Super Resolution Microscopy Notes

Introduction to Super Resolution Microscopy

• Super resolution microscopy is a technique for obtaining highly resolved images at the molecular level.
• It is essentially a modified confocal microscope.

Confocal Microscope Basics

• Uses a laser as the excitation light source.
• The laser scans the specimen in the X and Y directions, focusing on small regions.
• Images from each scanned area are combined through software into a single image.

Limitations of Confocal Microscopy

• Zooming in on a confocal image reveals blurriness due to closely spaced fluorescence molecules that cannot be resolved.
• The blurriness is attributed to the limitations of the laser's excitation spot size.

Understanding Point Spread Function

• The point spread function defines the minimum size of the excitation spot that can be achieved.
• Example: Light focused through a convex lens cannot be smaller than its wavelength (e.g., 500 nm for light with a 500 nm wavelength).
• The same limitation applies to confocal microscopy.

Achieving Super Resolution

• To achieve super resolution, the goal is to break the point spread function to create a smaller excitation spot.
• Physically, breaking the point spread function is considered impossible.

Use of Stimulant Emission

• Fluorescence involves electrons absorbing radiation, getting excited, and then emitting fluorescence upon returning to the ground state.
• Stimulant emission: An excited electron can be re-excited to a higher energy level, emitting fluorescence of a different wavelength when it returns to the ground state.
• An emission filter in the microscope allows only specific wavelengths to pass through (e.g., green light), blocking others and leading to fluorescence depletion.

Fluorescence Depletion Process

1. Excitation laser focuses on the specimen, exciting fluorescent molecules within the excitation spot.
2. Depletion lasers target the area around the excitation spot, depleting the surrounding fluorescence.
3. Only the fluorescent molecule in the center remains, reducing image blurriness caused by the point spread function.

Image Processing and Resolution

• Even if a molecule's actual size is a few nanometers, the smallest focused spot remains limited by the wavelength of emitted fluorescence.
• Software is used to consider only the central pixels of the point spread function to accurately locate a single molecule.
• The resulting image appears as a single spot due to the diffraction-limited resolution.