Correlative cathodoluminescence and near-infrared fluorescence imaging for bridging from nanometer to millimeter scale bioimaging.

Abstract

Correlative light and electron microscopy (CLEM) is one attractive method of observing biological specimens because it combines the advantages of both light microscopy (LM) and electron microscopy (EM). In LM, specimens are fully hydrated, and molecular species are distinguished based on the fluorescence colors of probes. EM provides both high-spatial-resolution images superior to those obtained with LM and ultrastructural information of cellular components. The combination of LM and EM gives much more information than either method alone, which helps us to analyze cellular function in more detail.We propose a Y2O3:Tm,Yb phosphor nanoparticle which allows upconversion luminescence (UCL) imaging with near-infrared (NIR) light excitation and cathodoluminescence (CL) imaging [1], where the light emission induced by an electron beam is called cathodoluminescence (CL). Due to electron beam excitation, the spatial resolution of CL microscopy is on the order of nanometers [2,3]. Upconversion is a process in which lower energy, longer wavelength excitation light is transduced to higher energy, shorter wavelength emission light. So far, in LM observation for CLEM, ultraviolet (UV) or visible light has been used for excitation. However, UV and visible light have limited ability to observe deep tissue regions due to absorption, scattering, and autofluorescence. On the other hand, NIR light does not suffer from these problems. Rare-earth-doped upconversion nanophosphors have been applied to biological imaging because of the advantages of NIR excitation [4].We investigated the UCL and CL spectra of Y2O3:Tm,Yb nanophosphors. Y2O3:Tm,Yb nanophosphors that emit visible and near-infrared UCL under 980nm irradiation and blue CL via electron beam excitation. To confirm bimodality of our nanophosphors, correlative UCL/CL images of the nanophosphors were obtained for the same region. The nanophosphors were poured onto a P doped Si substrate (Fig.1(a)) and were irradiated with 980 nm NIR CW laser light or an electron beam. Fig.1(b) shows the UCL image of the nanophosphors under 980 nm NIR CW laser irradiation, UCL spots were observed, but the individual nanophosphors in each spot were difficult to distinguish in the UCL image. On the other hand, the edges and the gap between the nanophosphors were clearly distinguished in the CL image (Fig.1(c)), showing that the spatial-resolution of CL imaging was enough higher than that of UCL image. We believe that upconversion phosphors of the type described here will allow the realization of new CLEM imaging techniques covering the nanometer to millimeter scale, i.e., the molecular to in vivo scale.jmicro;63/suppl_1/i29/DFU073F1F1DFU073F1Fig. 1.(a) SEM and correlative (b) UCL (intensity of 980 nm NIR CW laser 8 mW) and (c) CL images of Y2O3:Tm,Yb nanophosphors in same region (accelerating voltage 3 kV, exposure time 100 ms/pixel).