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).     

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     

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.

     

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.     

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.     

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.     

Chemico‐mechanical imaging of Barrett's oesophagus

Barrett's oesophagus is a condition characterized by a change in the lining of the oesophagus that markedly increases the risk of adenocarcinoma. We demonstrate the first site‐matched application of Brillouin microscopy, Raman microscopy and FTIR micro‐spectroscopic imaging to ex‐vivo epithelial tissue – Barrett's oesophagus. The mechanical and chemical characters of the epithelium were assessed in histological sections from a patient subjected to endoscopic oesophageal biopsy. Previous studies have shown that both these properties change within the oesophageal wall, owing to the presence of distinct cellular and extracellular constituents which are putatively affected by oesophageal cancer. Brillouin microscopy enables maps of elasticity of the epithelium to be obtained, whilst Raman and FTIR imaging provide ’chemical images' without the need for labelling or staining. This site‐matched approach provides a valuable platform for investigating the structure, biomechanics and composition of complex heterogeneous systems. A combined Brillouin‐Raman device has potential for in‐vivo diagnosis of pathology.

First application of site‐matched micro Brillouin, Raman and FTIR spectroscopic imaging to epithelial tissue in Barrett's oesophagus     

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.     

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.     

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.

     

Raman spectroscopy of blood serum for Alzheimer's disease diagnostics: specificity relative to other types of dementia

The key moment for efficiently and accurately diagnosing dementia occurs during the early stages. This is particularly true for Alzheimer's disease (AD). In this proof‐of‐concept study, we applied near infrared (NIR) Raman microspectroscopy of blood serum together with advanced multivariate statistics for the selective identification of AD. We analyzed data from 20 AD patients, 18 patients with other neurodegenerative dementias (OD) and 10 healthy control (HC) subjects. NIR Raman microspectroscopy differentiated patients with more than 95% sensitivity and specificity. We demonstrated the high discriminative power of artificial neural network (ANN) classification models, thus revealing the high potential of this developed methodology for the differential diagnosis of AD. Raman spectroscopic, blood‐based tests may aid clinical assessments for the effective and accurate differential diagnosis of AD, decrease the labor, time and cost of diagnosis, and be useful for screening patient populations for AD development and progression.

Multivariate data analysis of blood serum Raman spectra allows for the differentiation between patients with Alzheimer's disease, other types of dementia and healthy individuals.     

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.     

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.

     

Fiber probe for nonlinear imaging applications

Over the past years it had been demonstrated that multimodal imaging combining the nonlinear modalities coherent anti‐Stokes Raman scattering (CARS), two‐photon excited auto‐fluorescence (TPEF) and second harmonic generation (SHG) show a great potential for tissue diagnosis and tumor identification. To extend the applicability of this multimodal imaging approach for in‐vivo tissue screening of difficult to access body regions the development of suitable fiber optic probes is required. Here we report about a novel CARS imaging fiber probe consisting of 10,000 coherent light guiding elements preserving the spatial relationship between the entrance and the output of the fiber. Therefore the scanning procedure can be shifted from the distal to the proximal end of the fiber probe and no moving parts or driving current are required to realize in‐vivo CARS endoscopy.

Back scattered CARS image of rabbit aorta with plaques (white) using a laser scanning microscope and an imaging fiber.     

Comparing Raman and fluorescence lifetime spectroscopy from human atherosclerotic lesions using a bimodal probe

Fluorescence lifetime imaging (FLIm) and Raman spectroscopy are two promising methods to support morphological intravascular imaging techniques with chemical contrast. Both approaches are complementary and may also be used in combination with OCT/IVUS to add chemical specificity to these morphologic intravascular imaging modalities. In this contribution, both modalities were simultaneously acquired from two human coronary specimens using a bimodal probe. A previously trained SVM model was used to interpret the fluorescence lifetime data; integrated band intensities displayed in RGB false color images were used to interpret the Raman data. Both modalities demonstrate unique strengths and weaknesses and these will be discussed in comparison to histologic analyses from the two coronary arteries imaged.

     

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.

     

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.     

Label‐free,zeptomole cancer biomarker detection by surface‐enhanced fluorescence on nanoporous gold disk plasmonic nanoparticles

We experimentally demonstrate a label‐free biosensor for the ERBB2 cancer gene DNA target based on the distance‐dependent detection of surface‐enhanced fluorescence (SEF) on nanoporous gold disk (NPGD) plasmonic nanoparticles. We achieve detection of 2.4 zeptomole of DNA target on the NPGD substrate with an upper concentration detection limit of 1 nM. Without the use of molecular spacers, the NPGD substrate as an SEF platform was shown to provide higher net fluorescence for visible and NIR fluorophores compared to glass and non‐porous gold substrates. The enhanced fluorescence signals in patterned nanoporous gold nanoparticles make NPGD a viable material for further reducing detection limits for biomolecular targets used in clinical assays.

With patterned nanoporous gold disk (NPGD) plasmonic nanoparticles, a label‐free biosensor that makes use of distance‐dependent detection of surface‐enhanced fluorescence (SEF) is constructed and tested for zeptomole detection of ERBB2 cancer gene DNA targets.