This overview talk will focus on forward-looking scientific needs and physical limits to images of neuronal processes. The challenge in nervous systems is that the basic unit for "switching" events in the nervous system occurs on the one micrometer scale of synaptic spines, while computations involve communication between individual neurons across the full expanse of cortex, which is ten millimeters for mouse cortex. I will address hoped-for advances in optical microscopy, within the context of existing and proposed contrast mechanisms of neuronal function, that span the four orders of magnitude of length scales for neuronal processing
Blood is a limited resource that supplies neurons and glia with nutriments. How is blood distributed both in a fail-safe
way and in response to changing metabolic loads? We examine this issue in rodent cortex. First, in terms of topology,
all-optical histology is used to reconstruct the vasculature network. Second, in terms of dynamics, the flux of blood cells
in individual vessels is measured in response to neuronal activation and/or the perturbation of flow of a targeted vessel
via plasma-mediated ablation. Lastly, in terms of control, optical indicators of neurovascular signaling molecules that
alter vessel diameter are measured.
Femtosecond laser pulses have the unique ability to deposit energy into a microscopic volume in the bulk of a material that is transparent to the laser wavelength without affecting the surface of the material. Here we review the use of this capability to disrupt specifically targeted structures in live cells and animals with the goal of elucidating function and modeling disease states. Particular attention will be paid to recent work that uses femtosecond laser disruption to injure cerebral blood vessels that lie below the brain surface in a live, anesthetized rat. By varying the degree of injury, the vessel can be made to leak blood plasma, to rupture, or to clot. This technique thus provides a versatile model of cerebrovascular disorders such as small-scale stroke.
The recently developed technique of ultrafast third harmonic generation (THG) micro-spectroscopy is discussed. The approach is easily adapted to a standard laser scanning microscope and allows for two and three photon resonances to be identified in non-fluorescent unlabeled samples. This work provides nonlinear microscopists with a tool for further understanding the contrast and damage mechanisms they will encounter under nonlinear excitation. Here, we use THG micro-spectroscopy to investigate the nonlinear optical properties of hemoglobin over the spectral range of 770 -1000 nm with 100-fs duration, ~1-nJ energy laser pulses. We demonstrate the ability of this approach to distinguish different ligand binding states in physiological solutions of human hemoglobin.
Femtosecond lasers are very effective tools for three-dimensional micromachining of transparent materials. Nonlinear absorption of tightly focused femtosecond laser pulses allows energy to be deposited in a micrometer-sized volume in the bulk of the sample. If enough energy is deposited, localized changes in the material are produced (a change in refractive index, for example). These localized changes are the building blocks from which three-dimensional structures can be produced. With sufficiently tight focusing, the threshold for producing these changes can be achieved with pulse energies that are available directly from laser oscillators, offering greatly increased machining speeds and simpler, cheaper technology compared to using amplified lasers. In addition, the inter-pulse spacing from a laser oscillator is much shorter than the time required for energy deposited by one pulse to diffuse out of the focal volume. As a result, irradiation with multiple pluses on one spot in the sample leads to an accumulation of heat around the focal region. This localized heating provides another mechanism by which material properties can be altered. We demonstrate the three-dimensional fabrication of optical waveguides and microfluidic channels using pulse energies of only a few nanojoules to tens of nanojoules.
The study of neurovascular diseases such as vascular dementia and stroke require novel models of targeted vascular disruption in the brain. We describe a model of microvascular disruption in rat neocortex that uses ultrashort laser pulses to induce localized injury to specific targeted microvessels and uses two-photon microscopy to monitor and guide the photodisruption process. In our method, a train of high-intensity, 100-fs laser pulses is tightly focused into the lumen of a blood vessel within the upper 500 μm of cortex. Photodisruption induced by these laser pulses creates injury to a single vessel located at the focus of the laser, leaving the surrounding tissue intact. This photodisruption results in three modalities of localized vascular injury. At low power, blood plasma extravasation can be induced. The vessel itself remains intact, while serum is extravasated into the intercellular space. Localized ischemia caused by an intravascular clot results when the photodisruption leads to a brief disturbance of the vascular walls that initiates an endogenous clotting cascade. The formation of a localized thrombus stops the blood flow at the location of the photodisruption. A hemorrhage, defined as a large extravasation of blood including plasma and red blood cells, results when higher laser power is used. The targeted vessel does not remain intact.
Photothrombotic microstrokes are produced in rat cortex by 532-nm single-photon optical excitation of an intravenously injected photosensitizer, rose bengal. The dynamics of blood flow and clot formation in the cortical vasculature are observed using two-photon laser scanning microscopy of an intravenously injected fluorescent dye. Flowing and clotted vessels are clearly distinguishable in both large and small vessels, down to individual capillaries, using this technique. We find that by tightly focusing the laser light used to excite the photosensitizer, clots can be formed in individual blood vessels without affecting neighboring vessels tens of micrometers away. We observe many changes in blood flow as a result of localized clot formation, including upstream vascular dilation, clot clearing, i.e. recanalization, and complete reversal of blood flow direction downstream.
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