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  3. Devices Required for Fluorescence Observation: Fluorescence Observation

Basics of Microscopes

To perform fluorescence observation

Devices Required for Fluorescence Observation

In line with the advancement of research in life sciences, technologies for observing cells and proteins in a living state have also advanced. Fluorescence observation has played an important role in this evolution.
This technology uses a fluorescent dye or protein as a label to emphasize and project the target area. After a particular light (excitation light) is emitted to molecules of a substance, light energy is absorbed and the electrons in them are excited. Then, light called fluorescence is emitted in the process of returning to the ground state.

Using this phenomenon of exciting electrons, fluorescence microscopy uses a light source, an objective lens, and a fluorescent filter to pass a specific wavelength of light, amongst other equipment.

Selecting an appropriate fluorescent filter is important to obtain better images. The filters consists of an excitation filter to pass light with a specific wavelength from the light source, and an emission filter to pass the fluorescence emitted from the sample. A dichroic mirror is also included to reflect light at a particular wavelength and to pass light of a different wavelength to separate excitation light from fluorescence.
Recently, fluorescent filter sets (fluorescent filter cubes) are available from each manufacturer according to characteristics of fluorescent reagents, so users can select an appropriate combination based on their specimens.

Key points when selecting fluorescent filters

When selecting a fluorescent filter set (fluorescent filter cube), the user must closely match the characteristics of a fluorescent dye with the wavelength range of the set's excitation and emission filters. Characteristics of each filter are as follows:

- Excitation filter

There are narrow-band and broad-band filters according to the width of the wavelength band of light to support. Basically, the wider the band, the brighter image you can get. However, a narrow-band filter is generally used to reduce damage to living samples.

- Emission filter

This type of filter is roughly classified into long-pass (LP), short-pass (SP), and band-pass (BP) filters. LP filters only transmit wavelengths longer than a specific wavelength while SP filters only transmit wavelengths shorter than a specific wavelength. BP filters only transmit light in a specific wavelength band.
When using a single fluorescent reagent, you can obtain a brighter image by using an LP filter. On the other hand, when using several fluorescent dyes, you can obtain fluorescence images for each by using different BP filters.

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