FLUORESCENCE MICROSCOPY PRESENTED BY KAARTHIK J

INTRODUCTION A fluorescence microscope is a optical microscope that uses fluorescence and phosphorescence instead of , or I addition to reflection and absorption to study properties of organic or inorganic substances. Fluorescence – Fluorescence is the emission of Light by a substance that has absorbed light or other electromagnetic radiation. Phosphorescence- phosphorescence is specific type of photoluminescence related to fluorescence. Unlike fluorescence, a phosphorescent material doesn’t re-emit the radiation it absorbs.

Principle of fluorescence microscopy The specimen is illuminated with light of a specific wavelength,which is absorbed by the flurophores, causing them to emit light of longer wavelengths(i.e. of a principle different color than the absorbed light).

Differences between conventional and fluorescence microscope Conventional microscope uses light to illuminate the sample and produce a magnified image of the sample whereas fluorescence microscope uses higher intensity light to illuminate the sample. and also produces magnified image of the sample but the image is based on the second light source.

Typical components of fluorescence microscopy Light source(xenon arc lamp or mercury vapor lamp or lasers) The excitation filter The dichoric mirror The emission filter

WORKING The microscope has a high power lamp source, usually a mercury or xenon arc lamp. An excitation filter transmits the band of the excitation radiation. The excitation radiation is reflected by the dichroic mirror towards the condenser/objective lens that focuses the light on the specimen. The illumination of the specimen as well as the collection of the fluorescence light is achieved by a the single lens. This has become possible due to the incorporation of dichroic mirror in the optics. A dichroic mirror is largely reflective for the light below a threshold wavelength and transmissive for the light above that wavelength. Light emitted by the fluorescent molecules (higher wavelength due to Stokes shift) is collected by the same lens and is transmitted by the dichroic mirror towards the ocular lens.

Applications Used for the study of biological molecules Imaging structural components of small specimens, such as cells Conducting viability studies on cell populations (are they alive or dead?) Imaging the genetic material within a cell (DNA and RNA)

Advanced models Confocal Laser Scanning Microscope(CLSM) : Confocal fluorescence microscopy overcomes this problem by using a pinhole to reject out-of-focus light, producing high-resolution optical slices, or projections with extended depth of focus Total Internal Reflection Fluorescence Microscopy(TIRFM) exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices

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