Super-Resolution Imaging Notes

Super-Resolution Imaging: Beyond the Diffraction Limit

Introduction

  • Super-resolution imaging overcomes the diffraction limit in microscopy, enabling visualization of structures at a nanoscale level. Pointillism, exemplified by Georges-Pierre Seurat, serves as an analogy.
  • Techniques like dSTORM (direct Stochastic Optical Reconstruction Microscopy) enhance resolution significantly. Myocyte confocal and dSTORM are key examples.

Nobel Prize in Chemistry 2014

  • The Nobel Prize in Chemistry 2014 was awarded to Eric Betzig, Stefan W. Hell, and William E. Moerner for their development of super-resolved fluorescence microscopy.

The Diffraction Limit

  • The diffraction limit restricts the resolution of traditional light microscopy.
  • Abbe Limit: Defines the resolution limit based on wavelength (λ)(λ) and numerical aperture (NA).
  • Formula: \α = \frac{λ}{2NA}, where (λ=488nm)(λ = 488nm) and (NA=1.4)(NA = 1.4), resulting in resolution around ~250nm to ~600nm. The point spread function (PSF) describes how a point light source is spread out in the image.
  • Super-resolution microscopy allows us to see beyond this diffraction limit.

Diffraction-Limited Resolution

  • In diffraction-limited resolution, images of relatively large beads reflect the true size of the object.
  • As bead size decreases, the diffraction-limited spot does not become smaller, limiting resolution.

Fluorescence Imaging Fundamentals

  • Image formation is described by the convolution of the object with the point spread function (PSF):
    • Object⊗PSF=Image\text{Object} \otimes \text{PSF} = \text{Image}

Numerical Aperture and Airy Disc Size

  • The numerical aperture (NA) and Airy disc size are critical factors influencing resolution.

4Pi Microscopy

  • 4Pi microscopy uses two lenses to enhance resolution. Confocal microscopy is often used in conjunction.

Deconvolution

  • Deconvolution algorithms can improve image resolution by computationally removing the effect of the PSF.

Why Super-Resolution Microscopy?

  • Electron microscopy (EM) offers sub-nanometer resolution but lacks contrast and is not suitable for living specimens.
  • Fluorescence microscopy provides good contrast, color/multichannel imaging, flexibility, and ease of labeling, making it suitable for living and thick 3D specimens.
  • Biological structures of interest (genes, macromolecular protein machines) often fall within the 100-300 nm range, tantalizingly close to the classical resolution limit.
    • Genes: ~100-300nm (in interphase nucleus).
    • Macromolecular protein machines: ~100-300nm.
    • Cardiac dyad (RyR and DHPR protein clusters).
    • Replication foci.
    • Protein N-mers, large proteins, individual channels.
    • RyR 4-Mer ~30nm, nucleosomes ~10nm.

Advantages of Super-Resolution/Nanoscopy

  • Resolution: ~20 nm.
  • High contrast.
  • Macromolecular complex imaging.
  • 2D and 3D imaging.
  • Live cell imaging.
  • Correlative imaging.
  • Ease of use and sample preparation.

dSTORM

  • dSTORM (direct Stochastic Optical Reconstruction Microscopy) is a super-resolution technique.
  • It involves acquiring a confocal image run and performing stochastic optical reconstruction.

Technical Specifications of dSTORM

  • Highest resolution: ~10 nm.
  • Limited to: 10 µm imaging depth.
  • Imaging time: 20-40 minutes or 24,000 to 48,000 frames.
  • Requires specialized fluorophores.

Factors Affecting Localization Accuracy

  • Quantization: Minimized by good design.
  • Statistical Noise: Fundamental limit.
  • Background: Practically very important.
  • Localization Accuracy: σ=psf std. dev.N\sigma = \frac{\text{psf std. dev.}}{\sqrt{N}}, where N = number of photons. It's also related to pixel size (a) and background (b).

Photo Activated Localization Microscopy (PALM) and STORM

  • Photo Activated Localization Microscopy (PALM).
  • STORM: J Rust, M Bates, X Zhuang, Nature Methods, Aug 2006.
  • CD63 Lyosomal transmembrane protein
  • Effective resolution < 25nm Betzig et Al, Science, Aug. 2006
  • FPALM - S Hess, T Girirajan, M Mason, Biophysical Journal, Sept 2006
  • PALMIRA - Egner, A. et al., Biophys. J. , 2007

Process of Photoactivated Localization Microscopy

  • Normal dyes produce a diffraction-limited image.
  • Photoactivatable dyes are turned on by photoactivation.
  • After many cycles, positions of activated fluorophores are measured.
  • A generated image is produced.

PALM Simplified

  • Activate fluorophores.
  • Measure positions.
  • Bleach fluorophores.
  • 'Paint' the result.

Photo-switchable Fluorescent Proteins (FPs)

  • Photoactivation: Dark -> Bright (irreversible).
    • Examples: PA-GFP, PA-CFP, PA-mCherry.
  • Photoconversion: Eg. Green -> Red (usually irreversible).
    • Examples: mEos, Kaede.
  • Photoswitching: Dark

Carbocyanine Dyes

  • Used in STORM (e.g., Cy3-Cy5 pairs) and direct STORM (e.g., Cy5, Cy5.5, Alexa 647).
  • Entry to dark state: covalent attachment of thiol, e.g., mercapto-ethyl amine (MEA).
  • Exit from dark state: short wavelength light, thermal oxidation, or energy transfer from activator dye.
  • Switching improved by oxygen removal.

Rhodamine and Oxazine Derivatives

  • Switching is based on different oxidation states.
  • Dark state: partially reduced radical.
  • Entry: reduction of triplet (usually via a Thiol eg MEA).
  • Exit: thermal oxidation, or optically.
  • Examples: Alexa 488, Alexa 568, Atto 655.

Application of Super-Resolution Microscopy

  • Imaging RyR clusters in cardiac myocytes.

Near Single-Protein Resolution

Multicolor Imaging

  • Sequential: measure red fluorophore, then green. Issues with bleaching & crosstalk.
  • Simultaneous: use ratio to categorize fluorophores. Adjustable aperture and dichroic beamsplitter cube.

Multi-Color Localization Microscopy

Application of Super-Resolution in Disease

  • Confocal and super-resolution imaging of fibrosis of transverse tubules in human heart failure.

3D Localization

  • Dye molecules switch & bleach, preventing normal z-stacks.
  • Need to acquire all information for 3D position simultaneously.
  • Encode 3D position in PSF shape by recording multiple 2D images at different defocuses.

Encoding 3D Position in Shape

  • Change PSF so lateral shape gives information on defocus.
  • 3D position information is obtained from a single frame.
  • Methods include: Astigmatism, Double helix, Phase ramp.

Combining Confocal and Localization Microscopy

Analysis of Combined Confocal and Localization Microscopy Data

Importance of High Labeling Density

  • High labeling density is critical for achieving good resolution in single-molecule super-resolution imaging.

dSTORM of WGA Labeled Cardiac Myocyte

Drift Correction and Rendering

Label Size

  • The size of the label affects resolution.
  • Examples: Synthetic Dye (Cy5), IgG Antibody, Quantum Dot, Fluorescent Protein.

Down Sizing Labels

  • Use smaller labels such as nanobodies (~1/10 size).
  • SNAP-tag and DNA paint.

SMLM using DNA-PAINT

  • SMLM using DNA-PAINT covalent linker docking strand, green dye (permanent) imager strand PAINT dye.
  • Transient immobilization makes you stand out.

DNA-PAINT

  • DNA-PAINT involves transient binding and unbinding of imager strands to docking strands.

Spectrally Multiplexed DNA-PAINT

  • Spectrally multiplexed DNA-PAINT super-resolution imaging of microtubules and mitochondria inside fixed cells.

Ten Colour Sequential Exchange-PAINT

  • Exchange-PAINT schematic showing sequential imaging of multiple targets using imager strands labeled with the same fluorophore.

True Molecular Scale Visualization

  • True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine using Jayasinghe et al. Cell Rep; 2018;22:557–567. dSTORM and PAINT.

Comparison of Systems Available

  • Comparison of systems available through the BIRU: Confocal, Airyscan Confocal, dSTORM, STED.
  • Resolutions: 250 nm, 120 nm, 30 nm, 20 nm, respectively.

Confocal Resolution and Pinhole Diameter

  • Influence on image quality.

Zeiss Airyscan

  • Resolution: 120 nm.
  • Uses conventional fluorophores.
  • Suitable for live cell imaging.

HeLa Cells Imaged with Airyscan

  • HeLa cells, red: mitochondria membrane, green: microtubuli, magenta: actin fibres; comparison between confocal and Airyscan image.

STED Microscopy

  • STED (Stimulated Emission Depletion) microscopy.
  • Uses a depletion laser to sculpt the PSF.
  • Resolution: 20 nm.
  • Requires specialized fluorophores (readily available).
  • Suitable for easy 3D and live cell imaging.

Coaligned Dual-Channel STED Nanoscopy

  • Study of Xenopus Nuclear-pore complex Gp210 = nuclear pore glycoprotein-210 Pan-FG = pan-specific FG repeat nucleoporin.

Simple to Use 3D Imaging with STED

  • Living T cell in suspension.
  • 3D reconstruction of confocal and STED stacks.

Cochlea Inner Ear Hair Cells Imaged with Super-Resolution

  • Imaged in all three dimensions using easy3D STED.

RESCue STED

  • Live-cell superresolution microscopy at very low light doses.
  • Reduces light exposure by turning the laser off when not needed.

Live Cell Imaging of SiR-tubulin

  • Comparison between confocal and STED.

Importance of Refractive Index Matching

  • Necessary for optimal image quality in deep tissue imaging.

Adaptive Optics for Deep Tissue Imaging

  • Compensates for aberrations to form an optimal focal spot.

MINFLUX

  • MINFLUX (Minimal Fluxes) microscopy achieves ~1nm resolution by maximizing resolution with minimal emission.

Summary

  • Diffraction is no longer a limit to resolution.
  • Localization microscopy allows fundamental (~10 fold) improvement in imaging resolution through switchable dyes and stochastic switching.
  • Airyscan closes pinhole 0.2 but collects 1.2 A with honey comb detector.
  • STED microscopes sculpture the PSF with depletion laser.
  • Opens up nanoscale live cell imaging.