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S. Toyouchi et al.
In Fig. 7.4b, a fluorescence image of an Alexa647-labeled AgNW under widefield excitation at 632.8 nm is shown. The fluorescence spots appear most clearly
as satellites along the AgNW, which also exhibit significant background emission
(Fig. 7.4b). The large background obscured the single-molecule emission so that
it had to be assumed that each fluorescent spot was due to a single molecule at
its centroid. A time-averaged emission spectrum from an Alexa647-bound AgNW
clearly matches that of Alexa647 in buffer solution (Fig. 7.4c). In contrast, the timeaveraged spectrum from an Alexa-free AgNW is broad, typical of metallic nanostructures, and attributed to Raman scattering and/or luminescence of the metal or other
impurities (Fig. 7.4c). This broad emission cannot be filtered from and is clearly
of a similar order of magnitude to the Alexa emission during wide-field excitation,
a fundamental problem for the precise measurement of fluorescence PSFs. Superresolution reconstructed images are shown in Fig. 7.4d–g for AgNWs of diameters
110 and 250 nm, respectively. The reconstruction imaging suggests the diameter of
the 110 nm nanowire is 205 nm and completely fails to represent the true form of
the nanowire with 250 nm diameter.
When the same experiment is done utilizing remote excitation, much clearer
images of the fluorescence PSFs emerge (Fig. 7.5). They appear as bright spots
clearly discernable against the weak background emission, demonstrating once again
the benefit of using remote excitation for reducing spectroscopic background. Importantly, some groups of spots clearly blink on and blink off again at the same time,
indicating that they are actually multi-spot emission PSFs due to a single Alexa647
molecule. Finite-difference time-domain (FDTD) calculations confirm that these
multi-spot PSFs are particularly sensitive to the fluorophores position and orientation of adsorption on the nanowire, and dependent further on the dimensions of the
nanowire itself. While this is problematic for accurate super-resolution reconstruction imaging of metallic nanowires, it highlights the need for rigorous checks to
ensure correct PSF fitting for molecules near metallic nanoparticles generally. With
such knowledge, the complex PSFs could be used predictively in far-field imaging
assays of nanoparticle size/shape, or of molecular adsorption site/orientation, given
some prior knowledge of the NP sample.
These experiments show once again the advantage of using remote spectroscopy
on metallic NWs for minimizing background emission/scattering. In this case, remote
excitation revealed a complex array of multi-spot emission PSFs for single molecules
that were otherwise obscured during direct excitation. The results also suggest
a fundamental paradox for the goal of taking super-resolution optical images of
metallic NPs. Normally the technique is used to assess the size of an unknown object
of interest. Unfortunately, for molecules near metallic NPs, it is not possible to know
the form of the PSF without some a priori knowledge of the NP dimensions. This
work has consequences also for counting (for instance) single-molecule catalytic
turn over events on metallic NPs using fluorescence imaging.
S. Toyouchi et al.
In Fig. 7.4b, a fluorescence image of an Alexa647-labeled AgNW under widefield excitation at 632.8 nm is shown. The fluorescence spots appear most clearly
as satellites along the AgNW, which also exhibit significant background emission
(Fig. 7.4b). The large background obscured the single-molecule emission so that
it had to be assumed that each fluorescent spot was due to a single molecule at
its centroid. A time-averaged emission spectrum from an Alexa647-bound AgNW
clearly matches that of Alexa647 in buffer solution (Fig. 7.4c). In contrast, the timeaveraged spectrum from an Alexa-free AgNW is broad, typical of metallic nanostructures, and attributed to Raman scattering and/or luminescence of the metal or other
impurities (Fig. 7.4c). This broad emission cannot be filtered from and is clearly
of a similar order of magnitude to the Alexa emission during wide-field excitation,
a fundamental problem for the precise measurement of fluorescence PSFs. Superresolution reconstructed images are shown in Fig. 7.4d–g for AgNWs of diameters
110 and 250 nm, respectively. The reconstruction imaging suggests the diameter of
the 110 nm nanowire is 205 nm and completely fails to represent the true form of
the nanowire with 250 nm diameter.
When the same experiment is done utilizing remote excitation, much clearer
images of the fluorescence PSFs emerge (Fig. 7.5). They appear as bright spots
clearly discernable against the weak background emission, demonstrating once again
the benefit of using remote excitation for reducing spectroscopic background. Importantly, some groups of spots clearly blink on and blink off again at the same time,
indicating that they are actually multi-spot emission PSFs due to a single Alexa647
molecule. Finite-difference time-domain (FDTD) calculations confirm that these
multi-spot PSFs are particularly sensitive to the fluorophores position and orientation of adsorption on the nanowire, and dependent further on the dimensions of the
nanowire itself. While this is problematic for accurate super-resolution reconstruction imaging of metallic nanowires, it highlights the need for rigorous checks to
ensure correct PSF fitting for molecules near metallic nanoparticles generally. With
such knowledge, the complex PSFs could be used predictively in far-field imaging
assays of nanoparticle size/shape, or of molecular adsorption site/orientation, given
some prior knowledge of the NP sample.
These experiments show once again the advantage of using remote spectroscopy
on metallic NWs for minimizing background emission/scattering. In this case, remote
excitation revealed a complex array of multi-spot emission PSFs for single molecules
that were otherwise obscured during direct excitation. The results also suggest
a fundamental paradox for the goal of taking super-resolution optical images of
metallic NPs. Normally the technique is used to assess the size of an unknown object
of interest. Unfortunately, for molecules near metallic NPs, it is not possible to know
the form of the PSF without some a priori knowledge of the NP dimensions. This
work has consequences also for counting (for instance) single-molecule catalytic
turn over events on metallic NPs using fluorescence imaging.