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Single Molecule Techniques

Basic Concepts

  • Light: Fundamental principle utilized in microscopy applications.

Types of Microscopy

  • High then Super-Resolution Microscopy: Methods that enhance the resolution of images captured through microscopic techniques.

Labelling Cellular Components

  • Green Fluorescent Protein (GFP): A widely used marker in biological research that allows visualization of protein location within living organisms.

  • Fluorescent Dyes: 如 Alexa Fluor 488, which can be coupled with certain components (e.g., proteins) for visualization.

Molecular Structures and Genetic Elements

  • PUC ori (5612-6287), AmpR (4608-5465), Various Restriction Sites: Sequence numbers indicate specific regions relevant for DNA manipulation and cloning protocols. Examples include:

    • BglII (6696), Ndel (6463), XbaI (4208) – enzymes used in cutting DNA sequences.

    • PAL4-Lifeact (6862 bp): Gene sequences relevant to actin visualization in live cells.

  • GS-TagRFP and other fluorescent tags are also mentioned.

How Does Fluorescence Work?

  • Energy Levels of Fluorescent Molecules:

    • Ground State: Low energy state of an electron in an atom.

    • Excited State: Higher energy state obtained through absorption of light.

    • Excitation: Process by which energy is absorbed causing electrons to move to an excited state.

    • Emission: The release of energy in the form of light when electrons return to the ground state, typically resulting in longer wavelengths (lower energy) than the excitation light.

    • Energy Loss through Vibrational Relaxation: Energy dissipates through molecular vibrations before light is emitted.

Anatomy of a Fluorescence Microscope

  • Diagram Representation: The anatomy includes a light source, detector, and dichroic mirror components that play crucial roles in image acquisition.

  • Confocal vs. Widefield:

    • Confocal Microscopy: Utilizes a point scanning method allowing optical sectioning and reduces background noise.

    • Widefield Microscopy: Simultaneously illuminates the entire sample, leading to lower signal-to-noise ratios.

Biological Size Scales

  • Cellular Dimensions:

    • Mammalian cells: Hundreds of microns.

    • Mitochondria: Ranging from single microns to hundreds of nanometers.

    • Viruses: Generally fall within the range of tens to hundreds of nanometers.

    • Proteins: Typically a few nanometers to sub-Ångstroms in size (e.g., 1 Ångstrom = $10^{-10}$ m).

Super-Resolution Microscopy

  • Need for Resolution Enhancement: Traditional techniques cannot resolve structures smaller than $ ext{approx. } 250 ext{ nm}$; hence, super-resolution techniques are essential.

Microscopy Techniques Comparison

  • Fluorescence Microscopy vs. Electron Microscopy:

    • Availability of Labels: Fluorescence microscopy has a wide range; electron microscopy is limited.

    • Live-cell Compatibility: Fluorescence microscopy allows live imaging; electron microscopy requires fixation.

    • Sample Preparation: Relatively straightforward for fluorescence, but complicated for electron microscopy.

    • Temporal Resolution: Fluorescence can achieve down to milliseconds; electron microscopy has no applicable time resolution.

    • Spatial Resolution: Maximum of ~250 nm for fluorescence vs. ~0.5 nm for electron microscopy.

Diffraction Limit in Fluorescence Microscopy

  • Point Spread Function (PSF): Characterized by an Airy Disk Pattern, demonstrating that fluorophores emit light that disperses, resulting in a diffraction limit affecting resolution.

    • Rayleigh Limit Resolution Calculation: The formula for resolution d=rac0.61imesextλextNAd = rac{0.61 imes ext{λ}}{ ext{NA}} indicates the limitations where:

    • λ = wavelength of the light (e.g., 532 nm)

    • NA = numerical aperture of the lens, typically ~1.5.

Super-Resolution Approaches

  • Structured Illumination Microscopy (SIM): Achieves doubled resolution using complex methodologies.

  • Stimulated Emission Depletion (STED) Microscopy: A challenging approach that can damage samples, yet achieves resolution of approximately 50 nm.

  • Single Molecule Localization Microscopy (SMLM): Clarified as straightforward with the highest potential resolution; however, it has limitations in imaging single molecules over noise.

Imaging Techniques Overcoming SMLM Limitations

  • Total Internal Reflectance Fluorescence (TIRF) Microscopy: Effective for fast imaging at surfaces, allows single-molecule sensitivity, and is suitable forMembrane studies.

  • Light Sheet Microscopy and Oblique Angle (HiLo) Fluorescence Microscopy are techniques allowing deeper imaging solutions.

Evanescent Waves in TIRF

  • Evanescent Wave: Characterized as a decay pattern used to excite fluorophores close to the surface, where the exponential decrease improves signal-to-noise.

    • Light Behavior in TIRF: When light transitions from a high to low-density medium, specific angles lead to total internal reflection rather than transmission, governed by Snell's Law:

    • n<em>t/n</em>i=extsin(qc)n<em>t/n</em>i = ext{sin}(q_c)

    • For instance, for glass to water, n<em>extglass=1.52n<em>{ ext{glass}} = 1.52 and n</em>extwater=1.33n</em>{ ext{water}} = 1.33.

Combining Techniques

  • Implementation of TIRF in conjunction with SMLM for studies such as actin-associated proteins, highlighting progressive imaging techniques.

Summary of Imaging Techniques

  • Technique Comparisons:

    • Widefield: >220 nm spatial resolution; temporal resolution >10 ms; no special optics required.

    • Confocal: >220 nm spatial resolution; temporal resolution >100 ms; requires special optics and computational processing.

    • SMLM: <10 nm spatial resolution; time processing can exceed 10 seconds, requires computational post-processing with low dependence on special optics.