Abstract
Correlative light and electron microscopy exploits the advantages of optical methods, such as multicolour probes and their use in hydrated live biological samples, to locate functional units, which are then correlated with structural details that can be revealed by the superior resolution of electron microscopes. One difficulty is locating the area imaged by the electron beam in the much larger optical field of view. Multifunctional probes that can be imaged in both modalities and thus register the two images are required. Phosphor materials give cathodoluminescence (CL) optical emissions under electron excitation. Lanthanum phosphate containing thulium or terbium or europium emits narrow bands in the blue, green and red regions of the CL spectrum; they may be synthesised with very uniform-sized crystals in the 10- to 50-nm range. Such crystals can be imaged by CL in the electron microscope, at resolutions limited by the particle size, and with colour discrimination to identify different probes. These materials also give emissions in the optical microscope, by multiphoton excitation. They have been deposited on the surface of glioblastoma cells and imaged by CL. Gadolinium oxysulphide doped with terbium emits green photons by either ultraviolet or electron excitation. Sixty-nanometre crystals of this phosphor have been imaged in the atmospheric scanning electron microscope (JEOL ClairScope). This probe and microscope combination allow correlative imaging in hydrated samples. Phosphor probes should prove to be very useful in correlative light and electron microscopy, as fiducial markers to assist in image registration, and in high/super-resolution imaging studies.
Highlights
Most antibody localisation imaging techniques in biology have been based on optical microscopy, where the Rayleigh criterion d 1⁄4 0:6 λ = NA relates resolution (d) to the wavelength (λ) and numerical aperture (NA) of the objective lens
Another application where the bifunctional nature of phosphor probes can be useful is in correlative light and electron microscopy (CLEM) [1, 27, 4, 11] where ultrastructural information is obtained by electron microscopes (EMs) after optical microscopy has obtained the lower resolution data required for full description of the biological sample
We have found that commercial phosphor samples optimised for light microscopy are too weakly emitting in the electron beam and have high shape factors
Summary
Many protocols are available that have been developed for use with particles such as quantum dots [9] that rely on silanisation to coat the particle surface with, e.g. aminopropyl triethoxysilane as shown on the left, conjugation of the bioprobe with a carboxyl-to-amine cross-linking agent such as ethyl (dimethylaminopropyl) carbodiimide Another application where the bifunctional nature of phosphor probes can be useful is in correlative light and electron microscopy (CLEM) [1, 27, 4, 11] where ultrastructural information is obtained by EM after optical microscopy has obtained the lower resolution data required for full description of the biological sample. With the fluorescence microscope mounted above the dish, using a water dipping lens, before switching to the inverted SEM mode for correlative imaging of areas of interest in the sample on the silicon nitride film
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