Multispectral optoacoustic and MRI coregistration for molecular imaging of orthotopic model of human glioblastoma

Multi‐modality imaging methods are of great importance in oncologic studies for acquiring complementary information, enhancing the efficacy in tumor detection and characterization. We hereby demonstrate a hybrid non‐invasive in vivo imaging approach of utilizing magnetic resonance imaging (MRI) and Multispectral Optoacoustic Tomography (MSOT) for molecular imaging of glucose uptake in an orthotopic glioblastoma in mouse. The molecular and functional information from MSOT can be overlaid on MRI anatomy via image coregistration to provide insights into probe uptake in the brain, which is verified by ex vivo fluorescence imaging and histological validation.

In vivo MSOT and MRI imaging of an orthotopic glioma mouse model injected with IRDye800‐2DG. Image coregistration between MSOT and MRI enables multifaceted (anatomical, functional, molecular) information from MSOT to be overlaid on MRI anatomy images to derive tumor physiological parameters such as perfusion, haemoglobin and oxygenation.     

Intravascular magnetomotive optical coherence tomography of targeted early‐stage atherosclerotic changes in ex vivo hyperlipidemic rabbit aortas

We report the development of an intravascular magnetomotive optical coherence tomography (IV‐MM‐OCT) system used with targeted protein microspheres to detect early‐stage atherosclerotic fatty streaks/plaques. Magnetic microspheres (MSs) were injected in vivo in rabbits, and after 30 minutes of in vivo circulation, excised ex vivo rabbit aorta samples specimens were then imaged ex vivo with our prototype IV‐MM‐OCT system. The alternating magnetic field gradient was provided by a unique pair of external custom‐built electromagnetic coils that modulated the targeted magnetic MSs. The results showed a statistically significant MM‐OCT signal from the aorta samples specimens injected with targeted MSs.

Representative magnetomotive signal (green) using targeted and non‐targeted magnetomotive microspheres in atherosclerotic diseased rabbit aortas.     

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     

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).     

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.     

Oxygenation measurement by multi‐wavelength oxygen‐dependent phosphorescence and delayed fluorescence: catchment depth and application in intact heart

Oxygen delivery and metabolism represent key factors for organ function in health and disease. We describe the optical key characteristics of a technique to comprehensively measure oxygen tension (PO₂) in myocardium, using oxygen‐dependent quenching of phosphorescence and delayed fluorescence of porphyrins, by means of Monte Carlo simulations and ex vivo experiments. Oxyphor G2 (microvascular PO₂) was excited at 442 nm and 632 nm and protoporphyrin IX (mitochondrial PO₂) at 510 nm. This resulted in catchment depths of 161 (86) µm, 350 (307) µm and 262 (255) µm respectively, as estimated by Monte Carlo simulations and ex vivo experiments (brackets). The feasibility to detect changes in oxygenation within separate anatomical compartments is demonstrated in rat heart in vivo.

Schematic of ex vivo measurements.     

Label‐free in vivo imaging of peripheral nerve by multispectral photoacoustic tomography

Unintentional surgical damage to nerves is mainly due to poor visualization of nerve tissue relative to adjacent structures. Multispectral photoacoustic tomography can provide chemical information with specificity and ultrasonic spatial resolution with centimeter imaging depth, making it a potential tool for noninvasive neural imaging. To implement this label‐free imaging approach, a multispectral photoacoustic tomography platform was built. Imaging depth and spatial resolution were characterized. In vivo imaging of the femoral nerve that is 2 mm deep in a nude mouse was performed. Through multivariate curve resolution analysis, the femoral nerve was discriminated from the femoral artery and chemical maps of their spatial distributions were generated.

The femoral nerve was discriminated from the femoral artery by multivariate curve resolution analysis.     

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.     

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.

     

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.     

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.     

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.

     

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     

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.

     

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.     

Multicolor multiphoton microscopy based on a nanosecond supercontinuum laser source

Multicolor multiphoton microscopy is experimentally demonstrated for the first time on a spectral bandwidth of excitation of 300 nm (full width half maximum) thanks to the implementation a nanosecond supercontinuum (SC) source compact and simple with a low repetition rate. The interest of such a wide spectral bandwidth, never demonstrated until now, is highlighted in vivo: images of glioma tumor cells stably expressing eGFP grafted on the brain of a mouse and its blood vessels network labelled with Texas Red^® are obtained. These two fluorophores have a spectral bandwidth covering the whole 300 nm available. In parallel, a similar image quality is obtained on a sample of mouse muscle in vitro when excited with this nanosecond SC source or with a classical high rate, femtosecond and quasi monochromatic laser. This opens the way for (i) a simple and very complete biological characterization never performed to date with multiphoton processes, (ii) multiple means of contrast in nonlinear imaging allowed by the use of numerous fluorophores and (iii) other multiphoton processes like three‐photon ones.

     

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.     

Closed loop tracked Doppler optical coherence tomography based heart monitor for the Drosophila melanogaster larvae

This paper presents a novel instrument for biosciences, useful for studies of moving embryos. A dual sequential imaging/measurement channel is assembled via a closed‐loop tracking architecture. The dual channel system can operate in two regimes: (i) single‐point Doppler signal monitoring or (ii) fast 3‐D swept source OCT imaging. The system is demonstrated for characterizing cardiac dynamics in Drosophila melanogaster larva. Closed loop tracking enables long term in vivo monitoring of the larvae heart without anesthetic or physical restraint. Such an instrument can be used to measure subtle variations in the cardiac behavior otherwise obscured by the larvae movements.

A fruit fly larva (top) was continuously tracked for continuous remote monitoring. A heartbeat trace of freely moving larva (bottom) was obtained by a low coherence interferometry based doppler sensing technique.     

Ex vivo investigations on the potential of optical coherence tomography (OCT) as a diagnostic tool for reproductive medicine in a bovine model

Routine infertility investigations in the male and female include imaging techniques such as ultrasonography and endoscopy (fertiloscopy). However, these techniques lack the resolution to localize vital sperm or to reveal detailed morphological analysis of the oviduct which is often the cause of infertility in females. Therefore we set out to evaluate the efficiency of optical coherence tomography (OCT) as a diagnostic imaging tool for micron‐scale visualization of the male and female genital tract. Using the bovine as a model, the optical features of the Telesto^TM, Ganymede^TM (both Thorlabs) and Niris^TM (Imalux) OCT imaging systems were compared.

Comparative visualization of ex vivo bovine testicular tissue by the Telesto^TM microscopic optical coherence tomography system (left) and corresponding H&E staining (right).     

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.