In vivo imaging allows for the observation of the behaviour of single cells in the diseased nervous system. This can provide insights into how neurological diseases emerge and how they can be treated.
In vivo microscopy with single-cell resolution is based on progress in two fields. First, multiphoton microscopy, which provides high resolution views from deep within the nervous system. Second, transgenic mice with fluorescent neurons and glial cells, which allow for long-term observations with low toxicity. Combining these techniques now make in vivo observations possible in many neurological disease models.
In vivo imaging in models of neurotrauma has been used to study axonal degeneration and regeneration. One insight from these studies is the importance of glial guidance for successful regeneration in the PNS. By contrast, in the CNS, growing axons appear to lack such guidance, and degeneration outweighs axonal regrowth.
In the area of neurodegenerative diseases, in vivo microscopy studies have mainly focused on Alzheimer's disease models. Fluorescent dyes, which bind to amyloid-β, allow the analysis of the kinetics of plaque formation and clearance, as well as the evolution of plaque-related neuronal damage.
Multiphoton imaging is a convenient tool with which to study the brain's microvasculature by enabling the measurement of local flow and providing the means to specifically occlude single microvessels. This method has revealed the degree of haemodynamic compensation that occurs after vessel occlusion. Furthermore, co-visualization of blood flow and neuronal morphology can help to define how neurons are damaged by varying degrees of ischaemia.
The transmigration of immune cells across the blood–brain barrier is a crucial step in the initiation of neuroinflammatory diseases such as multiple sclerosis. By labelling immune cells ex vivo and co-visualizing the nervous system vasculature, transmigration can be readily studied with in vivo imaging. Imaging of immune cell invasion has helped to define the role of specific adhesion molecules in transmigration and has guided the development of therapies that restrict immune cell influx.
Functional microscopy techniques, such as intrinsic optical imaging and calcium imaging, allow the measurment of neuronal activity in vivo. Using these approaches, epileptogenic foci can be mapped, and aberrant patterns of activity can be recorded in single cells.
Important challenges remain for the future development of optical in vivo imaging in neurological diseases. One challenge is to address the molecular mechanisms that cause disease and provide targets for therapy. Another is to relate insights derived from imaging animal models at cellular resolution with the clinical presentation of human patients.
In vivo microscopy is an exciting tool for neurological research because it can reveal how single cells respond to damage of the nervous system. This helps us to understand how diseases unfold and how therapies work. Here, we review the optical imaging techniques used to visualize the different parts of the nervous system, and how they have provided fresh insights into the aetiology and therapeutics of neurological diseases. We focus our discussion on five areas of neuropathology (trauma, degeneration, ischaemia, inflammation and seizures) in which in vivo microscopy has had the greatest impact. We discuss the challenging issues in the field, and argue that the convergence of new optical and non-optical methods will be necessary to overcome these challenges.
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We wish to express our gratitude to J. Lichtman for his mentorship and support. Our thanks also go to J. Sanes, H. Wekerle and R. Hohlfeld for continued support. J. Lichtman, R. Hohlfeld and L. Godinho made valuable suggestions after reading a previous version of this manuscript. Work in our laboratories is supported by the Christopher Reeve Paralysis Foundation, the 'Brain-Immune Imaging Program' of the Dana-Foundation, the 'Emmy-Noether-Program' of the Deutsche Forschungsgemeinschaft, and the 'Verein Therapieforschung für MS-Kranke e.V.' We acknowledge B. Engelhardt (University of Bern, Switzerland), D. Kleinfeld (University of California, San Diego, USA) and B. Hyman (Harvard Medical School, Boston, USA) for permission to reproduce figure material from their work, and apologize to colleagues whose work we had no space to cover.
The authors declare no competing financial interests.
- Optical sectioning
The exclusion of signal from out-of-focus planes. In confocal microscopy this is achieved by placing a pinhole in front of the detector. In multiphoton microscopy, optical sectioning is an intrinsic property, as excitation is limited to the perifocal volume, where photon density peaks.
- Diffraction limit
Fundamental limitation of the resolution achievable with conventional light microscopy. As a result of light diffraction, the focal 'spot' cannot be made smaller than 100–200 nm in the plane of focus and 0.5 μm in depth (depending on the objective, immersion media and wavelength).
- Charge-coupled device cameras
(CCD cameras). Digital cameras used for wide-field microscopy that incorporate silicon chips as detectors; light that hits the photo-sensitive areas of the chip generates charge, which is measured to generate a digital image.
- Photomultiplier tubes
Point detectors used in many scanning microscopes (for example, confocal laser scanning or multiphoton microscopes), which have a photocathode as a front surface that releases electrons on illumination, and a secondary electrode array that multiplies the signal before readout.
- Perifocal volume
The volume surrounding the focus of an objective lens; real lenses do not focus light into a geometrical 'point' but into a small spheroid volume, the size of which depends on the wavelength of the light and the characteristics of the objective.
Toxic effects of light on cells that limits most in vivo microscopy experiments; caused at least in part by bleaching and associated photochemical generation of free radicals.
Green fluorescent proteins or coral fluorescent proteins and their spectral variants, such as yellow and cyan fluorescent proteins.
- Knock-in strategy
Technique by which an endogenous gene is replaced with a novel sequence; for example, to inactivate the gene and/or to characterize its expression pattern. Knock in strategies differ from 'transgenic' approaches in which genetic material is randomly inserted into the genome.
- Vital dye
Used in microscopy to denote any compound that can be applied to living organisms and cells to stain specific structures.
- Intrinsic contrast
All features of normal tissue that allow the detection of structures without additional labelling. Techniques that use intrinsic contrast are phase contrast microscopy and skeletal X-rays.
- Multicell bolus loading
Recent innovation in the field of calcium imaging in which large groups of neurons or glial cells are loaded with calcium indicator dyes through the injection of concentrated solutions of cell-permeant dye esters into brain tissue.
- Fluorescence resonance energy transfer
(FRET). Non-radiative transfer of photons between molecules with overlapping excitation–emission spectra; depends strongly on the proximity of molecules and can therefore indicate molecular interactions.
- Transcranial imaging
In the context of in vivo microscopy, this refers to imaging through the skull; multiphoton imaging can image 'through' thin slivers of bone, so that a craniotomy can be avoided by drilling away all but the inner table of the skull.
- Negative contrast
Form of contrast in which the object of interest is not labelled but surrounded by stained 'background'— for example, an unlabelled erythrocyte in fluorescently labelled serum.
- Photothrombotic stroke
Form of small vessel occlusion that depends on 'photosensitizers' — that is, molecules like rose bengal that cause vessel damage in response to being illuminated. By injecting such molecules intravenously, local clotting ('thrombosis') can be targeted by light ('optical' micro-occlusion models).
- Micro-embolic stroke
Miniature stroke model that is induced by injecting small particles into the blood stream. These particles lodge in microvessels and occlude them ('emboli'). This model can be acheived by injecting microspheres or fragmented clots into a major artery.
- Tomographic approaches
Describes imaging approaches in which the object is imaged from multiple angles and mathematical algorithms are used to reconstruct the three-dimensional structure of the signal source.
- Bioluminescence resonant energy transfer
(BRET). Energy transfer in which the donor is a bioluminescent molecule that emits light as part of a biochemical reaction and transfers it to a fluorescent acceptor. Green fluorescent protein is the acceptor in a BRET cascade that makes some jellyfish glow.
Optical tool used to measure differences in path length or travel time of light by interference — that is, the addition or cancellation of light waves as a consequence of phase shifts.
- Contrast agent
Used in clinical context for agents that generate a strong signal in an imaging technique and can be injected into the blood stream, swallowed by a patient or introduced into a body cavity.
- Bimodal contrast agent
A compound that enhances contrast in more than one imaging modality, for example, a substance that generates an MRI signal and is fluorescent at the same time.
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Misgeld, T., Kerschensteiner, M. In vivo imaging of the diseased nervous system. Nat Rev Neurosci 7, 449–463 (2006). https://doi.org/10.1038/nrn1905
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