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.     

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).     

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.     

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.     

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.     

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.

     

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     

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.

     

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.     

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.

     

Superpulsed (Ga‐As, 904 nm) low‐level laser therapy (LLLT) attenuates inflammatory response and enhances healing of burn wounds

Low‐level laser therapy (LLLT) using superpulsed near‐infrared light can penetrate deeper in the injured tissue and could allow non‐pharmacological treatment for chronic wound healing. This study investigated the effects of superpulsed laser (Ga‐As 904 nm, 200 ns pulse width; 100 Hz; 0.7 mW mean output power; 0.4 mW/cm² average irradiance; 0.2 J/cm² total fluence) on the healing of burn wounds in rats, and further explored the probable associated mechanisms of action. Irradiated group exhibited enhanced DNA, total protein, hydroxyproline and hexosamine contents compared to the control and silver sulfadiazine (reference care) treated groups. LLLT exhibited decreased TNF‐α level and NF‐kB, and up‐regulated protein levels of VEGF, FGFR‐1, HSP‐60, HSP‐90, HIF‐1α and matrix metalloproteinases‐2 and 9 compared to the controls. In conclusion, LLLT using superpulsed 904 nm laser reduced the inflammatory response and was able to enhance cellular proliferation, collagen deposition and wound contraction in the repair process of burn wounds.

Photomicrographs showing no, absence inflammation and faster wound contraction in LLLT superpulsed (904 nm) laser treated burn wounds as compared to the non‐irradiated control and silver sulfadiazine (SSD) ointment (reference care) treated wounds     

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.     

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).     

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.     

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.

     

In vivo autofluorescence in the biological windows: the role of pigmentation

Small animal deep‐tissue fluorescence imaging in the second Biological Window (II‐BW, 1000–1350 nm) is limited by the presence of undesirable infrared‐excited, infrared‐emitted (900–1700 nm) autofluorescence whose origin, spectral properties and dependence on strains is still unknown. In this work, the infrared autofluorescence and laser‐induced whole body heating of five different mouse strains with distinct coat colors (black, grey, agouti, white and nude) has been systematically investigated. While neither the spectral properties nor the magnitude of organ autofluorescence vary significantly between mouse strains, the coat color has been found to strongly determine both the autofluorescence intensity as well as the laser‐induced whole body heating. Results included in this work reveal mouse strain as a critical parameter that has to be seriously considered in the design and performance of small animal imaging experiments based on infrared‐emitting fluorescent markers.

     

Femtosecond pulsed laser ablation to enhance drug delivery across the skin

Laser poration of the skin locally removes its outermost, barrier layer, and thereby provides a route for the diffusion of topically applied drugs. Ideally, no thermal damage would surround the pores created in the skin, as tissue coagulation would be expected to limit drug diffusion. Here, a femtosecond pulsed fiber laser is used to porate mammalian skin ex vivo. This first application of a hollow core negative curvature fiber (HC‐NCF) to convey a femtosecond pulsed, visible laser beam results in reproducible skin poration. The effect of applying ink to the skin surface, prior to ultra‐short pulsed ablation, has been examined and Raman spectroscopy reveals that the least, collateral thermal damage occurs in inked skin. Pre‐application of ink reduces the laser power threshold for poration, an effect attributed to the initiation of plasma formation by thermionic electron emission from the dye in the ink. Poration under these conditions significantly increases the percutaneous permeation of caffeine in vitro. Dye‐enhanced, plasma‐mediated ablation of the skin is therefore a potentially advantageous approach to enhance topical/transdermal drug absorption. The combination of a fiber laser and a HC‐NCF, capable of emitting and delivering femtosecond pulsed, visible light, may permit a compact poration device to be developed.

Using a femtosecond pulsed, visible laser beam to create an array of micropores in dyed mammalian skin, with little collateral, thermal damage, leads to an enhancement in the percutaneous permeation of caffeine in vitro.     

Front Cover: A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution (J. Biophotonics 2/2018)

A new type of high‐throughput imaging flow cytometer (>20 000 cells s^‐1) based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. FACED imaging flow cytometers enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. It critically empowers largescale and deep characterization of single cells and their heterogeneity with high statistical power— an ability to become increasingly critical in single‐cell analysis adopted in a wide range of biomedical and life‐science applications. Further details can be found in the article by Wenwei Yan et al. ( e201700178 )