Extended Data Fig. 1: Characterization of the LMI fluorescence microscope for transcranial whole-cortex imaging at single plaque resolution.
From: Multiscale optical and optoacoustic imaging of amyloid-β deposits in mice
a, LMI image of fluorescent beads (1-5 μm-diameter) on a microscope slide; Repetition n = 3. b, Comparison of magnified views of wide-field (WF) and LMI images in the red boxed regions together with their signal profiles along the corresponding green lines. The Gaussian fitted full-width-at-half-maximum (FWHM) of the bead in region #1 is 18.2 μm and 8.3 μm for the WF and LMI images, respectively. The corresponding values for region #2 are 16.4 μm (WF) and 7.1 μm (LMI); For region #3: 18.9 μm (WF) and 7.4 μm (LMI); For region #4: 27.0 μm (WF) and 11.8 μm (LMI). An averaged FWHM of 20 different beads across the field-of-view (FOV) was calculated as 7.7 ± 1.4 μm (mean ± SD). c–e, The horizontal and vertical intensity profiles of one of the optical foci in the excitation illumination pattern measured with a beam profiler (SP628, Ophir Optronics). The signal intensity (represented as mean ± SD) was normalized and fitted by Gaussian function. The spot sizes along the horizontal and vertical directions were measured at 14 μm and 11 μm, respectively; Repetition n = 3. f, Curve of the spot size along the light path (z-axis). The depth-of-focus for excitation was larger than 1 mm within a spot size of 20 μm. LMI - large-field multifocal illumination. g–i, Dynamic contrast-enhanced LMI fluorescence microscopy reveals different probe dynamics in cerebral and calvarian blood vessels. To showcase the performance of the LMI fluorescence microscopy method, time-lapse imaging of the bolus passage after intravenous tail-vein injections of Cy5.5 solution was performed. (g) Cranial and intracranial vessel network shown in maximum intensity projection (MIP) over multiple image frames; (h) Analysis of perfusion dynamics (time to peak post dye injection) allows to discriminate between cerebral (fast time to peak) and calvarian (slower time to peak) vessels. As expected, the LMI approach exhibits excellent contrast and spatial resolution through the intact skull while also accurately resolving the perfusion process in the deeply embedded cerebral microvasculature. Major cerebral vessels such as anterior cerebral artery (ACA) and middle cerebral artery (MCA) were pronounced at an early stage followed by venous vessels, such as inferior cerebral vein (ICV) and superior sagittal sinus (SSS). (i) LMI image acquired at 30 min post Cy5.5 fluorescent dye injection, showing the probe retention in the suture areas. LMI - large-field multifocal illumination.
