Abstract
Particles of diamond in the 5–100 nm size range, known as nanodiamond (ND), have shown promise as robust fluorophores for optical imaging. We demonstrate here that, due to their photostability, they are not only suitable for two-photon imaging, but also allow significant resolution enhancement when combined with computational super-resolution techniques. We observe a resolution of 42.5 nm when processing two-photon images with the Super-Resolution Radial Fluctuations algorithm. We show manipulation of the point-spread function of the microscope using adaptive optics. This demonstrates how the photostability of ND can also be of use when characterizing adaptive optics technologies or testing the resilience of super-resolution or aberration correction algorithms.
Highlights
The biocompatibility of diamond [1], combined with the ability to make nanoscopic particles of less than 100 nm diameter, has led to research into the use of nanodiamond (ND) for a variety of biological applications [2], including drug delivery [3,4] and use as a fluorescent marker for microscopy [5,6]
We further demonstrate two-photon excitation (TPE) imaging of NV centres in ND and show how ND is suited to computational super-resolution techniques, such as that enabled by the superresolution radial fluctuations (SRRF) [32] approach
The final lens before the optical fibre was chosen to set an effective pinhole size of 1 Airy unit. This may give a slight increase in contrast in two-photon imaging mode, the effect of the pinhole will be dominated by the inherent optical sectioning provided by the TPE process
Summary
The biocompatibility of diamond [1], combined with the ability to make nanoscopic particles of less than 100 nm diameter, has led to research into the use of nanodiamond (ND) for a variety of biological applications [2], including drug delivery [3,4] and use as a fluorescent marker for microscopy [5,6]. Neighbouring carbon atoms are replaced by a nitrogen atom and a vacant space in the crystal lattice This 2 defect is most commonly found in a negatively charged state, referred to as NV−, and is efficiently imaged with a single photon excitation of 532 nm that results in emission over a wide wavelength band from 637 nm to approximately 800 nm. While multi-photon microscopy is inherently suited to imaging in optically aberrating samples, it benefits from adaptive optical image correction [35,36,37] With this in mind, we demonstrate adaptive optic control of the excitation focal volume and the resulting changes in the images obtained from single ND crystals. By combining adaptive-optics and computational super-resolution imaging, we show that ND is a superb fluorophore with significant potential for applications in which efficient correction of aberrations deep within an aberrating medium (such as tissue) is essential
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