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.     

Front Cover: Dual‐wavelength hybrid optoacoustic‐ultrasound biomicroscopy for functional imaging of large‐scale cerebral vascular networks (J. Biophotonics 9/2018)

We present a hybrid dual‐wavelength optoacoustic and ultrasound bio‐microscope capable of rapid transcranial visualization of morphology and oxygenation status of large‐scale cerebral vascular networks. Imaging of entire cortical vasculature in mice is achieved with single capillary resolution and complemented by simultaneously acquired pulse‐echo ultrasound microscopy scans of the mouse skull. The new approach holds potential to facilitate studies into neurological and vascular abnormalities of the brain. Further details can be found in the article by Johannes Rebling, Héctor Estrada, Sven Gottschalk, et al. ( e201800057 ).

     

Label‐free and non‐invasive discrimination of HaCaT and melanoma cells in a co‐culture model by hyperspectral confocal reflectance microscopy

A novel hyperspectral confocal microscopy method to separate different cell populations in a co‐culture model is presented here. The described methodological and instrumental approach allows discrimination of different cell types using a non‐invasive, label free method with good accuracy with a single cell resolution. In particular, melanoma cells are discriminated from HaCaT cells by hyperspectral confocal imaging, principal component analysis and optical frequencies signing, as confirmed by fluorescence labelling cross check. The identification seems to be quite robust to be insensitive to the cellular shape within the studied samples, enabling to separate cells according to their cytotype down to a single cell sensitivity.

Set of hyperspectral images of melanoma‐keratinocytes co‐culture model (left), score plot of principal component analysis and spectral analysis of principal components coefficients (center), label‐free spectral identification of cell populations (right).     

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.     

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     

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.     

Three‐dimensional multispectral optoacoustic mesoscopy reveals melanin and blood oxygenation in human skin in vivo

Optical imaging plays a major role in disease detection in dermatology. However, current optical methods are limited by lack of three‐dimensional detection of pathophysiological parameters within skin. It was recently shown that single‐wavelength optoacoustic (photoacoustic) mesoscopy resolves skin morphology, i.e. melanin and blood vessels within epidermis and dermis. In this work we employed illumination at multiple wavelengths for enabling three‐dimensional multispectral optoacoustic mesoscopy (MSOM) of natural chromophores in human skin in vivo operating at 15–125 MHz. We employ a per‐pulse tunable laser to inherently co‐register spectral datasets, and reveal previously undisclosed insights of melanin, and blood oxygenation in human skin. We further reveal broadband absorption spectra of specific skin compartments. We discuss the potential of MSOM for label‐free visualization of physiological biomarkers in skin in vivo.

Cross‐sectional optoacoustic image of human skin in vivo. The epidermal layer is characterized by melanin absorption. A vascular network runs through the dermal layer, exhibiting blood oxygenation values of 50–90%. All scale bars: 250 µm     

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.     

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.

     

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.

     

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.

     

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.     

Effects of the murine skull in optoacoustic brain microscopy

Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small‐animal neuroimaging methods, a variety of acoustic and light‐related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high‐resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically‐ and optically‐determined resolution scenarios. It is shown that strong low‐pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments.

(a) Experimental setup for hybrid acoustic and optical resolution optoacoustic microscopy. (b) Transcranial scan of an adult mouse brain using the optical resolution mode. Scale bar is 375 µm.     

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.

     

Rapid SERS monitoring of lipid‐peroxidation‐derived protein modifications in cells using photonic crystal fiber sensor

We proposed a side channel photonic crystal fiber (SC‐PCF) based Surface enhanced Raman spectroscopy (SERS) platform which is able to accurately monitor lipid peroxidation derived protein modifications in cells. This platform incorporates linoleamide alkyne (LAA), which is oxidized and subsequently modifies proteins in cells with alkyne functional group upon lipid peroxidation. By loading the side channel of SC‐PCF with a mixture of gold nanoparticles and LAA treated cells, and subsequently measuring the interference‐free alkyne Raman peak from these proteins in cells, strong SERS signal was obtained. The platform provides a method for the rapid monitoring of lipid peroxidation derived protein modification in cells.

     

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 )

     

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.     

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.     

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.     

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.