Label-free, multi-scale imaging of ex-vivo mouse brain using spatial light interference microscopy

Abstract

Brain connectivity spans over broad spatial scales, from nanometers to centimeters. In order to understand the brain at multi-scale, the neural network in wide-field has been visualized in detail by taking advantage of light microscopy. However, the process of staining or addition of fluorescent tags is commonly required, and the image contrast is insufficient for delineation of cytoarchitecture. To overcome this barrier, we use spatial light interference microscopy to investigate brain structure with high-resolution, sub-nanometer pathlength sensitivity without the use of exogenous contrast agents. Combining wide-field imaging and a mosaic algorithm developed in-house, we show the detailed architecture of cells and myelin, within coronal olfactory bulb and cortical sections, and from sagittal sections of the hippocampus and cerebellum. Our technique is well suited to identify laminar characteristics of fiber tract orientation within white matter, e.g. the corpus callosum. To further improve the macro-scale contrast of anatomical structures, and to better differentiate axons and dendrites from cell bodies, we mapped the tissue in terms of its scattering property. Based on our results, we anticipate that spatial light interference microscopy can potentially provide multiscale and multicontrast perspectives of gross and microscopic brain anatomy.

Figure 1

(a) The schematics of the spatial light interference microscope (SLIM). SLIM combines conventional phase contrast microscopy (left) and a SLIM module (right). The SLIM module consists of a 4f lens system, and spatial light modulator (SLM). The SLM is for phase modulation which is required for phase extraction. The yellow beam represents the unscattered light and the red beam presents the scattered light. (b) The four successive intensity images are taken when the phase of the unscattered beam is shifted by the SLM. The phase is shifted in increments of π/2 rad. (c) The SLIM image obtained by combining the four intensity images. (d) Wide-field SLIM image obtained by using an automatic wide-field acquisition and mosaic algorithm. There is 8% overlap between adjacent images.

Figure 2

SLIM images of coronal (A,B) and sagittal (C) sections of the mouse brain at different spatial scales. The location of the brain being imaged is indicated by the white boxed inserts in A, B, and C. The location of the cross-section is depicted in the 3D atlas on the right. The red boxed insets include: isocortex (a), anterior commissure (aco,b), olfactory bulb (OB,c), Caudoputamen (CP,d), corpus callosum (cc, e and f), Hippocampus (HP, g), Thalamus (TH, h), Cerebellum (CB, i) and Choroid Plexus (chpl, j) at increasing levels of magnification. Row 1 scale bar = 100 μm, row 2 scale bar = 20 μm, and row 3 scale bar = 3 μm. The dotted black line drawn from left to right across each figure in row 3 is plotted to show the phase distribution and variation across the region imaged in row 4.

Figure 3

Histological coronal (A,B) and sagittal (C) section images of the mouse brain stained by Nissl-LFB (a-e), and Holzer (f-j) stains at different spatial scales. The location of the brain being imaged is indicated by the boxed inserts in A, B, and C. The location of the cross-section is depicted in the 3D atlas on the right. The red boxed insets include isocortex (a), anterior commissure (aco,b), olfactory bulb (OB,c), Caudoputamen (CP,d), corpus callosum (cc, e and f), Hippocampus (HP, g), Thalamus (TH, h), Cerebellum (CB, i) and Choroid Plexus (chpl, j) at increasing levels of magnification. Row 1 scale bar = 100 μm, row 2 scale bar = 20 μm, and row 3 scale bar = 3 μm. The dotted black line drawn from left to right across each figure in row 3 is plotted in row 4. Similar to SLIM contrast, the regions where cell bodies are densely packed have relatively lower Nissl-FLB and higher Holzer stain values in plot.

Figure 4

Laminar structure of isocortex of ex-vivo mouse, somatosensory cortex (A and B) and motor cortex (C and D) taken by SLIM (A and C) and stained with Nissl-LFB stain (B and D). Magnified images of layers I, II/III, IV, V, VIa and VIb: (a) molecular, (b) external granular and pyramidal, (c) internal granular, (d) internal pyramidal, (e), (f) multiform layer are also shown in the panels on the right. Magnified SLIM and Nissl stained images of Motor cortex boxed inserts (g), (h), (i), (j), (k) and (l) that correspond to Motor cortex layers I, II/III, V, VIa, VIb and WM in (C) are shown on the right. The layers are differentiated by the density and the distribution of the cells and by the types of cell present. WM, white matter.

Figure 5. Anatomical structure of olfactory bulb (OB).

(A) Laminar structure of OB taken by SLIM. (B) Nissl-LFB stained image corresponding to (A). (C) Enlarged SLIM images of OB layers which correspond to each layer outlined in (A). (D) Magnified topology of cells indicated by the white boxes in (C). (E) Enlarged Nissl-LFB stained images of OB layers which correspond to each layer outlined in (B). (F) Magnified topology of cells indicated by the black boxes in (E). GCL, granule cell layers; IPL, internal plexiform layer; MCL, Mitral cell layer; EPL, external plexiform layer; GL, glomerular layer; ONL, olfactory nerve layer, SEZ, subependymal zone.

Figure 6. SLIM images of sagittal sections of the mouse brain.

The location of the brain being imaged is indicated by the boxed inserts (a), (b) and (c), and the location of the cross-section is depicted in the mouse brain 3D atlas. Regions (a) hippocampus, (b) cerebellum and (c) choroid plexus are shown at increasing magnification. The green box in (a) shows the area of the dentate gyrus. The red box in (b) shows a cerebellum lobule. The layered structure for each region is analyzed in terms of phase value. The red arrows in (c) are pointing to stromal capillaries within the choroid plexus.

Figure 7

(a) Flow chart for orientation extraction from the corpus callosum (cc). See text for more details. (b) Comparison of the cc fiber orientation angles in coronal and sagittal sections. To estimate the 2D orientation angle, we applied Fourier transform (FT) and Radon transform (RT), and the orientation angle is estimated from RT generated intensity profiles.

Figure 8. Myeloarchitecture of corpus callosum (cc).

The orientation angle of the myelin fibers was calculated for several regions (a–i) and analyzed by orientation angle histograms in polar coordinate plots. In the histograms, the frequency distribution, as a percent of the fibers, with respect to the orientation angle are displayed. In the middle of the cc, most of fibers (80%) are aligned in parallel (f) and almost no cell bodies are shown, whereas, some cell bodies are distributed in other region of cc. The alignment of the fiber becomes irregular as it nears the cingulum bundle (c,h) and at the fiber terminal (a,b,i).

Figure 9. Comparison between SLIM and mean free path for the regions of the olfactory bulb (OB, row 1), corpus callosum (cc, row 2) and hippocampus (HP, row 3).

(a) The SLIM image and the corresponding (b) mean free path map. Mean free path maps (b) of the OB, cc and HP laminar structure match the anatomical structure shown in the corresponding SLIM images (a), but with macroscopic contrast. (c) The phase and mean free path for the area marked by the dotted line drawn from top to bottom are analyzed and plotted. (d,e) Comparison between the phase and mean free path for the regions indicated by the black and white boxes in (a) and (b), respectively using box plots. The anatomical boundaries of tissue are more clearly identifiable by the mean free path (e) than phase value (d).