Fluorescence

First described in the 1800s by Sir George Stokes

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Fluorescence
Certain molecules absorbing the energy of electromagnetic radiation will jump to a higher energy level When these excited molecules return to the ground state they emit radiation This phenomenon is known as fluorescence Fluorescent molecules are known as fluorochromes or fluorophores
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Fluorescence microscope

an optical microscope used to study properties of organic Click to edit Master subtitle style or inorganic substances using the phenomena of fluorescence

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A fluorescence microscope is much the same as a conventional light microscope with added features to enhance its capabilities conventional microscope uses visible light (400-700 nanometers) to illuminate and produce a magnified image of a sample fluorescence microscope, on the other hand, uses a much higher intensity light source which excites a fluorescent species in a sample . This fluorescent species in turn emits a lower energy light of a longer wavelength
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The

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 There

are two main types of light source used; xenon arc lamp or mercury-vapor lamps

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The Dichroic Mirror

In a fluorescence microscope, a dichroic mirror is used to separate the excitation and emission light paths
The The

excitation light reflects off the surface of the dichroic mirror into the objective. fluorescence emission passes through the dichroic to the eyepiece or detection system.
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Two filters : Filter : ensures the illumination is near monochromatic and at the correct wavelength Emission filter : ensures none of the excitation light source reaches the detector.

Excitation

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 Excitation

filter -- In order to select the excitation wavelength, an excitation filter is placed in the excitation path just prior to the dichroic mirror. filter only transmitts the emission wavelength of the light emitted from the sample and remove traces of excitation light

Emission

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Sample preparation
In

order for a sample to be suitable for fluorescence microscopy it must be fluorescent. There are several methods of creating a fluorescent sample; the main techniques are labelling with fluorescent stains

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fluorescent stains
These

are small molecules which are intrinsically fluorescent and bind a biological molecule of interest. Major examples of these are nucleic acid stains like DAPI and Hoechst which bind the minor groove of DNA, thus labelling the nuclei of cell are many fluorescent reported molecules, called fluorophores such as fluorescein and DyLight 488, which can be
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There

Uses
Imaging

structural components of small specimens, such as cells viability studies on cell populations (are they alive or dead?) the genetic material within a cell (DNA and RNA)

Conducting

Imaging

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Limitations
Unlike

transmitted and reflected light microscopy techniques fluorescence microscopy only allows observation of the specific structures which have been fluorescently labeled. lose their ability to fluoresce as they are illuminated in a process called photobleaching. Photobleaching occurs as the fluorescent molecules accumulate chemical damage from the electrons excited during fluorescence
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Fluorophores

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