How does a fluorescence microscopy works?

Overview of Fluorescence Microscopy

Fluorescence microscopy is a powerful technique used to visualize specimens that can emit light (fluoresce) upon being illuminated with light of a specific wavelength. This type of microscopy is widely used in biological sciences to observe and study various structures within cells and tissues.

Key Components of a Fluorescence Microscope

1. Light Source:

   • Often a high-intensity light source like a mercury or xenon lamp, or LEDs, which provides the excitation light of specific wavelengths.

2. Excitation Filter:

   • This filter selects the specific wavelength of light that will excite the fluorescent molecules in the specimen.

3. Dichroic Mirror:

   • A special mirror that reflects light of the excitation wavelength towards the specimen but allows the emitted fluorescent light (longer wavelength) to pass through.

4. Objective Lens:

   • Gathers light from the specimen and is designed to focus both the excitation and emitted light accurately.

5. Emission Filter:

   • This filter blocks the excitation light and allows only the emitted fluorescence light to reach the detector or the eyepiece.

6. Detector or Camera:

   • Captures the fluorescent light emitted from the specimen. This could be an eyepiece for direct viewing or a camera for capturing images.

Working Principle

1. Excitation:

   • The light source emits light that passes through the excitation filter, which selects the wavelength suitable for exciting the fluorescent dye or protein in the sample.

2. Fluorescence:

   • The excitation light hits the sample, and the fluorescent molecules absorb this light and then emit light at a longer wavelength (lower energy).

3. Detection:

   • The emitted light passes through the dichroic mirror and emission filter, which ensures that only the fluorescent light is detected, removing any scattered excitation light.

4. Image Formation:

   • The objective lens collects the emitted light, which is then captured by the detector or camera, forming an image of the fluorescent structures within the specimen.

Applications

• Cell Biology: Observing the localization and movement of proteins, nucleic acids, and other molecules within cells.
• Medical Diagnostics: Identifying and studying pathogens, cancer cells, and other disease markers.
• Neuroscience: Mapping neural circuits and studying brain function.
• Material Science: Examining the properties of various materials and nanostructures.

Types of Fluorescence Microscopy

• Widefield Fluorescence Microscopy: Traditional method where the entire specimen is illuminated.
• Confocal Microscopy: Uses point illumination and a pinhole to eliminate out-of-focus light, providing clearer images.
• Two-Photon Microscopy: Uses near-infrared light to excite fluorophores, allowing deeper tissue penetration and reduced phototoxicity.
• Super-Resolution Microscopy: Techniques like STED, PALM, and STORM that surpass the diffraction limit of light, providing extremely high-resolution images.