicron bubbles around the AuNPs is the necessary condition for the cell membrane perforation. AuNP clustering increased drastically the bubble generation efficiency, thus enabling an effective laser treatment using low energy dose in the NIR optical therapeutical window.

Schematic representation of single femtosecond laser pulse plasmonic bubble generation in the vicinity of a cell.     

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.     

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.

     

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.     

A composite fibre optic catheter for monitoring peristaltic transit of an intra‐luminal bead

A fibre optic motion sensor has been developed for monitoring the proximity and direction of motion of a ferrous bead travelling axial to the sensor. By integrating an array of these sensors into our previously developed fibre optic manometry catheters we demonstrate simultaneous detection of peristaltic muscular activity and the associated motion of ferrous beads through a colonic lumen. This allows the motion of solid content to be temporally and spatially related to pressure variations generated by peristaltic contractions without resorting to videoflouroscopy to track the motion of a radio opaque bolus. The composite catheter has been tested in an in‐vitro animal preparation consisting of excised sections of rabbit colon.

Cut‐away image of the fibre optic motion sensor showing the location of the fibre Bragg gratings and the rare earth magnet.