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. 2010;475:27-59.
doi: 10.1016/S0076-6879(10)75002-3.

Molecules and Methods for Super-Resolution Imaging

Free PMC article

Molecules and Methods for Super-Resolution Imaging

Michael A Thompson et al. Methods Enzymol. .
Free PMC article


By looking at a fluorescently labeled structure one molecule at a time, it is possible to side-step the optical diffraction limit and obtain "super-resolution" images of small nanostructures. In the Moerner Lab, we seek to develop both molecules and methods to extend super-resolution fluorescence imaging. Methodologies and protocols for designing and characterizing fluorophores with switchable fluorescence required for super-resolution imaging are reported. These fluorophores include azido-DCDHF molecules, covalently linked Cy3-Cy5 dimers, and also the first example of a photoswitchable fluorescent protein, enhanced yellow fluorescent protein (EYFP). The imaging of protein superstructures in living Caulobacter crescentus bacteria is used as an example of the power of super-resolution imaging by single-molecule photoswitching to extract information beyond the diffraction limit. Finally, a new method is described for obtaining three-dimensional super-resolution information using a double-helix point-spread function.


Figure 2.1
Figure 2.1. Photochemistry and spectroscopy of an azido DCDHF fluorogen
(A) Various products resulting from photochemical conversion of an azido DCDHF fluorogen. (B) Absorption curves in ethanol (bubbled with N2) showing photoactivation of 1 (λabs = 424 nm) over time to fluorescent product 2 (λabs = 570 nm). Different colored curves represent 0, 10, 90, 150, 240, 300, 480, and 1320 s of illumination by 3.1 mW cm−2 of diffuse 407-nm light, where the arrows show the direction of increasing time. The sliding isosbestic point may indicate a build-up of reaction intermediates. Dashed line is the absorbance of pure, synthesized 2. (Inset) Dotted line is weak preactivation fluorescence of 1 excited at 594 nm; solid line is strong postactivation fluorescence resulting from exciting 2 at 594 nm, showing > 100-fold turn-on ratio. (Adapted and reproduced with permission from Lord et al.,2008. Copyright 2008 American Chemical Society.)
Figure 2.2
Figure 2.2. Photoactivation of the azido DCDHF fluorogen in live mammalian cells
(A) Three Chinese Hamster Ovary cells incubated with azido DCDHF fluorogen are dark before activation. (B) The fluorophore lights up in the cells after activation with a 10-s flash of diffuse, low-irradiance (0.4 W cm−1) 407-nm light. The white-light transmission image is merged with the fluorescence images, excited at 594 nm (~1 kW cm−1). Scalebar: 20 μm. (C) Single molecules of the activated fluorophore in a cell under higher magnification. Scalebar: 800 nm. (Adapted with permission from Lord et al., 2008. Copyright 2008 American Chemical Society.)
Figure 2.3
Figure 2.3. Synthesis and bulk characterization of covalently linked Cy3–Cy5 dimers
(A) Structures of reactive cyanine dyes and covalent heterodimers. (B) Absorption (solid) and fluorescence emission (hollow, λex = 516 nm) spectra of Cy3–Cy5 covalent heterodimers 4 and 5 (in water; 3.7 μM for absorption; 37 nM for fluorescence) before photodarkening. (Adapted and reproduced with permission from Conley et al., 2008. Copyright 2008 American Chemical Society.)
Figure 2.4
Figure 2.4. Photoswitching behavior of and super-resolution imaging using the Cy3–Cy5 covalent dimer
(A) Representative single-molecule fluorescence time trace of 5-labeled bovine serum albumin showing reactivation cycles 12–16, denoted by the dashed lines. (B) Fluorescence images at times 1, 2, and 3 corresponding to the times labeled in panel (A). Scale bar, 1 μm. (C) Super-resolution fluorescence image of C. crescentus stalks with 30 nm resolution superimposed on a white-light image of the cells. The C. crescentus cells were incubated in 4 μM of Cy3–Cy5 NHS ester for 1 h and then washed five times before imaging to remove free fluorophores. The data were acquired over 2048 100-ms imaging frames with 633 nm excitation at 400 W cm−2. After initial imaging and photobleaching of the Cy3–Cy5 dimers, the molecules were reactivated every 10 s for 0.1 s with 532-nm light at 10 W cm−2. (Adapted and reproduced with permission from Conley et al., 2008. Copyright 2008 American Chemical Society.)
Figure 2.5
Figure 2.5. Reactivation of EYFP-MreB fusions in live C. crescentus cells
(A) Single 100-ms acquisition frames showing isolated EYFP-MreB molecules (a, c, and e) upon photo-activation and no single-molecule emission (b, d, f ) after photobleaching. The spot in the bottom left of each image is an imaging artifact. (B) Bulk reactivation of a sample of 22 cells. The grey lines indicate 2-s pulses of 407 nm light. (Adapted and reproduced with permission from Biteen et al., 2008. Copyright 2008 Nature Publishing Group.)
Figure 2.6
Figure 2.6. Super-resolution images of MreB in live C. crescentus cells
(A–B) Images taken using standard PALM (C–D) images taken using time-lapse imaging to obtain higher labeling density using. (A) Image of MreB forming a midplane ring in a predivisional cell. (B) Banded MreB structure in a stalk cell. (C) Quasi-helical MreB structure at 40 nm resolution observed using time-lapse PALM. (D) Structure in panel (C) displayed without white-light image in order to highlight the continuity of the structure. (E) Time-lapse PALM image of MreB midplane ring in a predivisional cell. (Adapted and reproduced with permission from Biteen et al., 2008. Copyright 2008 Nature Publishing Group.)
Figure 2.7
Figure 2.7. Localization of 200-nm fluorescent beads using the DH-PALM microscope
(A) Schematic illustrating the collection path in a DH-PALM setup, IL is imaging lens, L1 and L2 are 150 mm focal length achromat lenses, P is a linear polarizer, and SLM is a phase-only spatial light modulator. (B) Three-dimensional representation of the experimentally observed DH-PSF (created with VolumeJ (Abrámoff and Viergever, 2002) with slices taken at z positions of approximately (1) −450 nm, (2) 0 nm, and (3) 500 nm where 0 is taken to be the designed focal plane of the microscope. (C) Calibration of angle between the two lobes as a function of the distance between the objective surface and the bead with 0 being the position when the lobes are horizontal with respect to one another. (D) Plot of angle between the lobes versus frame for a fluorescent bead as the objective is scanned through 50 nm steps showing clear steps in the angle with a low standard deviation in each step.
Figure 2.8
Figure 2.8. Single-molecule superlocalization using the DH-PSF
(A) Image of a single DCDHF-V-PF4-azide molecule coming through the DH-PALM imaging system. (B) Histograms of 44 positions of the single molecule in (A) with standard deviations of 12.8 nm in x, 12.1 nm in y, and 19.5 nm in z. For these nonoptimized measurements, the average number of photons detected was 9300 with background noise of 48 photons/pixel and a pixel size of 160 nm. (Adapted and reproduced with permission from Pavani et al., 2009. Copyright 2009 National Academy of Sciences.)
Scheme 2.1
Scheme 2.1. Schematic showing the key idea of super-resolution imaging of a structure by PALM
(A) It is not possible to resolve the underlying structure in a conventional widefield fluorescence image because the fluorescent labels are in high concentration and the images overlap. (B) Using controllable fluorophores, it is possible to turn on and image a sparse subset of molecules which then can be localized with nanometer precision (black line is the underlying structure being sampled). Once the first subset of molecules photobleaches, another subset is turned on and localized. This process is repeated and the resulting localizations summed to give a super-resolution image of the underlying structure.

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