Fluorescence microscope

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

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

Principle

A fluorescence microscope uses special light to make an object glow a different color. This glowing light is separated from the original light using special filters. Common parts include a light source like a lamp or laser, and filters that choose the right colors of light.

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

Light sources

Fluorescence microscopes need very bright, single-colored light. Regular lights like halogen lamps cannot do this. 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 advanced techniques like confocal 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 limit to how small a spot it can focus on, called the diffraction limit. This was found in the 1800s and stops microscopes from showing very tiny details.

Fluorescence microscopes help scientists see past this limit. In the 1900s, new methods were made to improve microscopes, but they still couldn’t beat the diffraction limit. Later, ideas like the 4Pi microscope were made to try to overcome this limit by focusing light from many directions.

One of the first methods to beat the diffraction limit was STED microscopy, made 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. They make different parts of cells or tiny structures inside them glow in special colors. This helps us learn more about how living things work.

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