Cathodoluminescence imaging for identifying uptaken fluorescence materials in Kupffer cells using scanning electron microscopy

Abstract

Cathodoluminescence (CL) is the light that is emitted from a material irradiated by an electron beam. The present study was undertaken to show the applicability to biological studies of a scanning electron microscope (SEM) equipped with a high-sensitive cathodoluminescence detection system. For this purpose, we injected inorganic fluorescent powders (P43) suspended in phosphate buffered saline into rat blood circulation, fixed the animals with glutaraldehyde within a day, and observed the hepatic tissues with a SEM. Our instrument enabled the simultaneous collection of both secondary electron (SE) and CL images of these tissues. Backscattered electron (BSE) images of the same portion were also able to be obtained with this microscope. SE and BSE images clearly showed the three-dimensional structure of the hepatic tissues including hepatocytes, Kupffer cells, Ito cells, and sinusoidal epithelial cells, while CL images visualized cathodoluminescence signals emitted from P43 as bright spots. We observed non-coated tissues under a low-vacuum condition and metal-coated tissues under a high-vacuum condition, and found that the high-vacuum observation of metal-coated tissues provided high quality CL images of P43 in the Kupffer cells. The superimposition of the CL images onto the corresponding SE or BSE images revealed that bright spots in the CL images were produced by the fluorescent powders uptaken by Kupffer cells. These findings indicate that the detection of CL as well as SE or BSE signals by SEM all provide us with useful information on the distribution of fluorescent tracers in tissues and cells in three-dimensional images.

Correlative near-infrared light and cathodoluminescence microscopy using Y2O3:Ln, Yb (Ln = Tm, Er) nanophosphors for multiscale, multicolour bioimaging

“…Although various imaging probes have been designed for NIR imaging, including fluorescent proteins 24 25 , carbon nanotubes 26 , quantum dots 27 28 , and rare-earth complexes 29 , only a few candidates enable selection of the excitation and emission light in the NIR wavelength range. Some probes have also been studied for CL bioimaging, including fluorescent proteins 30 , inorganic fluorescent powders 31 , rare-earth complexes 32 , quantum dots 33 , and nanodiamonds 10 34 ; in particular, quantum dots and nanodiamonds are promising imaging probes that combine different microscopy observations with photoluminescence under UV-visible light excitation and cathodoluminescence under accelerated electron excitation. The multimodalities of our rare-earth NPs will allow their use in multiscale correlative NIR excitation–NIR emission and CL imaging, which can be used to trace the region of interest at different scales, ranging from cellular dynamics to molecular distributions.…”

Multicolour correlative imaging using phosphor probes

“…An alternative methodology that combines the resolution of electron microscopes with the wavelength discrimination of optical detection is cathodoluminescence (CL) [13]. This employs phosphor particles as probes and detects the photons emitted under electron bombardment.…”

Multi‐Color Imaging of Fluorescent Nanodiamonds in Living HeLa Cells Using Direct Electron‐Beam Excitation

“…To improve the image contrast, fluorescent materials that yield a strong CL emission can be used to label specific bio-molecules. CL imaging using quantum dots, organic materials, [8,19] and phosphors composed of metal oxides, [15,20,21] have been reported.…”