Chapter 3
Label-Free, Ultrahigh-Speed, Direct
Imaging and Tracking
of Bionanoparticles in Live Cells by Using
Coherent Brightfield Microscopy
Chia-Lung Hsieh
Abstract Many important biological phenomena, ranging from cell signaling to
viral infection, are accomplished by transportation of biological substances encapsulated in native nano-sized particles. Thermal fluctuation drives nanoparticles through
cellular environments; this movement is facilitated by their small size. To understand how a specific cell function can be achieved through random collisions, it is
useful to know the interactions between single particles and the local environment,
as determined by measuring cell dynamics at high spatial and temporal resolutions.
In this chapter, a simple yet powerful wide-field optical technique, coherent brightfield (COBRI) microscopy, is presented. COBRI microscopy detects linearly scattered light from a nanoparticle through imaging-based interferometry, which enables
direct observation of small biological nanoparticles in live cells without labels. Proper
image post-processing further improves the detection sensitivity of small particles by
removing the scattering background of cell structures. COBRI microscopy can easily
operate at a high speed due to its wide-field nature and stable, indefinite scattering
signal. Using COBRI, the dynamics of single virus particles and cell vesicles in live
cells can be successfully captured at a microsecond temporal resolution and nanometer spatial precision in three dimensions. The ultrahigh spatiotemporal resolution and
shot-noise-limited sensitivity of COBRI microscopy provide an opportunity to study
the biophysics and biochemistry of live cells at the nanoscale.
3.1 Introduction to Label-Free Imaging in Live Cells
Through Linear Scattering
A biological cell is a highly dynamic system. Cell organelles and other subcellular
structures constantly evolve and migrate to facilitate cell functions. Active transportation is an effective method for the redistribution of substance in live cells [1,
C.-L. Hsieh (B)
Institute of Atomic and Molecular Sciences (IAMS), Academia Sinica, No. 1, Roosevelt Road,
Section 4, Taipei 10617, Taiwan
e-mail: clh@gate.sinica.edu.tw
© Springer Nature Switzerland AG 2019
V. Astratov (ed.), Label-Free Super-Resolution Microscopy,
Biological and Medical Physics, Biomedical Engineering,
https://doi.org/10.1007/978-3-030-21722-8_3
67
Label-Free, Ultrahigh-Speed, Direct
Imaging and Tracking
of Bionanoparticles in Live Cells by Using
Coherent Brightfield Microscopy
Chia-Lung Hsieh
Abstract Many important biological phenomena, ranging from cell signaling to
viral infection, are accomplished by transportation of biological substances encapsulated in native nano-sized particles. Thermal fluctuation drives nanoparticles through
cellular environments; this movement is facilitated by their small size. To understand how a specific cell function can be achieved through random collisions, it is
useful to know the interactions between single particles and the local environment,
as determined by measuring cell dynamics at high spatial and temporal resolutions.
In this chapter, a simple yet powerful wide-field optical technique, coherent brightfield (COBRI) microscopy, is presented. COBRI microscopy detects linearly scattered light from a nanoparticle through imaging-based interferometry, which enables
direct observation of small biological nanoparticles in live cells without labels. Proper
image post-processing further improves the detection sensitivity of small particles by
removing the scattering background of cell structures. COBRI microscopy can easily
operate at a high speed due to its wide-field nature and stable, indefinite scattering
signal. Using COBRI, the dynamics of single virus particles and cell vesicles in live
cells can be successfully captured at a microsecond temporal resolution and nanometer spatial precision in three dimensions. The ultrahigh spatiotemporal resolution and
shot-noise-limited sensitivity of COBRI microscopy provide an opportunity to study
the biophysics and biochemistry of live cells at the nanoscale.
3.1 Introduction to Label-Free Imaging in Live Cells
Through Linear Scattering
A biological cell is a highly dynamic system. Cell organelles and other subcellular
structures constantly evolve and migrate to facilitate cell functions. Active transportation is an effective method for the redistribution of substance in live cells [1,
C.-L. Hsieh (B)
Institute of Atomic and Molecular Sciences (IAMS), Academia Sinica, No. 1, Roosevelt Road,
Section 4, Taipei 10617, Taiwan
e-mail: clh@gate.sinica.edu.tw
© Springer Nature Switzerland AG 2019
V. Astratov (ed.), Label-Free Super-Resolution Microscopy,
Biological and Medical Physics, Biomedical Engineering,
https://doi.org/10.1007/978-3-030-21722-8_3
67