Fluorescence Microscope

FLUORESCENCE MICROSCOPE

 
A fluorescence microscope is basically a conventional light microscope with added features and components that extend its capabilities.

·         A conventional microscope uses light to illuminate the sample and produce a magnified image of the sample.

·         A fluorescence microscope uses a much higher intensity light to illuminate the sample. This light excites fluorescence species in the sample, which then emit light of a longer wavelength. A fluorescent microscope also produces a magnified image of the sample, but the image is based on the second light source -- the light emanating from the fluorescent species -- rather than from the light originally used to illuminate, and excite, the sample.

Working Principle:

In most cases the sample of interest is labelled with a fluorescent substance known as a fluorophore and then illuminated through the lens with the higher energy source. The illumination light is absorbed by the fluorophores (now attached to the sample) and causes them to emit a longer lower energy wavelength light. This fluorescent light can be separated from the surrounding radiation with filters designed for that specific wavelength allowing the viewer to see only that which is fluorescing.

The basic task of the fluorescence microscope is to let excitation light radiate the specimen and then sort out the much weaker emitted light from the image. First, the microscope has a filter that only lets through radiation with the specific wavelength that matches your fluorescing material. The radiation collides with the atoms in your specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light. To become detectable (visible to the human eye) the fluorescence emitted from the sample is separated from the much brighter excitation light in a second filter. This works because the emitted light is of lower energy and has a longer wavelength than the light that is used for illumination.

Most of the fluorescence microscopes used in biology today are epi-fluorescence microscopes, meaning that both the excitation and the observation of the fluorescence occur above the sample. Most use a Xenon or Mercury arc-discharge lamp for the more intense light source.

Instrumentation:

Nearly all fluorescence microscopes have following basic parts below.

·         Focus Illumination

·         Collector for emitted fluorescence

·         Dichroic mirror

·         Filter:

1.       Focus the illumination (excitation) light on the sample.

In order to excite fluorescent species in a sample, the optics of a fluorescent microscope must focus the illumination (excitation) light on the sample to a greater extent than is achieved using the simple condenser lens system found in the illumination light path of a conventional microscope.

2.       Collect the emitted fluorescence:

This type of excitation-emission configuration, in which both the excitation and emission light travel through the objective, is called epifluorescence. The key to the optics in an epifluorescence microscope is the separation of the illumination (excitation) light from the fluorescence emission emanating from the sample. In order to obtain either an image of the emission without excessive background illumination, or a measurement of the fluorescence emission without background "noise", the optical elements used to separate these two light components must be very efficient.

3.      The Dichroic Mirror

In a fluorescence microscope, a dichroic mirror is used to separate the excitation and emission light paths. Within the objective, the excitation emission share the same optics.

The excitation light reflects off the surface of the dichroic mirror into the objective.

The fluorescence emission passes through the dichroic to the eyepiece or detection system.

The dichroic mirror's special reflective properties allow it to separate the two light paths. Each dichroic mirror has a set wavelength value -- called the transition wavelength value -- which is the wavelength of 50% transmission. The mirror reflects wavelengths of light below the transition wavelength value and transmits wavelengths above this value. This property accounts for the name given to this mirror (dichroic, two color). Ideally, the wavelength of the dichroic mirror is chosen to be between the wavelengths used for excitation and emission.

The dichroic mirror is a key element of the fluorescence microscope, but it is not able to perform all of the required optical functions on its own. Typically, about 90% of the light at wavelengths below the transition wavelength value are reflected and about 90% of the light at wavelengths above this value are transmitted by the dichroic mirror. When the excitation light illuminates the sample, a small amount of excitation light is reflected off the optical elements within the objective and some excitation light is scattered back into the objective by the sample. Some of this "excitation" light is transmitted through the dichroic mirror along with the longer wavelength light emitted by the sample. This "contaminating" light would otherwise reach the detection system if it were not for another wavelength selective element in the fluorescence microscope: an emission filter.

Figure 1: Optical diagram of Fluorescent Microscope

4.      Excitation and Emission Filters

Two filters are used along with the dichroic mirror:

Excitation filter -- In order to select the excitation wavelength, an excitation filter is placed in the excitation path just prior to the dichroic mirror.

Emission filter -- In order to more specifically select the emission wavelength of the light emitted from the sample and to remove traces of excitation light, an emission filter is placed beneath the dichroic mirror. In this position, the filter functions to both select the emission wavelength and to eliminate any trace of the wavelengths used for excitation.

These filters are usually a special type of filter referred to as an interference filter, because of the way in which it blocks the out of band transmission. Interference filters exhibit an extremely low transmission outside of their characteristic bandpass. Thus, they are very efficient in selecting the desired excitation and emission wavelengths.

Applications:

The refinement of epi-fluorescent microscopes and advent of more powerful focused light sources, such as lasers, has led to more technically advanced scopes such as the confocal laser scanning microscopes and total internal reflection fluorescence microscopes (TIRF).

CLSM's are invaluable tools for producing high resolution 3-D images of sub-surfaces in specimens such as microbes. Their advantage is that they are able to produce sharp images of thick samples at various depths by taking images point by point and reconstructing them with a computer rather than viewing whole images through an eyepiece.

These microscopes are often used for -

·         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)

·         Viewing specific cells within a larger population with techniques such as FISH