3 Label-Free, Ultrahigh-Speed, Direct Imaging and Tracking …
69
Label-free imaging of endogenous bio-nanoparticle is complicated in live cells
because of the scattering background of cell structures [30–34]. The smallest particle
that can be detected and tracked in a live cell is often limited by imaging specificity,
not by optical sensitivity. Having a strategy for distinguishing the signal from the
background is therefore highly valuable. In addition, a cell is a three-dimensional
(3D) object, and the dynamics of small particles are also 3D. Projection of 3D dynamics onto two-dimensional (2D) can cause misinterpretation. Thus, a label-free 3D
imaging and tracking technique is highly desirable.
In this chapter, a simple, yet powerful imaging modality, namely coherent brightfield (COBRI) microscopy, is presented for sensitive and high-speed direct imaging
of biological nanoparticles [31, 32]. By using a highly coherent light source (i.e., a
laser) for illumination, COBRI microscopy facilitates rapid and sensitive imaging
beyond what conventional brightfield microscopy can offer. Through interference,
COBRI microscopy also enables tracking in 3D. Section 3.2 describes the concepts,
technical details and characterization of COBRI microscopy. In Sect. 3.3, strategies
of background estimation and correction for COBRI imaging in live cells are discussed. Section 3.4 presents two examples of COBRI imaging and tracking of single
bio-nanoparticles in live cells. Specifically, Sect. 3.4.1 describes how the highly diffusive motion of single virus particles on a cell’s surface is captured with nanometer
spatial precision and microsecond temporal resolution; Sect. 3.4.2 describes how
active transport of individual native cell vesicles inside a living cell is resolved with
ultrahigh clarity. Finally, Sect. 3.5 concludes the chapter by discussing the sensitivity limit and possible applications of ultrasensitive and ultrahigh-speed label-free
imaging.
3.2 COBRI Microscopy
COBRI microscopy essentially works by employing a brightfield microscope and
a highly coherent laser light source [31]. The high temporal coherence maximizes
imaging contrast and thus sensitivity. The high spatial coherence enables versatile
beam shaping (e.g., focusing and structured illumination). Laser also provides sufficient illumination intensity at the sample for high-speed measurements. A schematic
of COBRI microscopy is shown in Fig. 3.1. A laser beam is focused onto the sample
through a condenser lens. The choice of the condenser lens determines the available spatial frequency bandwidth in illumination. For example, when using a lownumerical-aperture (low-NA) condenser (as plotted in Fig. 3.1), the illumination has
a Gaussian-like profile of a few to tens of micrometers [31]. When using a high-NA
condenser, a sharp submicrometer focus can be created at the sample [35]. Although
stationary illumination is sufficient for the operation of COBRI microscopy, a rapid,
2D scan of the illumination often makes the measurement easier [31]. Such 2D beam
scanning is achieved by using a 2-axis acousto-optic defector (AOD) that typically
scans at approximately 100 kHz. The beam scanning is synchronized with image
acquisition, which ensures identical beam scanning is performed within each image
69
Label-free imaging of endogenous bio-nanoparticle is complicated in live cells
because of the scattering background of cell structures [30–34]. The smallest particle
that can be detected and tracked in a live cell is often limited by imaging specificity,
not by optical sensitivity. Having a strategy for distinguishing the signal from the
background is therefore highly valuable. In addition, a cell is a three-dimensional
(3D) object, and the dynamics of small particles are also 3D. Projection of 3D dynamics onto two-dimensional (2D) can cause misinterpretation. Thus, a label-free 3D
imaging and tracking technique is highly desirable.
In this chapter, a simple, yet powerful imaging modality, namely coherent brightfield (COBRI) microscopy, is presented for sensitive and high-speed direct imaging
of biological nanoparticles [31, 32]. By using a highly coherent light source (i.e., a
laser) for illumination, COBRI microscopy facilitates rapid and sensitive imaging
beyond what conventional brightfield microscopy can offer. Through interference,
COBRI microscopy also enables tracking in 3D. Section 3.2 describes the concepts,
technical details and characterization of COBRI microscopy. In Sect. 3.3, strategies
of background estimation and correction for COBRI imaging in live cells are discussed. Section 3.4 presents two examples of COBRI imaging and tracking of single
bio-nanoparticles in live cells. Specifically, Sect. 3.4.1 describes how the highly diffusive motion of single virus particles on a cell’s surface is captured with nanometer
spatial precision and microsecond temporal resolution; Sect. 3.4.2 describes how
active transport of individual native cell vesicles inside a living cell is resolved with
ultrahigh clarity. Finally, Sect. 3.5 concludes the chapter by discussing the sensitivity limit and possible applications of ultrasensitive and ultrahigh-speed label-free
imaging.
3.2 COBRI Microscopy
COBRI microscopy essentially works by employing a brightfield microscope and
a highly coherent laser light source [31]. The high temporal coherence maximizes
imaging contrast and thus sensitivity. The high spatial coherence enables versatile
beam shaping (e.g., focusing and structured illumination). Laser also provides sufficient illumination intensity at the sample for high-speed measurements. A schematic
of COBRI microscopy is shown in Fig. 3.1. A laser beam is focused onto the sample
through a condenser lens. The choice of the condenser lens determines the available spatial frequency bandwidth in illumination. For example, when using a lownumerical-aperture (low-NA) condenser (as plotted in Fig. 3.1), the illumination has
a Gaussian-like profile of a few to tens of micrometers [31]. When using a high-NA
condenser, a sharp submicrometer focus can be created at the sample [35]. Although
stationary illumination is sufficient for the operation of COBRI microscopy, a rapid,
2D scan of the illumination often makes the measurement easier [31]. Such 2D beam
scanning is achieved by using a 2-axis acousto-optic defector (AOD) that typically
scans at approximately 100 kHz. The beam scanning is synchronized with image
acquisition, which ensures identical beam scanning is performed within each image