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.

     

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.     

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.     

Distinction between breast cancer cell subtypes using third harmonic generation microscopy

Third Harmonic Generation (THG) microscopy as a non‐invasive, label free imaging methodology, allows linkage of lipid profiles with various breast cancer cells. The collected THG signal arise mostly from the lipid droplets and the membrane lipid bilayer. Quantification of THG signal can accurately distinguish HER2‐positive cells. Further analysis using Fourier transform infrared (FTIR) spectra reveals cancer‐specific profiles, correlating lipid raft‐corresponding spectra to THG signal, associating thus THG to chemical information.

THG imaging of a cancer cell.     

Alterations in plasma biochemical composition in NO deficiency induced by L‐NAME in mice analysed by Fourier Transform Infrared Spectroscopy

Mouse model of nitric oxide deficiency, induced by prolonged treatment with N^G‐nitro‐L‐arginine methyl ester (L‐NAME) was used for infrared spectroscopy (FTIR) analysis of plasma. L‐NAME leads to increased peripheral resistance and systemic hypertension. Classification of spectral response was by principal component analysis (PCA) and linear discriminant analysis (LDA). PCA allowed to separate each animal group showing that FTIR spectra are sensitive to development of NO‐deficiency on contrary to blood pressure values indicating hypertension. Globally, the most pronounced spectral alternations were observed in the second and third week of L‐NAME treatment indicating that infrared signature of blood plasma can serve as indicator of early and late stages of the disease. The PLS‐DA method provided >95% classification accuracy. Spectral features characteristic for L‐NAME treatment were mainly associated with an elevated level of proteins accompanied by a decrease of a tyrosine content and changes in lipids/phospholipid concentration. In our work we discuss these changes for which statistically significant differences (p < 0.05 – 0.005) were observed between spectra collected for each time‐point of the L‐NAME treatment versus control subjects. We demonstrated for the first time that NO‐deficiency and hypertension resulted in changes in biochemical profile of plasma that was detected by FTIR spectroscopy.

     

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.     

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.     

A virtually imaged defocused array (VIDA) for high‐speed 3D microscopy

We report a method to capture a multifocus image stack based on recording multiple reflections generated by imaging through a custom etalon. The focus stack is collected in a single camera exposure and consequently the information needed for 3D reconstruction is recorded in the camera integration time, which is only 100 µs. We have used the VIDA microscope to temporally resolve the multi‐lobed 3D morphology of neutrophil nuclei as they rotate and deform through a microfluidic constriction. In addition, we have constructed a 3D imaging flow cytometer and quantified the nuclear morphology of nearly a thousand white blood cells flowing at a velocity of 3 mm per second. The VIDA microscope is compact and simple to construct, intrinsically achromatic, and the field‐of‐view and stack number can be easily reconfigured without redesigning diffraction gratings and prisms.

     

Multi‐modal acousto‐optic/ultrasound imaging of ex vivo liver tumors at 790 nm using a Sn2P2S6 wavefront adaptive holographic setup

Biological tissues are very strong light‐scattering media. As a consequence, current medical imaging devices do not allow deep optical imaging unless invasive techniques are used. Acousto‐optic imaging is a light‐ultrasound coupling technique that takes advantage of the ballistic propagation of ultrasound in biological tissues to access optical contrast with a millimeter resolution. We have developed a photorefractive‐crystal‐based system that performs self‐adaptive wavefront holography and works within the optical therapeutic window. As it works at an appropriate wavelength range for biological tissues imaging, it was tested on ex vivo liver samples containing tumors as a pre‐clinical study. Optical contrast was obtained even if acoustical one was not significant.

Ultrasound image (left) and acousto‐optic image (right) of a liver biopsy with tumors. Acousto‐optic imaging exhibits tumors that are not detected through ultrasound.     

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).     

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 )

     

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.     

Inside Back Cover: In vivo full‐field functional optical hemocytometer (J. Biophotonics 2/2018)

Full‐field functional optical hemocytometer (FFOH), based on the absorption intensity fluctuation modulation (AIFM) effect, is in vivo label‐free image method for capillaries of near‐transparent live biological specimens. FFOH can provide a flow video, flow velocity measurement and RBC count, simultaneously. The zebrafish experimental result shows the potential to study the physiological mechanisms of the blood circulation systems. Further details can be found in the article by Fuli Zhang et al. ( e201700039 )

     

Quantitative and rapid estimations of human sub‐surface skin mass using ultra‐high‐resolution spectral domain optical coherence tomography

Non‐invasive and quantitative estimations for the delineation of sub‐surface tumor margins could greatly aid in the early detection and monitoring of the morphological appearances of tumor growth, ensure complete tumor excision without the unnecessary sacrifice of healthy tissue, and facilitate post‐operative follow‐up for recurrence. In this study, a high‐speed, non‐invasive, and ultra‐high‐resolution spectral domain optical coherence tomography (UHR‐SDOCT) imaging platform was developed for the quantitative measurement of human sub‐surface skin mass. With a proposed robust, semi‐automatic analysis, the system can rapidly quantify lesion area and shape regularity by an en‐face‐oriented algorithm. Various sizes of nylon sutures embedded in pork skin were used first as a phantom to verify the accuracy of our algorithm, and then in vivo, feasibility was proven using benign human angiomas and pigmented nevi. Clinically, this is the first step towards an automated skin lesion measurement system.

In vivo optical coherence tomography (OCT) image of angioma (A). Thin red arrows point to a blood vessel (BV).     

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.

     

Statistically quantified measurement of an Alzheimer's marker by surface‐enhanced Raman scattering

Fibrillar forms of the Amyloid‐β (Aβ) protein have been implicated in the early stages of Alzheimer's disease (AD), however there are no standardised assays for soluble Aβ oligomer biomarkers that provide the best indication of the disease progression [1,2]. As a step towards a fast and label‐free method for testing different AD biomarkers, we have combined laser nano‐textured substrates with a SERS mapping technique and validated it using soluble Aβ‐40 oligomers [3‐5]. The nano‐textured SERS substrates provide fast (&5 min), label‐free spectra associated with soluble Aβ‐40 oligomers down to a concentration of 10 nM. Statistical analysis of the spectral intensities mapped over the substrate surface shows a quantitative correlation with the oligomer concentration.

Schematics of experiments: SERS mapping of Aβ‐40 (left figure: measured SERS intensity overlayed with an SEM image of ripples) was carried out on the laser nano‐textured (ripple) surface of sapphire and statistical analysis of the SERS intensity was carried out for qualitative (a high SERS intensity at low probability) and quantitative (a moderate SERS intenisty at the highest probability) measures. Quantitative statistical analysis of SERS mapping data can be performed off line for cross correlations with other known SERS signatures.     

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.     

Surface functionalization allowing repetitive use of optical sensors for real‐time detection of antibody‐bacteria interaction

In this study, sensor surface functionalization allowing the repetitive use of a sensing device was evaluated for antibody‐based detection of living bacteria using an optical planar Bragg grating sensor. To achieve regenerable immobilization of bacteria specific antibodies, the heterobifunctional cross‐linker N‐succinimidyl 3‐(2‐pyridyldithio) propionate (SPDP) was linked to an aminosilanized sensor surface and subsequently reduced to expose sulfhydryl groups enabling the covalent conjugation of SPDP‐activated antibodies via disulfide bonds. The immobilization of a capture antibody specific for Staphylococcus aureus on the sensor surface as well as specific binding of S. aureus could be monitored, highlighting the applicability of optical sensors for the specific detection of large biological structures. Reusability of bacteria saturated sensors was successfully demonstrated by cleaving the antibody along with bound bacteria through reduction of disulfide bonds and subsequent re‐functionalization with activated antibody, resulting in comparable sensitivity towards S. aureus.