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.     

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     

Autofluorescence flow sorting of breast cancer cell metabolism

Clinical cancer treatment aims to target all cell subpopulations within a tumor. Autofluorescence microscopy of the metabolic cofactors NAD(P)H and FAD has shown sensitivity to anti‐cancer treatment response. Alternatively, flow cytometry is attractive for high throughput analysis and flow sorting. This study measures cellular autofluorescence in three flow cytometry channels and applies cellular autofluorescence to sort a heterogeneous mixture of breast cancer cells into subpopulations enriched for each phenotype. Sorted cells were grown in culture and sorting was validated by morphology, autofluorescence microscopy, and receptor expression. Ultimately, this method could be applied to improve drug development and personalized treatment planning.

     

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.

     

Significance of the follicular pathway for dermal substance penetration quantified by laser Doppler flowmetry

The understanding of transdermal substance penetration pathways remains an important field for the development of future topical drugs and cosmetics. Laser Doppler flowmetry is a well‐established method for evaluating cutaneous perfusion. In a study on 6 healthy male volunteers, we topically applied the vasoactive substance benzyl nicotinate on two test areas with open and obturated hair follicles and measured changes in the blood flow by Doppler flowmetry. Contrary to occluded follicles, the application onto the test area with open follicles led to a statistically significant perfusion increase within the first 5 minutes, emphasizing the importance of the follicular pathway for epidermal penetration.

     

Raman spectroscopy of serum: A study on ‘pre’ and ‘post’ breast adenocarcinoma resection in rat models

Risk of recurrence is a major problem in breast cancer management. Currently available prognostic markers have several disadvantages including low sensitivity and specificity, highlighting the need for new prognostic techniques. One of the candidate techniques is serum‐based Raman spectroscopy (RS). In this study, feasibility of using RS to distinguish ‘pre’ from ‘post’ breast tumor resection serum in rats was explored. Spectral analysis suggests change in proteins and amino acid profiles in ‘post’ compared to ‘pre‐surgical’ group. Principal‐Component‐Linear‐Discriminant‐Analysis shows 87% and 91% classification efficiency for ‘pre’ and ‘post‐surgical’ groups respectively. Thus, the study further supports efficacy of RS for theranostic applications.

     

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.     

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.     

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.

     

Kinetic analysis of nanoparticle‐protein interactions using a plasmon waveguide resonance

A plasmon waveguide resonance (PWR) sensor is proposed for studying the interaction between gold nanoparticles and proteins. The ability of the PWR sensor to operate in both TM and TE Polarizations, i.e. its polarization diversity, facilitates the simultaneous spectroscopy of the nanoparticles surface reactions using both polarizations. The response of each polarization to streptavidin‐biotin binding at the surface of gold nanoparticles is investigated in real time. Finally, using the principles of multimode spectroscopy, the nanoparticle's surface reactions are decoupled from the bulk solution refractive index variations.

Schematic diagram of the NP‐modified PWR sensor     

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.

     

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.

     

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.     

An infrared sensor analysing label‐free the secondary structure of the Abeta peptide in presence of complex fluids

The secondary structure change of the Abeta peptide to beta‐sheet was proposed as an early event in Alzheimer's disease. The transition may be used for diagnostics of this disease in an early state. We present an Attenuated Total Reflection (ATR) sensor modified with a specific antibody to extract minute amounts of Abeta peptide out of a complex fluid. Thereby, the Abeta peptide secondary structure was determined in its physiological aqueous environment by FTIR‐difference‐spectroscopy. The presented results open the door for label‐free Alzheimer diagnostics in cerebrospinal fluid or blood. It can be extended to further neurodegenerative diseases.

An immunologic ATR‐FTIR sensor for Abeta peptide secondary structure analysis in complex fluids is presented.     

Label‐free in vivo imaging of peripheral nerve by multispectral photoacoustic tomography

Unintentional surgical damage to nerves is mainly due to poor visualization of nerve tissue relative to adjacent structures. Multispectral photoacoustic tomography can provide chemical information with specificity and ultrasonic spatial resolution with centimeter imaging depth, making it a potential tool for noninvasive neural imaging. To implement this label‐free imaging approach, a multispectral photoacoustic tomography platform was built. Imaging depth and spatial resolution were characterized. In vivo imaging of the femoral nerve that is 2 mm deep in a nude mouse was performed. Through multivariate curve resolution analysis, the femoral nerve was discriminated from the femoral artery and chemical maps of their spatial distributions were generated.

The femoral nerve was discriminated from the femoral artery by multivariate curve resolution analysis.     

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 )

     

Brillouin spectroscopy as a new method of screening for increased CSF total protein during bacterial meningitis

Bacterial meningitis is a disease of pronounced clinical significance, especially in the developing world. Immediate treatment with antibiotics is essential, and no single test can provide a conclusive diagnosis. It is well established that elevated total protein in cerebrospinal fluid (CSF) is associated with bacterial meningitis. Brillouin spectroscopy is a widely used optical technique for noninvasive determination of the elastic moduli of materials. We found that elevated protein levels in CSF alter the fluid elasticity sufficiently to be measurable by Brillouin spectroscopy, with model healthy and diseased fluids distinguishable to marked significance (P = 0.014), which increases with sample concentration by dialysis.

Typical raw output of a 2‐stage VIPA Brillouin spectrometer: inelastically scattered Brillouin peaks (arrows) and elastically scattered incident radiation (center cross).     

Analysis of forward and backward Second Harmonic Generation images to probe the nanoscale structure of collagen within bone and cartilage

Collagen ultrastructure plays a central role in the function of a wide range of connective tissues. Studying collagen structure at the microscopic scale is therefore of considerable interest to understand the mechanisms of tissue pathologies. Here, we use second harmonic generation microscopy to characterize collagen structure within bone and articular cartilage in human knees. We analyze the intensity dependence on polarization and discuss the differences between Forward and Backward images in both tissues. Focusing on articular cartilage, we observe an increase in Forward/Backward ratio from the cartilage surface to the bone. Coupling these results to numerical simulations reveals the evolution of collagen fibril diameter and spatial organization as a function of depth within cartilage.

     

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.