A proof-of-concept study for developing integrated two-photon microscopic and magnetic resonance imaging modality at ultrahigh field of 16.4 tesla

Abstract

Functional magnetic resonance imaging (fMRI) based on the blood oxygen level dependent (BOLD) contrast has gained a prominent position in neuroscience for imaging neuronal activity and studying effective brain connectivity under working state and functional connectivity at resting state. However, the fundamental questions in regards to fMRI technology: how the BOLD signal inferences the underlying microscopic neuronal activity and physiological changes and what is the ultimate specificity of fMRI for functional mapping of microcircuits, remain unanswered. The capability of simultaneous fMRI measurement and functional microscopic imaging in a live brain thus holds the key to link the microscopic and mesoscopic neural dynamics to the macroscopic brain activity at the central nervous system level. Here we report the first demonstration to integrate high-resolution two-photon fluorescence microscopy (TPM) with a 16.4 tesla MRI system, which proves the concept and feasibility for performing simultaneous high-resolution fMRI and TPM imaging at ultrahigh magnetic field.

Figure 1

Schematic of the MRI-compatible TPM imaging system. The laser scanning module (upper left corner) was located outside the 16.4T magnet room and contained inside a magnetic field shielding enclosure. The excitation laser beam was guided to the in-MRI imaging module inside the magnet by two sets of relay lenses (RL1A, RL1B, RL2A and RL2B) and mirrors (M). The in-MRI imaging module (enclosed by the blue dotted box) comprised an objective lens, a dichroic beam splitter, an optical mirror, a filter and two lenses (L1 and L2) which focused the two-photon excited fluorescence emission onto the entrance port of a 9 meter long fiber light guide. The exit port of the light guide was in contact with the front window of a GaAsP PMT housed inside the enclosure of the remote scanning module.

Figure 2

Resolution characterization of the long-distance remote TPM imaging system. (a) Two-photon fluorescence images of beads of 1 μm in diameter. Scale bar: 50 μm. (b) Plot across the diameter of a bead and its Gaussian fitting (blue line) to determine the FWHM values.

Figure 3

TPM imaging inside the 16.4T MRI magnet. (a) TPM images of microglia cells in a fixed CX3CR1 mouse brain (scale bar: 50 μm). (b–d) The cross-correlation peak value and peak positions of the time lapsed TPM images during which we performed MRI recording. The cross-correlation shows that the operation of MRI had no influence on TPM. (e) Cross-sectional plot of the process of microglia and its Gaussian fitting (blue line) to determine the FWHM values. (f) Ex vivo ultrahigh-resolution spin-echo multiple-slice MRI of the same fixed mouse brain (axial orientation, 117 μm × 117 μm in plane resolution, 1 mm slice thickness, and 2 signal averages) acquired at 16.4 T. (g) In vivo ultrahigh-resolution mouse brain image (102 μm × 102 μm in plane resolution, 0.5 mm slice thickness and 4 signal averages) acquired from a different mouse at 9.4T for comparison.