Multi-photon excitation microscopy in intact animals.

Two-photon excitation fluorescence microscopy and backscattered-second harmonic generation microscopy permit the investigation of the subcellular events within living animals but numerous aspects of these experiments need to be optimized to overcome the traditional microscope geometry, motion and optical coupling to the subject. This report describes a stable system for supporting a living instrumented mouse or rabbit during endogenous reduced nicotinamide adenine dinucleotide and exogenous dye two-photon excitation fluorescence microscopy measurements, and backscattered-second harmonic generation microscopy measurements. The system was a modified inverted LSM510 microscope (Carl Zeiss, Inc., Thornwood, NY, U.S.A.) with a rotating periscope that converted the inverted scope to an upright format, with the objective located approximately, 15 cm from the centre of the microscope base, allowing easy placement of an instrumented animal. An Olympus 20x water immersion objective was optically coupled to the tissue, without a cover glass, via a saline bath or custom hydrated transparent gel. The instrumented animals were held on a specially designed holder that poised the animal under the objective as well as permitted different ventilation schemes to minimize motion. Using this approach, quality images were routinely collected in living animals from both the peripheral and body cavity organs. The remaining most significant issue for physiological studies using this approach is motion on the micrometre scale. Several strategies for motion compensation are described and discussed.

Whole-brain 3D perfusion MRI at 3.0 T using CASL with a separate labeling coil.

A variety of continuous and pulsed arterial spin labeling (ASL) perfusion MRI techniques have been demonstrated in recent years. One of the reasons these methods are still not routinely used is the limited extent of the imaging region. Of the ASL methods proposed to date, continuous ASL (CASL) with a separate labeling coil is particularly attractive for whole-brain studies at high fields. This approach can provide an increased signal-to-noise ratio (SNR) in perfusion images because there are no magnetization transfer (MT) effects, and lessen concerns regarding RF power deposition at high field because it uses a local labeling coil. In this work, we demonstrate CASL whole-brain quantitative perfusion imaging at 3.0 T using a combination of strategies: 3D volume acquisition, background tissue signal suppression, and a separate labeling coil. The results show that this approach can be used to acquire perfusion images in all brain regions with good sensitivity. Further, it is shown that the method can be performed safely on humans without exceeding the current RF power deposition limits. The current method can be extended to higher fields, and further improved by the use of multiple receiver coils and parallel imaging techniques to reduce scan time or provide increased resolution.