Abstract
We report the use of fluorescent nanodiamonds (FNDs) as a photostable fluorescent probe for high resolution saturated excitation (SAX) microscopy. We confirmed that FNDs show a nonlinear fluorescence response under saturated excitation conditions generated by intense excitation light. Using FNDs, we quantified the spatial resolution improvement inherent in SAX microscopy, and experimentally demonstrated the scalability of the spatial resolution of SAX microscopy. The photostability of the FNDs allowed us to perform nanoparticle imaging of a multicolor-stained macrophage cell with a spatial resolution beyond the diffraction limit.
Highlights
In optical microscopy, the spatial resolution is restricted by the diffraction limit of light
We report the use of fluorescent nanodiamonds (FNDs) as a photostable fluorescent probe for high resolution saturated excitation (SAX) microscopy
We confirmed that FNDs show a nonlinear fluorescence response under saturated excitation conditions generated by intense excitation light
Summary
The spatial resolution is restricted by the diffraction limit of light. We have reported the use of saturated excitation (SAX) for improving the spatial resolution of confocal microscopy [12,13]. SAX microscopy exploits the nonlinear fluorescence response which occurs when fluorescence molecule excited states become saturated. 2. Nonlinear fluorescence response of FNDs We calculated fluorescence signal intensities using the electronic state model shown in Fig. 1 b) and the rate equations for the population probability of each level. Nonlinear fluorescence response of FNDs We calculated fluorescence signal intensities using the electronic state model shown in Fig. 1 b) and the rate equations for the population probability of each level In this calculation, we treated 1E and 1A as a single level [20] because the lifetime of 1E (~30 ns) is almost 10 times shorter than the time-scale for non-radiative decay from 1A to 3A (~300 ns). The calculated relationship between demodulated fluorescence from the FNDs and the excitation intensity
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