2 Interferometric Scattering (iSCAT) Microscopy and Related Techniques
49
plane of the microscope objective to achieve uniform illumination of an area with
lateral extension in the order of 5–10 µm and used CMOS cameras for imaging.
Aside from the simultaneous study of many particles, an advantage of a camera as
detector is that the total power recorded over all pixels can be conveniently used to
register the incident power and thus account for its fluctuations. Moreover, recent
camera technologies easily allow imaging at speeds of several tens of kHz up to MHz,
which is far faster than scanning schemes, whether they use piezoelectric actuators
[73, 115, 133] or acousto-optical deflectors [116, 120].
The first sensing experiments were reported in 2014 on single proteins as light as
about 50 kDa [119], shown in Fig. 2.12a. A careful analysis of the obtained iSCAT
contrast for several proteins of different size and mass as well as comparison with
single-molecule fluorescence measurements demonstrated the potential of iSCAT for
label-free detection of single proteins. This method has several decisive advantages
over other biosensing solutions. First, the ability to count single proteins brings
sensing to its absolute limit. Second, this happens over a large surface area as opposed
to methods relying on plasmonic antennas [165] or optical microcavities [166] with
very limited active area. Third imaging provides invaluable information about the
spatial distribution and position of each protein, and finally, the essential setup is very
simple. As in the great majority of biosensing platforms based on surface plasmons,
mechanical oscillators, or microcavities, however, specificity has to be reached via
surface functionalization.
The linearity of iSCAT contrast with protein mass allows classification of the
detected proteins according to their size (see Fig. 2.12b). This feature has been
recently used to demonstrate the application of iSCAT to quantitative mass spectrometry [108]. Moreover, this work nicely shows the ability of iSCAT to watch
the dynamics of molecular processes, such as protein aggregation, cross-linking
and oligomerization. Indeed, iSCAT has been successfully applied to a range of
related investigations such as self-assembly of individual tubulin dimers to a growing
microtubule [167], disassembly of a single microtubule [168], real-time monitoring
of 28 nm viral capsid self-assembling around a viral RNA scaffold [169], and the
growth, attachment, and retraction of bacterial pili [170].
Another recent application of label-free single-protein detection was showcased
in the context of real-time investigation of cellular secretion [134, 164], illustrated in Fig. 2.12c. Secretion is the basis of intercellular communication and has
been a subject of single-cell studies using different methods [171], however singleprotein sensitivity was only possible with iSCAT. In a proof of principle experiment,
immunoglobulin G (IgG) antibodies of mass 150 kDa (4 nm size) were detected in a
spatiotemporally resolved fashion following secretion by Laz388 cells [134, 164].
49
plane of the microscope objective to achieve uniform illumination of an area with
lateral extension in the order of 5–10 µm and used CMOS cameras for imaging.
Aside from the simultaneous study of many particles, an advantage of a camera as
detector is that the total power recorded over all pixels can be conveniently used to
register the incident power and thus account for its fluctuations. Moreover, recent
camera technologies easily allow imaging at speeds of several tens of kHz up to MHz,
which is far faster than scanning schemes, whether they use piezoelectric actuators
[73, 115, 133] or acousto-optical deflectors [116, 120].
The first sensing experiments were reported in 2014 on single proteins as light as
about 50 kDa [119], shown in Fig. 2.12a. A careful analysis of the obtained iSCAT
contrast for several proteins of different size and mass as well as comparison with
single-molecule fluorescence measurements demonstrated the potential of iSCAT for
label-free detection of single proteins. This method has several decisive advantages
over other biosensing solutions. First, the ability to count single proteins brings
sensing to its absolute limit. Second, this happens over a large surface area as opposed
to methods relying on plasmonic antennas [165] or optical microcavities [166] with
very limited active area. Third imaging provides invaluable information about the
spatial distribution and position of each protein, and finally, the essential setup is very
simple. As in the great majority of biosensing platforms based on surface plasmons,
mechanical oscillators, or microcavities, however, specificity has to be reached via
surface functionalization.
The linearity of iSCAT contrast with protein mass allows classification of the
detected proteins according to their size (see Fig. 2.12b). This feature has been
recently used to demonstrate the application of iSCAT to quantitative mass spectrometry [108]. Moreover, this work nicely shows the ability of iSCAT to watch
the dynamics of molecular processes, such as protein aggregation, cross-linking
and oligomerization. Indeed, iSCAT has been successfully applied to a range of
related investigations such as self-assembly of individual tubulin dimers to a growing
microtubule [167], disassembly of a single microtubule [168], real-time monitoring
of 28 nm viral capsid self-assembling around a viral RNA scaffold [169], and the
growth, attachment, and retraction of bacterial pili [170].
Another recent application of label-free single-protein detection was showcased
in the context of real-time investigation of cellular secretion [134, 164], illustrated in Fig. 2.12c. Secretion is the basis of intercellular communication and has
been a subject of single-cell studies using different methods [171], however singleprotein sensitivity was only possible with iSCAT. In a proof of principle experiment,
immunoglobulin G (IgG) antibodies of mass 150 kDa (4 nm size) were detected in a
spatiotemporally resolved fashion following secretion by Laz388 cells [134, 164].