FLUORESCENCE MICROSCOPE

 FLUORESCENCE MICROSCOPE

·       The Fluorescence microscope is a specially modified Compound microscope furnished with an Ultraviolet (UV) radiation source and Filters that protect the viewer’s eye from injury by these dangerous rays.

·       The Fluorescence microscope was invented in the early part of the 19^th century by August Köhler, Carl Reichert and Heinrich Lehmann.

·       The most commonly used Fluorescence microscopy is Epifluorescence microscopy, also called Incident light or Reflected light Fluorescence microscopy.

·       Fluorescence microscopes employ an Objective lens that also acts as a Condenser. The Objective lens acts not only as a magnifier for the light emitted by the fluorescing object, but also as a Condenser for the Incident light.

·       Fluorescence microscopy uses Fluorescence dyes to examine the Structural organization and Spatial distribution of samples.

·       Fluorescence microscopy is particularly used to study Samples that are Complex and cannot be examined under conventional transmitted - Light microscope.

·       Fluorescence microscopy images helps to study the substances present in low concentrations where high-sensitivity is crucial to detect them.

Fluorescent dye molecules or Fluorochromes used for Fluorescence microscope

a)     Acridine orange (Fluoresces Orange colour)

b)    Fluorochrome Auramine O (Fluoresces Golden Yellow colour)

c)     Diamidino-2-phenyl indole (DAPI) (Fluoresces Green colour)

d)    Fluorescein Isothiocyanate (FITC) (Fluoresces Green colour)

e)     Tetramethyl rhodamine isothiocyanate (TRITC or Rhodamine) (Fluoresces Red colour).

Parts of Fluorescence Microscope

a)     Light Source (Xenon or Mercury vapor lamp)

b)    Exciter filter or Excitation filter

c)     Dichromatic mirror

d)    Objective lens

e)     Barrier filter or Emission filter

f)      Eye piece

Working Principle

·       A Mercury vapor arc lamp or other source produces an intense beam of light. The light emitted from the Mercury arc lamp is 10-100 times brighter than most Incandescent lamps and provides light in a wide range of wavelengths from Ultra-violet to the infrared.

·       The light from Mercury vapor passes through an Exciter filter or Excitation filter which removes Longer wavelength light and covert to Shorter wavelength light.  

·       The light passed through the Exciter filter is directed down the Microscope by a special mirror called the Dichromatic mirror.

·       The Dichromatic mirror reflects light of Shorter wavelengths (i.e., the Excitation light) through the Objective lens to the specimen, which is usually stained with special Fluorescent dye molecules called Fluorochromes.

·       The emitted Fluorescent light travels up through the Objective lens into the microscope.

·       The emitted Fluorescent light has a Longer wavelength, it passes through the Dichromatic mirror to a Barrier filter, which blocks out any residual Excitation light.

·       Finally, the Emitted light passes through the Barrier filter or Emission filter to the Eye pieces.

Epifluorescence Microscopy

Uses

a)     The Fluorescence microscope has become an essential tool in Medical Microbiology, Microbial Ecology and Material Science and various fields of Biosciences.

b)    The unique functionality of Fluorescence microscopes helps to identify cells and sub-microscopic cellular components with accuracy and details.

c)     Bacterial pathogens like Mycobacterium tuberculosis, the causative agent of Tuberculosis (Bright yellow colour) and Bacillus anthrasis, the causative agent of Anthrax (Fluoresces Green colour - FITC) can be identified.

d)    Microbial Ecologists use Epifluorescence microscopy to visualize photosynthetic microbes, as their pigments naturally fluoresce when excited by light of specific wavelengths.

e)     Fluorescence microscope is even possible to distinguish live bacteria from dead bacteria by the color they fluoresce after treatment with a special mixture of stains.

f)      In the field of Histochemistry, Fluorescence microscope is used to detect particles such as Neurotransmitter amines which cannot be seen by conventional Microscopes.

g)    In Food Chemistry, the Fluorescence microscope is used to assess the specific Food components in a product.

h)    In Mineralogical studies, the Fluorescence microscope is used to study the Minerals such as Coal, Graphene oxide and more.

i)      In Paper and Textile industry, the Fluorescence microscope is used to analyze the Fiber dimensions.

j)      Fluorescence microscopy is ideal for studies of porosity in ceramics, using a Fluorescent dye.

k)    Fluorescence microscopy is used for imaging the Genetic materials (DNA & RNA) within the cell.

l)      Fluorescence microscopy helps in viewing the specific cells within a larger population with techniques such as FISH (Fluorescence In situ Hybridization).

Fluorescence microscopy is a common tool for today’s life science research because it allows the use of multicolor staining, labeling of structures within cells, and the measurement of the physiological state of a cell.