L18 - Single molecule detection principles, STED

Single molecule enzymology

distributions of bound times

cholesterol oxidase, a 53-kDa flavoprotein that catalyzes the oxidation of cholesterol by oxygenMichaelis-Menten: equilibrium + catalysis

issue with detecting single molecules

ν is the Raman shift which is around 3400-3600 cm-1 for water\

volume effect

Total Internal Reflection (TIR)

suppresses background fluorescence and enables observation of surface attached molecules

Confocal microscopy on dilute samples

Near Field Scanning Optical Microscopy (NSOM)

RECAP of SMD

Why SMD?

Captures dynamic processes without the need for synchronization

Allows measurement of distributions, not just ensemble averages

Key Insight:

Use SMD to directly observe kinetic fluctuations of enzymes

Techniques for Reducing Background

Fluorescence often buried under scattering and autofluorescence

Raman scattering can mimic fluorescence

Solution: Limit the detection volume

Three Main SMD Modalities

  1. TIRF (Total Internal Reflection Fluorescence) - Excites only near-surface molecules (~200 nm)

  2. Confocal Microscopy - Spatial filtering (pinhole) to exclude out-of-focus light

  3. NSOM (Near-field Scanning Optical Microscopy) - Sub-diffraction illumination via nanoscopic tip

Diffraction Barrier

Stimulated Emission Depletion Microscopy (STED)

Fluorophores can label specific molecules of interestFluorescence microscopy is limited by the diffraction limit

Resolution enhancement

put the graph in the notes little bro 💔

STED setup

Limitations

  1. Photobleaching and Phototoxicity

    1. High-intensity depletion laser (~100–1000 mW) accelerates photobleaching.

    2. Fluorophores quickly lose their ability to fluoresce, especially under repeated scanning.

    3. The intense depletion and excitation light can damage live cells.

  2. Fluorophore Requirements

    1. Not all fluorophores are STED-compatible.

    2. Fluorophores must exhibit efficient stimulated emission and photostability.

    3. Careful spectral separation is required between excitation, emission, and depletion wavelengths.

  1. Complex Instrumentation

    1. Requires precise alignment of multiple laser beams (excitation, depletion).

    2. Beam shaping (e.g., donut-shaped depletion) demands advanced optics.

  1. Limited Imaging Speed

    1. STED is often performed as point-scanning, so it's relatively slow.

    2. This may not capture fast biological dynamics effectively.