Fluorescence imaging

What is fluorescence imaging?
What is fluorescence microscopy?
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What is fluorescence imaging?

Fluorescence imaging is an imaging technique that visualizes cells and molecules by fluorescently labeling them using fluorescent dyes, fluorescent proteins, and NV centers. Generally, cells and molecules are colorless and transparent, so they cannot be observed as they are. There are various staining methods other than fluorescent labeling to visualize cells; however, many of them can damage the cells. Fluorescent dyes, fluorescent proteins, and NV centers used in fluorescence imaging can label cells while they are alive, enabling live cell imaging of organisms. This allows us to obtain various information such as cell structure and function, and molecular interactions. Fluorescence imaging has become an important tool in the development of biology.

Confocal microscopy

Fluorescence imaging made significant advancements thanks to the Nobel Prize in Chemistry awarded in 2008 to three researchers: Professor Osamu Shimomura, Professor Martin Chalfie, and Professor Roger Y. Tsien. The prize recognized their discovery and development of the green fluorescent protein (GFP). GFP is a protein isolated from the bioluminescent jellyfish. Professor Shimomura was the first to successfully isolate GFP from jellyfish after observing that it emits green light when exposed to ultraviolet radiation. Subsequently, Professor Martin Chalfie contributed to understanding the fluorescence emission mechanism and successfully expressed GFP within living cells using genetic engineering techniques. Additionally, Professor Roger Y. Tsien elucidated the molecular mechanism of GFP chromophore formation, allowing the creation of artificial fluorescent proteins that emit various colors beyond green. This breakthrough enabled the expression of different colored fluorescent proteins within living organisms, facilitating simultaneous tracking of multiple biological phenomena, including the analysis of interactions between different proteins.

Structure of green fluorescent protein

Figure 1: Structure of green fluorescent protein (GFP)

Example of fluorescently stained cell imaging

Figure 2: Example of fluorescence-stained cells

What is fluorescence microscopy?

A fluorescence microscope is a type of microscope used for imaging cellular structures through fluorescence. A sample labeled with a fluorescent dye or protein is irradiated with excitation light, and the fluorescence emitted from the sample is focused by an objective lens and photographed by a camera.

A fluorescence microscope mainly consists of an optical system including a light source, filter, dichroic mirror, and objective lens. Figure 3 shows a schematic diagram of a commonly used epifluorescence microscope. In an epifluorescence microscope fluorescence microscope, light emitted from the light source (excitation light) has its unwanted wavelengths cut off by an excitation filter. The excitation light is then reflected by a dichroic mirror and irradiated onto the sample through an objective lens. The sample irradiated by the excitation light emits fluorescence, which is passed through the objective lens, dichroic mirror, and fluorescence filter (absorption filter) before being guided to a detector such as an eyepiece lens or camera.

In recent years, various fluorescence imaging techniques have been developed, including confocal microscopy, super-resolution microscopy, light-sheet microscopy, and multiphoton microscopy. These fluorescence imaging techniques enable observation at higher resolution, faster speeds, and greater depths than ever before.

Schematic diagram of an epifluorescence microscope

Figure 3: Schematic diagram of an epifluorescence microscope

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Confocal microscopy

An imaging technique that removes fluorescence generated outside the focal plane, resulting in high-resolution images with minimal blur.

Super-resolution microscopy

An imaging technique that is used to observe with resolution beyond the diffraction limit of conventional optical microscopes.

Light-sheet microscopy

An imaging technique that allows rapid image acquisition while minimizing phototoxicity by exciting the sample using sheet illumination.

Multiphoton microscopy

An imaging technique that enables deep image acquisition by utilizing multiphoton excitation phenomena.

Fluorescence lifetime imaging microscopy

An imaging technique that maps the spatial distribution of sample fluorescence lifetimes and acquires images.

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