Cardiac Light-Sheet Fluorescent Microscopy for Multi-Scale and Rapid Imaging of Architecture and Function

Abstract

Light Sheet Fluorescence Microscopy (LSFM) enables multi-dimensional and multi-scale imaging via illuminating specimens with a separate thin sheet of laser. It allows rapid plane illumination for reduced photo-damage and superior axial resolution and contrast. We hereby demonstrate cardiac LSFM (c-LSFM) imaging to assess the functional architecture of zebrafish embryos with a retrospective cardiac synchronization algorithm for four-dimensional reconstruction (3-D space + time). By combining our approach with tissue clearing techniques, we reveal the entire cardiac structures and hypertrabeculation of adult zebrafish hearts in response to doxorubicin treatment. By integrating the resolution enhancement technique with c-LSFM to increase the resolving power under a large field-of-view, we demonstrate the use of low power objective to resolve the entire architecture of large-scale neonatal mouse hearts, revealing the helical orientation of individual myocardial fibers. Therefore, our c-LSFM imaging approach provides multi-scale visualization of architecture and function to drive cardiovascular research with translational implication in congenital heart diseases.

Figure 1. Implementation of cardiac Light-Sheet Fluorescence Microscopy (LSFM).

(a) The optical setting of LSFM. A laser beam (purple) is collected and focused by the beam expander to optimize the beam size. A cylindrical lens (CL) converts the laser beam to a sheet of laser light to illuminate a thin layer of the sample. The sample is mounted at the intersection of the illumination lens (IL) and detection lens (DL). The illuminated 2-D layer (fluorescent detection in green) is captured by the high-frame rate CMOS camera. The illumination axis is orthogonal to the detection axis, and the illumination optics is designed to illuminate a very thin volume around the focal plane of the detection objective. The configurations of light-sheet illumination and fluorescence detection are highly tunable to accommodate for various heart samples. (b) The plane fluorescent images at different axial (z) depths are sequentially captured by the camera (c,d). For non-transparent fetal mouse hearts, multi-view (MV) techniques are applied to rotate the samples for multi-view imaging, followed by registering and fusing these views into a 3-D cardiac architecture. An iterative deconvolution technique is applied to the blurred sequence for high resolution. (e) A digitally reconstructed heart is accomplished by stacking the deconvolved images.

Figure 2. Light-sheet profiles for reconstructing cardiac architecture.

(a) The axial confinement of the light-sheet (LS) was used for sectioning the (i) embryonic zebrafish, (ii) adult zebrafish, and (iii) neonatal mouse hearts. The small aperture of the slit reduced the beam width to render the waist of laser sheet less focused and wider (shown in the inserts). LS: light sheets. (b) The changes in Rayleigh Range corresponded to the area available for light-sheet sectioning. The double-headed arrow line indicates the Rayleigh range (confocal region), in which the light-sheet is considered to be uniform. The scale bars are 100 μm in length for the sub-images in (i), (ii) and (iii). (c) Imaging a 400 nm fluorescent bead (sub-resolution point source) was compared with the (i) 5μm light-sheet (LS) detected by the 20X/0.5 detection objective (DO), (ii) 9 μm LS by 10X/0.3 DO, (iii) 18 μm LS by 4X/0.13 DO and (iv) 18 μm LS by 4X/0.13 DO, with resolution enhancement applied. The FWHM extent of image blurring from the point source in the x-y, x-z and y-z plane reflects the lateral and axial resolution.

Figure 3. 4-D synchronized images to quantify global longitudinal strain rates and volume change of the ventricle at 100 hours post fertilization (hpf).

(a) Changes in global longitudinal strain rates were quantified during the entire cardiac cycle. (b) The ventricular volume was measured in terms of EDV at 95.4 × 10⁵ μm³ and ESV at 1.5 × 10⁵ μm³, respectively. (c) LSFM images captured the zebrafish hearts in the x-y, x-z, and y-z planes during the cardiac cycle. (d) 4-D synchronized LSFM-acquired images revealed endocardial trabeculation in the x-y, x-z and y-z plane during the cardiac cycle. (e,f) 4-D zebrafish cardiac motion was captured during ventricular diastole and systole. A: Atrium, V: Ventricle. Scale bar: 50 μm

Figure 4. Rapid 3-D images to recapitulate trabeculated network in response to doxorubicin (Dox) treatment in the adult zebrafish.

(a) A representative wild-type zebrafish heart at 120 dpf. The coronal, sagittal and transverse planes of the heart displayed a compact trabecular network. The atrioventricular valve (AV) was identified (yellow arrows). Scale bars are 200 μm in length. In the rightmost column, a 3-D rendering of the “digital heart” was reconstructed by stacking 500 slices of plane images in volume. The 3-D structure of the “digital heart” can be assessed by arbitrary cropping. (b) A representative Dox-injected zebrafish heart at 120 dpf. The endocardial cavity appeared enlarged and the trabecular network was accentuated. (c) The quantified volume ratios of the myocardium (left) and the ventricle cavity (right) in the whole heart.

Figure 5. Cardiac LSFM (c-LSFM) imaging of a 1-day neonate mouse heart with enhanced cellular resolution.

(a) The coronal, sagittal, and transverse planes at different depths uncover 3-D architecture. Scale bars are 1 mm in length in all of the sub-graphs. (b) The boxes were cropped from the volume rendering of the reconstructed “digital heart” to reveal the endocardial architecture. (c) The cardiac architecture is compared with the (i) 18 μm light-sheet and 4X/0.13 objective, (ii) 4X/0.13 resolution enhanced images, and (iii) 9 μm light-sheet and 10X/0.3 objective. Magnification from left to right reveals the field of view, lateral, and axial resolving power, followed by the volumetric rendering effects of 3 configurations. Myocardial orientation was resolved in detail in the resolution-enhanced c-LSFM group. All scale bars are 500 μm, except for 50 μm in the rightmost column.

Figure 6. High resolution architecture of neonatal mouse hearts.

(a) 3-D LSFM revealed the distinct helical organization of individual cardiomyocyte fibers from the right ventricular wall to septum to left ventricular walls (zones 1, 2, and 3), providing insights into the mechanics of ventricular contraction in RV vs. LV. Endocardial structure of the left atrial appendage revealed the muscular ridge and muscular trabeculation (zone 4). The yellow curved arrows indicate the orientation of cardiomyocyte fibers. (b) Ultrastructure in the RV (zone 1) and LV cavity (zone 2) unravel trabeculation/papillary muscle (zone 1). LV: left ventricle; RV: right ventricle; LA: left atrium.