Ultraviolet photoacoustic microscopy with tissue clearing for high-contrast histological imaging

Abstract

Ultraviolet photoacoustic microscopy (UV-PAM) has been investigated to provide label-free and registration-free volumetric histological images for whole organs, offering new insights into complex biological organs. However, because of the high UV absorption of lipids and pigments in tissue, UV-PAM suffers from low image contrast and shallow image depth, hindering its capability for revealing various microstructures in organs. To improve the UV-PAM imaging contrast and imaging depth, here we propose to implement a state-of-the-art optical clearing technique, CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis), to wash out the lipids and pigments from tissues. Our results show that the UV-PAM imaging contrast and quality can be significantly improved after tissue clearing. With the cleared tissue, multilayers of cell nuclei can also be extracted from time-resolved PA signals. Tissue clearing-enhanced UV-PAM can provide fine details for organ imaging.

Keywords: Label-free imaging; Photoacoustic microscopy; Tissue clearing; Volumetric imaging.

Fig. 1

Two protocols of CUBIC. (a) Fast clearing protocol for mouse kidneys and hearts. (b) Simple immersion protocol for mouse brains.

Fig. 2

Schematic of the UV-PAM system. DAQ, data acquisition card.

Fig. 3

Mouse kidney, heart, and brain before and after clearing.

Fig. 4

Resolution measurement of the UV-PAM system. (a) A UV-PAM image of a gold nanoparticle. (b) and (c) Plots showing the lateral resolution and axial resolution measurements, respectively.

Fig. 5

A comparison of a mouse kidney slice without and with tissue clearing. (a) and (b) Photographs of a kidney slice before and after clearing, respectively. (c) A UV-PAM image of a kidney slice before clearing. (d)–(f) Zoomed-in images of the corresponding regions marked in (c), with scale bars: 100 µm. (g) A UV-PAM image of the same kidney slice after clearing. (h)–(j) Zoomed-in images of the corresponding regions marked in (g).

Fig. 6

UV-PAM imaging of kidney slice sectioned from a cleared kidney. (a) A photograph of a kidney slice. (b) A UV-PAM image of the kidney slice. (c)–(g) Zoomed-in images of the corresponding marked regions in (b), with scale bars: 100 µm. A kidney slice is stained by H&E and shown at the corner of (c)–(e) for comparison.

Fig. 7

A comparison of a mouse heart slice without and with tissue clearing. (a) and (b) A UV-PAM image and photograph of a formalin-fixed mouse heart slice without clearing, respectively. (c) and (d) A photograph and UV-PAM image of a heart slice sectioned from a cleared heart, respectively. (e)–(h) Zoomed-in images of the corresponding marked regions in (a), with scale bars: 100 µm. (i)–(l) Zoom-in images of the corresponding marked regions in (d).

Fig. 8

A comparison of a mouse brain slice without and with tissue clearing. (a) A slice sectioned from a formalin-fixed mouse brain without clearing. (b) A slice sectioned from an intact and cleared mouse brain. (c) and (k) UV-PAM images of the corresponding marked regions in (a). (d)–(f) and (l)–(n) Zoomed-in images of corresponding marked regions in (c) and (k), respectively, with scale bars: 100 µm. (g) and (o) UV-PAM images of the corresponding marked regions in (b). (h)–(j) and (p)–(r) Zoomed-in images of corresponding marked regions in (g) and (o), respectively.

Fig. 9

Depth-resolved image comparison of the mouse brain slice with and without clearing. (a) and (b) Image stacks and depth-encoded image of the mouse brain slice with clearing, respectively. Scale bar: 100 µm. (c) and (d) Depth-encoded image and image stacks of the mouse brain slice without clearing, respectively.