Fluorescent Nanodiamonds

10Nanoscopic Imaging

We have learned a great deal so far about what fluorescent nanodiamonds (FNDs) can do, for example, from detecting a single biomolecule (Chapter 7) to imaging a whole organism (Chapter 9). Now the question is how. How to make all these wonders of science? Specifically, in a tiny world of nanoparticles, what tools are necessary to study FNDs? What do we have to know in order to make the best use of these tools?

To “see” FNDs in action, researchers have been relying on fluorescence microscopy, which has made profound advance throughout past decades in enhancing our understanding of molecular and cellular biology. As powerful as it is, however, fluorescence microscopy is limited by the diffraction of light to elucidate detailed structure of the organelles in cells. Considering a microscopic object to consist of diffraction gratings, Ernst Abbe concluded in 1873 that the resolution limit of a microscope is one half the wavelength of the light used for illumination [1],

(10.1)

where d_min is the minimum resolvable distance, λ is the wavelength of the light, and α is the half aperture angle of the microscope’s objective [2]. For green light of 532 nm in wavelength as an example, the Abbe limit is d_min = λ/2 = 266 nm (or 0.266 μm), which is small compared to most biological cells (~10 μm), but large compared to viruses (~100 nm), proteins (~5 nm), and small biomolecules ...