32
R. W. Taylor and V. Sandoghdar
million-fold reduction in signal strength for dark-field microscopy. To address this
huge challenge, other techniques such as lock-in enhanced photothermal detection
[71, 72], interferometric scattering (iSCAT) [73] and lock-in enhanced transmission
[74] were soon introduced. The success of these measurements showed that sensitive
optical detection of nano-objects would indeed be possible through nonfluorescent
means. In the decade that followed right up to the present day we see a broad array
of research efforts, drawn from numerous communities, exploring interferometric
detection of single nano-objects such as viruses, DNA, microtubules, exosomes, and
proteins [75–91]. Interestingly, interferometric microscopies are also flourishing in
the general context of label-free imaging of cells and membranes even if nanoparticles are not at the center of attention [75, 76, 90, 92–105]. The underlying physics of
these methods remains the same although a plethora of acronyms such as interference
reflectance imaging sensing (IRIS) [77], rotating coherent scattering (ROCS) [98],
interference plasmonic imaging (iPM) [88], coherent bright-field imaging (COBRI)
[106], stroboscopic interference scattering imaging (stroboSCAT) [107], interferometric scattering mass spectrometry (iSCAMS) [108] are on the rise. In what follows,
we bring all these techniques under the umbrella of iSCAT, emphasizing the two central concepts and mechanisms of interference and scattering as the basis for recording
the extinction signal (nano-shadow) generated by nanoparticles.
2.3 Interferometric Scattering Microscopy (iSCAT)
2.3.1 Foundations
The principle concept in interferometric microscopy is to superpose a reference light
beam with the response of the sample, as illustrated in Fig. 2.3a. Let us consider a
field E s = E s e
iφ s scattered from the object. The signal on the detector reads:
I det ∝ |E r + E s |
2
= I r + I s + 2E r E s cos φ ,
(2.1)
where E r = E r e
iφ r denotes the complex electric field of the reference arm. The
three resulting components can be respectively identified as the contribution of the
reference field (I r = |E r |
2 ), the pure scattering recorded from the object (I s = |E s |
2 )
and finally the cross-term (2E r E s cos φ), wherein φ = φ r − φ s . In general, this phase
contains a component describing the sinusoidal modulation along the propagation
path, a Gouy phase stemming from variations of wavevectors and the scattering phase
imposed by the material properties of the object [109, 110].
As is common in holography, one can realize various iSCAT illumination and
detection schemes, which are all essentially described by (2.1). The simplest, oldest
and perhaps the most subtle version is shown in Fig. 2.3b, corresponding to conventional bright-field imaging. Although the intuitive explanation of this imaging modality is based on the concept of absorption and shadow, its mathematical essence is well
R. W. Taylor and V. Sandoghdar
million-fold reduction in signal strength for dark-field microscopy. To address this
huge challenge, other techniques such as lock-in enhanced photothermal detection
[71, 72], interferometric scattering (iSCAT) [73] and lock-in enhanced transmission
[74] were soon introduced. The success of these measurements showed that sensitive
optical detection of nano-objects would indeed be possible through nonfluorescent
means. In the decade that followed right up to the present day we see a broad array
of research efforts, drawn from numerous communities, exploring interferometric
detection of single nano-objects such as viruses, DNA, microtubules, exosomes, and
proteins [75–91]. Interestingly, interferometric microscopies are also flourishing in
the general context of label-free imaging of cells and membranes even if nanoparticles are not at the center of attention [75, 76, 90, 92–105]. The underlying physics of
these methods remains the same although a plethora of acronyms such as interference
reflectance imaging sensing (IRIS) [77], rotating coherent scattering (ROCS) [98],
interference plasmonic imaging (iPM) [88], coherent bright-field imaging (COBRI)
[106], stroboscopic interference scattering imaging (stroboSCAT) [107], interferometric scattering mass spectrometry (iSCAMS) [108] are on the rise. In what follows,
we bring all these techniques under the umbrella of iSCAT, emphasizing the two central concepts and mechanisms of interference and scattering as the basis for recording
the extinction signal (nano-shadow) generated by nanoparticles.
2.3 Interferometric Scattering Microscopy (iSCAT)
2.3.1 Foundations
The principle concept in interferometric microscopy is to superpose a reference light
beam with the response of the sample, as illustrated in Fig. 2.3a. Let us consider a
field E s = E s e
iφ s scattered from the object. The signal on the detector reads:
I det ∝ |E r + E s |
2
= I r + I s + 2E r E s cos φ ,
(2.1)
where E r = E r e
iφ r denotes the complex electric field of the reference arm. The
three resulting components can be respectively identified as the contribution of the
reference field (I r = |E r |
2 ), the pure scattering recorded from the object (I s = |E s |
2 )
and finally the cross-term (2E r E s cos φ), wherein φ = φ r − φ s . In general, this phase
contains a component describing the sinusoidal modulation along the propagation
path, a Gouy phase stemming from variations of wavevectors and the scattering phase
imposed by the material properties of the object [109, 110].
As is common in holography, one can realize various iSCAT illumination and
detection schemes, which are all essentially described by (2.1). The simplest, oldest
and perhaps the most subtle version is shown in Fig. 2.3b, corresponding to conventional bright-field imaging. Although the intuitive explanation of this imaging modality is based on the concept of absorption and shadow, its mathematical essence is well