2 Interferometric Scattering (iSCAT) Microscopy and Related Techniques
51
Fig. 2.12 Detection of single-protein extinction. a Schematic for the first demonstration of
single-protein detection: (i) A solution of proteins is pipetted upon a functionalized coverslip.
(ii) Background-corrected differential iSCAT images showing the coverslip as pipette flow begins
and ends, depositing single proteins which appear as dark spots. (iii) A super-localizing fit to one
protein PSF indicates how individual binding sites can be super-resolved (iv) [119]. Reproduced
with permission from Nature Publishing Group. Mass calibration (b): The contrast of single and
complexed proteins can be calibrated to their mass (i), with monomeric and oligomeric derivatives
also resolvable by this technique (ii) [108]. Reproduced with permission from the American Association for the Advancement of Science. c Single-cell secretion dynamics resolved at single-protein
sensitivity with iSCAT. (i) Schematic of the experimental arrangement wherein a secreting cell is
positioned near to the iSCAT field of view. The contrast distribution of proteins secreted by the cell
as they bind to an anti-IgG functionalized surface (blank) agrees with that expected from pure IgG
(red). iSCAT also reveals an abundance of other secreted products (blue) [164]. Reproduced with
permission from the American Chemical Society
This study paves the way for a wide range of future experiments e.g., the interaction
of immune cells. iSCAT investigation of secretion has also been recently extended
to plasmonic substrates, where the incident light is coupled to surface plasmons in a
thin gold surface [88].
2.4.2 Dynamics in Nanobiology
High-speed, high-spatial precision and a long measurement duration in imaging are
some of the key advantages of iSCAT that become particularly important when
investigating processes such as diffusion and transport of viruses, proteins, lipids or
other nanoscopic entities. In this section, we present a few case studies where iSCAT
was used in this context.
2.4.2.1 Protein Tracking
In the previous section, we discussed the power of iSCAT in detecting unlabeled
proteins. The precision and fast temporal imaging of iSCAT microscopy also lends
itself to investigation of the mobility of single proteins such as myosin-5 motor protein
on actin [120, 172] (see Fig. 2.13). In addition, the mobility of single microtubules can
be investigated to high precision when forming a gliding assay upon kinesin [168].
Furthermore, the motion of unlabeled small proteins upon landing on a surface has
been visualized using iSCAT [118].
2.4.2.2 Lipid Membranes
Lipid membranes are one of the most important substrates in biology, often composed of a bilayer of phospholipid molecules and integral and peripheral proteins
51
Fig. 2.12 Detection of single-protein extinction. a Schematic for the first demonstration of
single-protein detection: (i) A solution of proteins is pipetted upon a functionalized coverslip.
(ii) Background-corrected differential iSCAT images showing the coverslip as pipette flow begins
and ends, depositing single proteins which appear as dark spots. (iii) A super-localizing fit to one
protein PSF indicates how individual binding sites can be super-resolved (iv) [119]. Reproduced
with permission from Nature Publishing Group. Mass calibration (b): The contrast of single and
complexed proteins can be calibrated to their mass (i), with monomeric and oligomeric derivatives
also resolvable by this technique (ii) [108]. Reproduced with permission from the American Association for the Advancement of Science. c Single-cell secretion dynamics resolved at single-protein
sensitivity with iSCAT. (i) Schematic of the experimental arrangement wherein a secreting cell is
positioned near to the iSCAT field of view. The contrast distribution of proteins secreted by the cell
as they bind to an anti-IgG functionalized surface (blank) agrees with that expected from pure IgG
(red). iSCAT also reveals an abundance of other secreted products (blue) [164]. Reproduced with
permission from the American Chemical Society
This study paves the way for a wide range of future experiments e.g., the interaction
of immune cells. iSCAT investigation of secretion has also been recently extended
to plasmonic substrates, where the incident light is coupled to surface plasmons in a
thin gold surface [88].
2.4.2 Dynamics in Nanobiology
High-speed, high-spatial precision and a long measurement duration in imaging are
some of the key advantages of iSCAT that become particularly important when
investigating processes such as diffusion and transport of viruses, proteins, lipids or
other nanoscopic entities. In this section, we present a few case studies where iSCAT
was used in this context.
2.4.2.1 Protein Tracking
In the previous section, we discussed the power of iSCAT in detecting unlabeled
proteins. The precision and fast temporal imaging of iSCAT microscopy also lends
itself to investigation of the mobility of single proteins such as myosin-5 motor protein
on actin [120, 172] (see Fig. 2.13). In addition, the mobility of single microtubules can
be investigated to high precision when forming a gliding assay upon kinesin [168].
Furthermore, the motion of unlabeled small proteins upon landing on a surface has
been visualized using iSCAT [118].
2.4.2.2 Lipid Membranes
Lipid membranes are one of the most important substrates in biology, often composed of a bilayer of phospholipid molecules and integral and peripheral proteins