Fluorescence microscope

A fluorescence microscope is a special kind of optical microscope that helps scientists see tiny things by using a process called fluorescence. Instead of just using light that bounces off an object or passes through it, this microscope can make certain materials glow. This glowing light helps scientists study the properties of both natural and inorganic substances, like cells or tiny particles.

Fluorescence microscopes can be simple or very advanced. Some are basic setups, like an epifluorescence microscope, while others are more complex, such as a confocal microscope. These advanced microscopes use a method called optical sectioning to get clearer and more detailed pictures of the glowing materials. This helps scientists see smaller details and understand how things work at a very tiny level.

Principle

A fluorescence microscope shines special light on an object, which makes the object glow a different color. This glowing light is separated from the original light using special filters. Common parts of these microscopes include a light source like a lamp or laser, and filters that pick out the right colors of light.

Most fluorescence microscopes used today are called epifluorescence microscopes. In these, the same lens is used both to shine light on the object and to collect the glowing light. This design is popular in biology and forms the basis for more advanced microscopes.

Light sources

Fluorescence microscopes need very bright, single-colored light, which regular lights like halogen lamps cannot give. There are four main types of lights used: xenon arc lamps, mercury-vapor lamps with an excitation filter, lasers, supercontinuum sources, and high-power LEDs. Lasers are often used for more advanced techniques like confocal microscopy and total internal reflection fluorescence microscopy. Xenon lamps, mercury lamps, and LEDs with a dichroic excitation filter are usually used for widefield epifluorescence microscopes. Using two microlens arrays can make the light very even.

Sample preparation

For a sample to work with a fluorescence microscope, it needs to glow or fluoresce. There are a few ways to make this happen. One way is to use special glowing stains. For living things, scientists can even make cells produce their own glowing material.

Fluorescent stains can stick to specific parts of a cell, like the material that makes up the center of the cell or the tiny parts that help the cell move. Some glowing materials can be linked to other molecules to find exactly what scientists are looking for in a sample.

One special method uses tiny hooks that stick only to certain parts of a cell, making those parts glow. This helps scientists see where important cell pieces are, even in living cells.

Limitations

When using a fluorescence microscope, the special colors that help us see tiny parts can fade over time. This fading, called photobleaching, happens because the light can damage the molecules that glow. Scientists use special chemicals or stronger glowing colors to help slow this down.

Looking at living cells with this kind of microscope can also be tricky. The bright light can harm the cells, especially if it is very strong. Some new computer methods can help by guessing what the glowing parts look like without using the light, which keeps the cells safer. However, this type of microscope only shows what has been marked to glow, so it can only reveal specific details and not everything in the cells.

Sub-diffraction techniques

See also: Super resolution microscopy and Correlative Light-Electron Microscopy

Light has a natural limit to how small a spot it can focus on, called the diffraction limit. This was explained in the 1800s and stops microscopes from showing very tiny details clearly.

Fluorescence microscopes help scientists find ways to see past this limit. In the 1900s, new methods were created to improve how well microscopes could show details, but they still couldn’t beat the diffraction limit. Later, ideas like the 4Pi microscope were developed to try to overcome this limit by focusing light from many directions at once.

One of the first methods to really beat the diffraction limit was STED microscopy, created in the 1990s. This and other similar methods work by controlling special light interactions with glowing molecules to see much smaller details.

Other methods, like SPDM localization microscopy and photoactivated localization microscopy, also help scientists see very tiny parts inside cells by using special glowing markers and controlling how they light up.

Fluorescence micrograph gallery

These images show what scientists can see using a fluorescence microscope. Each picture highlights different parts of cells or tiny structures inside them, making them glow in special colors to help us learn more about how living things work.

The pictures include cells with their parts stained in blue, green, and red to show details like DNA, proteins, and tiny thread-like structures. Some images even let us see single molecules or very small details in cells.