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.

     

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     

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.     

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.

     

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.

     

Front Cover: Ultraviolet radiation oxidative stress affects eye health (J. Biophotonics 7/2018)

The role of ultraviolet radiation in oxidative stress‐related ocular pathologies is less known than its role in skin cancer. Excessive exposure to ultraviolet radiation is associated with increased oxidative stress in eye tissues, which may promote the development of photokeratitis, cataract, and retinal damages. Children are especially vulnerable: large pupils, transparent ocular media. Efficient everyday protection of the eye should be considered from early age. (Image: with permission from Carl Zeiss Vision International GmbH, Aalen, Germany) Further details can be found in the article by Iliya V. Ivanov, Timo Mappes, Patrick Schaupp, et al. ( e201700377 ).

     

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.

     

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.

     

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.     

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.     

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.

     

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.

     

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.     

Raman spectroscopic identification of Mycobacterium tuberculosis

In this study, Raman microspectroscopy has been utilized to identify mycobacteria to the species level. Because of the slow growth of mycobacteria, the per se cultivation‐independent Raman microspectroscopy emerges as a perfect tool for a rapid on‐the‐spot mycobacterial diagnostic test. Special focus was laid upon the identification of Mycobacterium tuberculosis complex (MTC) strains, as the main causative agent of pulmonary tuberculosis worldwide, and the differentiation between pathogenic and commensal nontuberculous mycobacteria (NTM). Overall the proposed model considers 26 different mycobacteria species as well as antibiotic susceptible and resistant strains. More than 8800 Raman spectra of single bacterial cells constituted a spectral library, which was the foundation for a two‐level classification system including three support vector machines. Our model allowed the discrimination of MTC samples in an independent validation dataset with an accuracy of 94% and could serve as a basis to further improve Raman microscopy as a first‐line diagnostic point‐of‐care tool for the confirmation of tuberculosis disease.

     

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 )

     

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     

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.

     

Maintaining polarization in polarimetric multiphoton microscopy

Polarimetric measurements in multiphoton microscopy can reveal information about the local molecular order of a sample. However, the presence of a dichroic through which the excitation beam propagates will generally scramble its polarization. We propose a simple scheme whereby a second properly‐oriented compensation dichroic is used to negate any alteration regardless of the wavelength and the initial polarization. We demonstrate how this robust and rapid approach simplifies polarimetric measurements in second‐harmonic generation, two‐photon excited fluorescence and coherent anti‐Stokes Raman scattering.

Illustration of the polarization maintaining strategy with the compensating dichroic oriented such that its s‐ and p‐axes are interchanged with these of the primary dichroic.     

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.