Recently, various in vivo optical brain imaging techniques have been developed. Here, the authors introduce some of these systems and their application to in vivo brain imaging in a mouse model of Alzheimer’s disease (AD). Two-photon laser scanning microscopy (TPLSM) is specialized for fluorescence imaging in deep tissue with sub-micron resolution and has scanning capabilities, intrinsic optical signal imaging detects the relative changes in oxy- and deoxy-hemoglobin concentration following sensory stimulation and voltage-sensitive dye imaging can directly image the changes of the membrane potential after neural stimulation.
TopI. Two-Photon Laser Scanning Microscopy
The concept of two-photon (or multi-photon) excitation of fluorescence was first introduced by Nobel laureate Maria Göppert-Mayer in 1963 (Peticolas, Goldsborough, & Rieckhoff, 1963). The first laser scanning microscope that used this concept was reported in 1990 (Denk, Strickler & Webb, 1990). Since then, TPLSM has been used for in vivo tissue imaging, particularly in brain imaging. This device utilizes the two-photon effect; a fluorescent probe is excited not by a single photon of visible light, but by nearly simultaneous two photons of lower-energy (infrared). Fluorescently labeled specimens are illuminated by a Ti:sapphire femto-second pulsed laser of near infrared light, and emit the specified emission wavelength light.
Because TPLSM uses a long-wavelength light source (over 700 nm to 1,000 nm) and because light with longer wavelengths can penetrate deeper into samples, deep tissue imaging (theoretically up to 1 mm) becomes possible. Furthermore, simultaneous photon absorption significantly reduces photo-bleach and/or photo-damage in peripheral planes that are not in focus. These features of TPLSM allow in vivo fluorescence imaging with high temporal and spatial resolution.
1) Cranial Window and Structural Imaging
For in vivo brain imaging by the above-mentioned TPLSM, a cranial window is required because the skull is not sufficiently transparent. The open-skull and thinned-skull cranial window methods are primarily used with various minor modifications. For an open-skull window, 2 × 2 mm or 3 × 3 mm craniotomy on the region of interest is performed, and the cortex is covered with 1-1.5% agarose and a glass cover slip. The margin is secured by cyanoacrylate glue, and dental cement is applied around the cover slip to provide a well for a water-immersion lens. In a thinned-skull cranial window, the region of interest is gently thinned with a high-speed hand-drill, typically to a thickness at which cerebral blood flow can be clearly visualized when water is applied. Once the skull is thinned, dental cement is applied to make a well for water immersion of the lens.
The vascular structure is imaged through the cranial window. To better visualize the vessel, a fluorescent probe tagged with dextran is injected into the tail vein. Dextran tagging prevents the dye from crossing the vessel wall. As seen in Figure 1(a), fluorescein isothiocyanate (FITC)-dextran (2 MDa) was injected, and the cerebral vasculature images were obtained through the cranial window.
Figure 1. (a) Cerebral blood vessel morphology as detected by TPLSM. Maximum projection image (left) and z-axis transparent projection (right), scale bar = 50 μm. (b) Amyloid plaques and perivascular amyloid deposition (arrow) detection using thioflavin S (left). FITC-dextran was injected via the tail vein to visualize cerebral vessels. Prominent cerebral amyloid angiopathy and dense amyloid plaques were detected with methoxy-X04 (right). (c) Time-lapse line scanning of RBCs in cerebral capillaries. Each column represents a capillary, and black streaks represent RBCs. The bottom of each column shows the RBC velocity (left). Velocities were mapped into a capillary network (right) that displays direction as well as velocity. (d) Calcium staining with Oregon-green BAPTA 1-AM and detection of spontaneous neuronal calcium spikes.
Amyloid plaques are one of hallmarks of the AD brain, and they can be imaged and quantified through cranial windows with appropriate probes. Thioflavin S and methoxy-X04 are common probes used for amyloid plaque detection. Figure 1(b) shows thioflavin S imaging and methoxy-X04 imaging for amyloid plaque and cerebrovascular amyloid angiopathy in a mouse model of AD. Methoxy-X04 is more advantageous than thioflavin S because it crosses the blood brain barrier; therefore, it can be systemically administered (Nagayama, Zeng, Xiong, Fletcher, Masurkar et al., 2007).