2 Interferometric Scattering (iSCAT) Microscopy and Related Techniques
43
2.4 iSCAT Showcase
2.4.1 Detection and Sensing of Nanoparticles
Since the debut of iSCAT in 2004, iSCAT and related techniques have revived interference and extinction microscopies in the context of detection and imaging of various
nanoparticles. In this section, we provide a brief overview of some of the exciting
application areas to which these methods have contributed.
2.4.1.1 Gold Nanoparticles
Gold nanoparticles in the colloidal form have a wide range of applications due to
key properties such as biocompatibility, inertness, ease of fabrication and possibility
for functionalization with different molecules. The recent developments of nanooptics, and in particular plasmonics, have brought about a strong drive for studying
and using these nanoparticles. Indeed, the first account of iSCAT was published on
the direct far-field imaging of single GNPs, to size as small as 5 nm. This was a
formidable task at the time as in dark-field imaging one begins to struggle to visualize gold colloids smaller than 40 nm. The GNPs, immobilized upon a coverslip,
were imaged when index-matched with oil [73] and also under ambient conditions
at the water–glass interface [113]—see Fig. 2.8a. The latter is especially important
as it begins to match biological conditions wherein ultra-small GNPs serve as useful super-resolution probes (discussed later). The sensitivity limit on the size of the
detectable GNPs in the early experiments was set by the speckle background that
results from slightest variations in the refractive index and topography of the underlying glass substrate.
To date, GNPs as small as 2 nm in diameter [145], smaller than a protein, have
been imaged and localized to a precision of 8 nm with short exposure times sufficient
to function as a scattering label—cementing iSCAT as a powerful means to image
and localize nanoscale colloids. At this point, it is perhaps interesting to mention that
we had actually started exploiting iSCAT already in 2001 when investigating gold
nanoparticles of diameter 100 nm [146]. In those studies, we detected individual gold
nanoparticles at the glass–air interface as dark spots in a scanning confocal reflection
measurement and were puzzled that particles which scattered well would actually
appear dark. Further studies then led to the appreciation of the role of interference
and the advent of iSCAT [73].
Given that iSCAT imaging intrinsically contains quantitative phase information
about the sample, proper interpretation of the interferometric scattering PSF allows
one to attain material and morphological features of the colloid. For example, calibration of the spherical colloid size from the extinction contrast has been demonstrated
[147], as well as the orientation of anisotropic ellipsoidal nanorods through polarized detection [148]. Moreover, the complete complex dielectric function of a single
43
2.4 iSCAT Showcase
2.4.1 Detection and Sensing of Nanoparticles
Since the debut of iSCAT in 2004, iSCAT and related techniques have revived interference and extinction microscopies in the context of detection and imaging of various
nanoparticles. In this section, we provide a brief overview of some of the exciting
application areas to which these methods have contributed.
2.4.1.1 Gold Nanoparticles
Gold nanoparticles in the colloidal form have a wide range of applications due to
key properties such as biocompatibility, inertness, ease of fabrication and possibility
for functionalization with different molecules. The recent developments of nanooptics, and in particular plasmonics, have brought about a strong drive for studying
and using these nanoparticles. Indeed, the first account of iSCAT was published on
the direct far-field imaging of single GNPs, to size as small as 5 nm. This was a
formidable task at the time as in dark-field imaging one begins to struggle to visualize gold colloids smaller than 40 nm. The GNPs, immobilized upon a coverslip,
were imaged when index-matched with oil [73] and also under ambient conditions
at the water–glass interface [113]—see Fig. 2.8a. The latter is especially important
as it begins to match biological conditions wherein ultra-small GNPs serve as useful super-resolution probes (discussed later). The sensitivity limit on the size of the
detectable GNPs in the early experiments was set by the speckle background that
results from slightest variations in the refractive index and topography of the underlying glass substrate.
To date, GNPs as small as 2 nm in diameter [145], smaller than a protein, have
been imaged and localized to a precision of 8 nm with short exposure times sufficient
to function as a scattering label—cementing iSCAT as a powerful means to image
and localize nanoscale colloids. At this point, it is perhaps interesting to mention that
we had actually started exploiting iSCAT already in 2001 when investigating gold
nanoparticles of diameter 100 nm [146]. In those studies, we detected individual gold
nanoparticles at the glass–air interface as dark spots in a scanning confocal reflection
measurement and were puzzled that particles which scattered well would actually
appear dark. Further studies then led to the appreciation of the role of interference
and the advent of iSCAT [73].
Given that iSCAT imaging intrinsically contains quantitative phase information
about the sample, proper interpretation of the interferometric scattering PSF allows
one to attain material and morphological features of the colloid. For example, calibration of the spherical colloid size from the extinction contrast has been demonstrated
[147], as well as the orientation of anisotropic ellipsoidal nanorods through polarized detection [148]. Moreover, the complete complex dielectric function of a single