1. Imaging Live-Cell Dynamics and Structure at the Single-Molecule Level
Zhe Liu, Luke D. Lavis, Eric Betzig 
Molecular Cell 
Volume 58, Issue 4, Pages (May 2015)
DOI: /j.molcel
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Figure 1
Basic Concepts of Single-Molecule Localization, Tracking, and Fluorescence Correlation Spectroscopy
(A) Schematics illustrating the relative size of a single emitter, the resulting Airy disk in the image plane, the PSF as observed on the microscope camera, and single-molecule probability distribution of position as determined by Gaussian fitting.
(B) An example of temporally separating single-molecule detections in densely labeled samples for super-resolution imaging. Photoactivable fluorescent proteins attached to a diffraction limited structure were repeatedly activated, imaged and bleached (Ba–Bd). Summing the molecular images across all frames results in a diffraction-limited image (Be and Bf). However, if the location of each molecule is first determined by fitting the expected molecular image given by the PSF of the microscope (Bg, center) to the actual molecular image (Bg, left), the molecule can be plotted (Bg, right) as a Gaussian that has a standard deviation equal to the precision in the fitted position. Repeating with all molecules across all frames (Ba′–Bd′) and summing the results yields a super-resolution image (Be′ and Bf′). Credit, Betzig et al., 2006.
(C) An example of 3D SPT of HaloTag-Sox2 expressed in a single embryonic stem cell and labeled with tetramethylrhodamine (TMR) using multifocus microscopy. Volume rendering of a Sox2 single-molecule image (purple) superimposed with single-molecule trajectories. Three molecules with distinct behaviors were selectively displayed on the top (from left to right, freely diffusing particle, particle undergoing a free/bound transition, and immobile molecule). Color bar shows the corresponding frame number. Scale bar, 2 μm. Credit, Chen et al., 2014b.
(D) Typical confocal FCS optical layout (top left) and the observation volume (pink, top right). Fluorescence fluctuation curves for fast (middle left) and slow (middle right) diffusion. Autocorrelation curves (bottom) for varied molecular concentrations and diffusion properties of a single-component system. DM, dichroic mirror.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Trade-Offs in Live-cell Imaging and Spatiotemporal Dimensions in Biology
(A) The limited photon budget from the specimen can be spent to improve spatial resolution, temporal resolution, or the depth of imaging. However, in some cases, it is used inefficiently, which only contributes to unnecessary phototoxicity.
(B) Photobleaching and phototoxicity depend not only on the total amount of excitation photons delivered to the sample but also nonlinearly on the instantaneous peak intensity applied to the specimen.
(C) (Upper) Low labeling density and poor incorporation of fluorescently tagged proteins into the structures of interest limit the ability to visualize them clearly. (Lower) Nyquist sampling criterion, to unambiguously resolve structure features with size close to d, in average one localization event in 1/2 d spatial unit need to be sampled. For example, when distances between closest labeling events are comparable to the size of the smaller πs (50% labeling density panel), they become illegible.
(D) π is legible again after particle averaging of partially labeled smaller πs in (C).
(E) General spatial and temporal length scales in biology.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Rapidly Advancing Illumination Methods
(A) Schematic comparisons on the optical sectioning power and the axial depth adjusting flexibility of different illumination techniques.
(B) Differences between a single scanned Bessel beam, scanned parallel, noninterfering Bessel beams, and lattice light-sheet illumination.
(C) Images of molecules that have displacements larger than the localization precision (Δ) within the illumination duration (δt) suffer from motion burring. Stroboscopic illumination reduces motion blurring effects by shortening the temporal width of the illumination. The maximum diffusion coefficient (Dmax) without inducing significant motion blurring is calculated according to Brownian diffusion model.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Detection Methods to Access the Axial Dimension
(A) The schematic shows the concept of multifocal microscopy.
(B) Examples of engineering axial depth sensitive PSFs. Credit, Huang et al., 2008; Jia et al., 2014; Pavani et al., 2009.
(C) Schematics and operating principle of multiphase interferometric microscope illustrating how z position is resolved. The electric field from a point source with z position δ propagates both upward and downward. These two fields interfere in a special beam splitter that produces three output beams with 120° relative phase shifts between the two input fields. (Bottom) The interfered beams are sent to the three color-coded CCD cameras, and the intensity of the point source on each oscillates out of phase with the others depending on the axial position of the point source. Credit, Shtengel et al., 2009.
(D) Reported performance summary of different single-molecule axial detection strategies.
(E) The relationship between illumination thickness, SBR, and allowed labeling density for single-molecule imaging.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Labeling Strategies and Chemical Fluorophores Used in Single-Molecule Assays
(A–E) Schematics of different labeling strategies useful for live-cell microscopy. The biomolecule components and fluorophores are represented by gray and green shapes, respectively.
(F) Structures and spectral properties of fluorophores useful for single-molecule imaging. The dyes are ordered according to the absorption maxima of the dye (dashed line). Structures are colored according to the emission maxima. Cell-impermeant fluorophores are above the wavelength scale, and cell-permeable dyes are below the wavelength scale.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Workflow and Applications
Shown is a unified and multifaceted imaging framework to study spatiotemporal controls and molecular mechanisms of key biological systems in live cells at the single-molecule level.
Molecular Cell  , DOI: ( /j.molcel )
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