Fluorescence microscopy

Summary

This lecture explores the fundamentals of fluorescence microscopy, focusing on the techniques, principles, and applications in imaging small biological structures. It covers key concepts like magnification, resolution, and the use of fluorescence labeling to observe molecular interactions. The lecture delves into various fluorescence microscopy methods such as epifluorescence, confocal, and TIRF microscopy, explaining their uses and limitations. Additionally, it highlights cutting-edge super-resolution techniques, including PALM, STORM, and SIM, which overcome diffraction limits to achieve higher spatial resolution. The presentation concludes with a discussion on combining these techniques for enhanced imaging and the challenges posed by long acquisition times and high laser powers in biological studies.

Takeaways

• 😀 Fluorescence microscopy is a powerful tool for studying small biological structures using fluorescently labeled molecules.
• 😀 Magnification increases the size of an object, while resolution determines the smallest detail that can be observed in microscopy.
• 😀 In fluorescence microscopy, a laser excites molecules to a higher state, and the resulting emitted light is collected to form an image.
• 😀 Proper labeling of molecules with fluorescent dyes or quantum dots is crucial for accurate imaging in fluorescence microscopy.
• 😀 Important factors in choosing a fluorescent label include photostability, brightness, and size of the label.
• 😀 Diffraction-limited techniques, such as epifluorescence, confocal, and TIRF microscopy, are commonly used to study biological samples.
• 😀 Confocal microscopy uses a laser and pinhole to eliminate out-of-focus light, improving image clarity.
• 😀 TIRF microscopy exploits total internal reflection to excite only surface-adjacent fluorophores, offering high signal-to-noise ratio.
• 😀 Super-resolution techniques like PALM and STORM surpass the diffraction limit by localizing individual fluorophores with high precision.
• 😀 STED microscopy uses a donut-shaped beam to selectively deplete fluorescence, allowing for higher resolution imaging.
• 😀 SIM (Structured Illumination Microscopy) uses patterned light grids to create interference patterns, providing super-resolution images through computational processing.

Q & A

What is fluorescence microscopy?

Fluorescence microscopy is a technique that uses the fluorescence of molecules to study biological samples. A laser beam excites molecules to a higher energy state, and as they return to their ground state, they emit photons, which are detected to create an image.

What is the difference between magnification and resolution in microscopy?

Magnification refers to how many times larger an object appears, while resolution is the smallest detail of the object that can be observed. Resolution defines the ability to distinguish closely spaced objects.

What are the main types of labels used in fluorescence microscopy?

The main types of labels are quantum dots, fluorescent proteins, and small organic dye molecules. These labels are used to tag biological molecules of interest and help in their visualization under the microscope.

How does a dichroic mirror function in fluorescence microscopy?

A dichroic mirror in fluorescence microscopy blocks the excitation light (higher energy) and only transmits the emitted light (lower energy) to prevent interference and enhance image clarity.

What is photostability, and why is it important in choosing fluorescent labels?

Photostability refers to how well a fluorescent label can resist photobleaching, which is the loss of fluorescence due to intense light exposure. Photostable labels are important for longer imaging sessions and clearer results.

What are diffraction-limited and super-resolution fluorescence microscopy techniques?

Diffraction-limited microscopy refers to techniques that are limited by the diffraction of light, such as epifluorescence and confocal microscopy. Super-resolution microscopy techniques, such as PALM and STORM, overcome this limit and provide higher resolution images by exploiting specific properties of fluorophores.

What is the principle behind confocal microscopy?

Confocal microscopy uses a laser to focus light on a sample and employs a pinhole to filter out out-of-focus light. Only fluorescence from the focal plane is detected, improving image clarity and reducing background noise.

How does total internal reflection fluorescence (TIRF) microscopy work?

TIRF microscopy utilizes an evanescent field created by total internal reflection of light at the interface of two media. This technique excites fluorophores near the surface of the sample, allowing for high signal-to-noise ratio imaging of surface-bound molecules.

What is the advantage of super-resolution techniques like PALM and STORM?

Super-resolution techniques like PALM and STORM allow for imaging at resolutions beyond the diffraction limit, providing the ability to observe structures at the nanoscale by localizing individual fluorophores with high precision.

How does stimulated emission depletion (STED) microscopy work?

STED microscopy uses a second laser beam to selectively deplete the fluorescence from the area surrounding the focal point of the first laser. This enables higher resolution by forcing fluorophores to emit only at the focal spot, improving the spatial resolution.