Multiphoton Microscopy

Structural proteins like collagen and elastin are major constituents of the extracellular matrix (ECM). Extracellular matrix degradation and remodeling in diseases significantly impact the micro-organization of these structural proteins. Therefore, tracking the changes of collagen and elastin fiber's morphological features within ECM impacted by disease progression could provide a valuable insight of pathological processes such as tissue fibrosis and atherosclerosis. Benefitted from its intrinsic high-resolution imaging power and superior biochemical specificity, nonlinear optical microscopy (NLOM) is capable of providing information critical to the understanding of ECM remodeling. In this study, alterations of structural fibrillar proteins, such as collagen and elastin in arteries excised from atherosclerotic rabbits were assessed by the combination of NLOM images and textural analysis methods such as fractal dimension (FD) and directional analysis (DA). FD and DA were tested for their performance in tracking the changes of extracellular elastin and fibrillar collagen remodeling resulting from atherosclerosis progression/aging. Although other methods of image analysis to study the organization of elastin and collagen structures have been reported, the simplified calculations of FD and DA presented in this work proves that they are viable strategies for extracting and analyzing fiber-related morphology from disease-impacted tissues. Furthermore, this study also demonstrated the potential utility of FD and DA in studying ECM remodeling caused by other pathological processes such as respiratory diseases, several skin conditions or even cancer.

An imaging platform based on broadband coherent anti-Stokes Raman scattering (BCARS) has been developed which provides an advantageous combination of speed, sensitivity and spectral breadth. The system utilizes a configuration of laser sources that probes the entire biologically-relevant Raman window (500 cm(-1) to 3500 cm(-1)) with high resolution (< 10 cm(-1)). It strongly and efficiently stimulates Raman transitions within the typically weak "fingerprint" region using intrapulse 3-colour excitation, and utilizes the nonresonant background (NRB) to heterodyne amplify weak Raman signals. We demonstrate high-speed chemical imaging in two- and three-dimensional views of healthy murine liver and pancreas tissues and interfaces between xenograft brain tumours and the surrounding healthy brain matter.

Implantable biosensors are valuable scientific tools for basic neuroscience research and clinical applications. Neurotechnologies provide direct readouts of neurological signal and neurochemical processes. These tools are generally most valuable when performance capacities extend over months and years to facilitate the study of memory, plasticity, and behavior or to monitor patients’ conditions. These needs have generated a variety of device designs from microelectrodes for fast scan cyclic voltammetry (FSCV) and electrophysiology to microdialysis probes for sampling and detecting various neurochemicals. Regardless of the technology used, the breaching of the blood–brain barrier (BBB) to insert devices triggers a cascade of biochemical pathways resulting in complex molecular and cellular responses to implanted devices. Molecular and cellular changes in the microenvironment surrounding an implant include the introduction of mechanical strain, activation of glial cells, loss of perfusion, secondary metabolic injury, and neuronal degeneration. Changes to the tissue microenvironment surrounding the device can dramatically impact electrochemical and electrophysiological signal sensitivity and stability over time. This review summarizes the magnitude, variability, and time course of the dynamic molecular and cellular level neural tissue responses induced by state-of-the-art implantable devices. Studies show that insertion injuries and foreign body response can impact signal quality across all implanted central nervous system (CNS) sensors to varying degrees over both acute (seconds to minutes) and chronic periods (weeks to months). Understanding the underlying biological processes behind the brain tissue response to the devices at the cellular and molecular level leads to a variety of intervention strategies for improving signal sensitivity and longevity.

A femtosecond CARS-based nonlinear optical microscope was used to simultaneously image extracellular structural proteins and lipid-rich structures within intact aortic tissue obtained from myocardial infarction- prone Watanabe heritable hyperlipidemic rabbits (WHHLMI). Clear differences in the NLO microscopic images were observed between healthy arterial tissue and regions dominated by atherosclerotic lesions. In the current ex-vivo study, we present a single parameter based on intensity changes derived from multi-channel NLO image to classify plaque burden within the vessel. Using this parameter we were able to differentiate between healthy regions of the vessel and regions with plaque, as well as distinguish plaques relative to the age of the WHHLMI rabbit.

Structural dynamics of dendritic spines is one of the key correlative measures of synaptic plasticity for encoding short-term and long-term memory. Optical studies of structural changes in brain tissue using con-focal microscopy face difficulties of scattering. This results in low signal-to-noise ratio and thus limiting the imaging depth to few tens of microns. Multiphoton microscopy (MpM) overcomes this limitation by using low-energy photons to cause localized excitation and achieve high resolution in all three dimensions. Multiple low-energy photons with longer wavelengths minimize scattering and allow access to deeper brain regions at several hundred microns. In this article, we provide a basic understanding of the physical phenomena that give MpM an edge over conventional microscopy. Further, we highlight a few of the key studies in the field of learning and memory which would not have been possible without the advent of MpM.

Label-free imaging of bulk arterial tissue is dem- onstrated using a multimodal nonlinear optical micro- scope based on a photonic crystal fiber and a single fem- tosecond oscillator operating at 800nm. Colocalized imaging of extracellular elastin fibers, fibrillar collagen, and lipid-rich structures within aortic tissue obtained from atherosclerosis-prone myocardial infarction-prone Wa- tanabe heritable hyperlipidemic 􏰀WHHLMI􏰁 rabbits is demonstrated through two-photon excited fluorescence, second harmonic generation, and coherent anti-Stokes Ra- man scattering, respectively. These images are shown to differentiate healthy arterial wall, early atherosclerotic le- sions, and advanced plaques. Clear pathological changes are observed in the extracellular matrix of the arterial wall and correlated with progression of atherosclerotic disease
as represented by the age of the WHHLMI rabbits.