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(First day)
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First day

  • Fluorescence Microscopy

Fluorescence Microscopy (Basic Concepts)

Fluorescence is an optical phenomenon that occurs when light is absorbed by a material or substance. As a result of this, light is emitted at a different wavelength. Usually, the emitted wavelength is longer than the incident one, unless the incident electromagnetic radiation is intense and one electron absorbs two photons causing a shorter wavelength emission. More specifically, when electrons absorb light, they get excited to higher energy states. Right after, the electrons fall back to the lowest excited energy state in a process known as internal conversion. They remain there for a considerable amount of time, in the order of 10 ns, and as they go back to the ground state they emit the photon.

Fluorescence microscopy

Summary of how a fluorescence microscope works

First, the sample needs to be dyed with fluorophores and be placed in the stage. Then, the light source sends the excitation light towards the excitation filter, which only lets through the excitation light. This is reflected by the dichroic mirror in a direction such that the reflected light passes through the objective to the sample. Then, the emitted fluorescence passes through the same objective in the same direction as the emitted light. Therefore, it once again reaches the dichroic mirror, which enables only the emitted light to go through. After this, the light hits the emission filter or blocking filter which blocks the excitation light and only lets the emission light through. Finally, the emitted light reaches the eyepiece or detection system and can be observed by the naked eye. The excitation and emission filters, along with the dichroic mirror, are placed in what is known as the filter cube.

Description of the main parts of a fluorescence microscope
  • Fluorophores are a type of dye or stains that are “attached” to the sample. They are characterized by being excited by specific wavelengths of irradiating light.
  • The light source generates high intensity light. For instance, arc-lamps, lasers and LEDs are used. The most common are the Mercury HBO and the Xenon HBO arc-discharge fluorescent lamps.
  • A dichroic mirror is a laser mirror that reflects a specific wavelength region and transmits other regions.
  • The excitation and emission filters are made of dyed glass or gelatin. Basically, in a glass substrate several layers are deposited, some of them are reflective and the others are transparent. Once the incident light hits the glass it will create constructive and destructive interference depending on the distances between the layers. The constructive interference will be yield to the pass of the light whereas the destructive interference will yield to the reflection of the incident light.
Epi-fluorescence

Epifluorescence microscopy, unlike regular fluorescence microscopy, allows the light source to direct light onto the specimen by first passing the excitation light through the microscope objective on its way towards the sample. Then, it is the same objective that collects the emitted fluorescence. In this case, the objective works as a condenser.

Stokes shift

There is a difference in the wavelength between the maximum of the emission spectrum and the maximum of the absorption spectrum. This difference is known as Stokes shift. Since the emitted light produces less energy than the excited light, the emission spectrum looks just like the absorption spectrum, but it is shifted to a higher wavelength.

Related Concepts

  • Incandescence is the emission of light due to high temperatures.
  • Phosphorescence has essentially the same process as fluorescence. However, the material does not immediately re-emit the absorbed light.

References

[1] [2] [3] [4] [5]



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