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Fluorescence Microscope SUSMITA CHAKRABARTY

INVERTED FLUORESCENCE MICROSCOPE

Fluorescence:

A process by which a photon is absorbed at one wavelength and released at a different wavelength or energy.

OR

The absorption and subsequent re-radiation of light by organic and inorganic specimens is typically the result of well-established physical phenomena described as being either fluorescence or phosphorescence.

Discovery

  • British scientist Sir George G. Stokes first described fluorescence in 1852.
  • He observed that the mineral fluorspar emitted red light when it was illuminated by ultraviolet excitation.
  • Stokes noted that fluorescence emission always occurred at a longer wavelength than of the excitation light.
  • This shift towards longer wavelength is known as Strokes Shift.
Sir George G. Stokes

Principle

  1. Most cellular components are colorless and cannot be clearly distinguished under a microscope. The basic premise of fluorescence microscopy is to stain the components with dyes.
  2. Fluorescent dyes, also known as fluorophores or fluorochromes, are molecules that absorb excitation light at a given wavelength (generally UV), and after a short delay emit light at a longer wavelength. The delay between absorption and emission is negligible, generally on the order of nanoseconds.
  3. The emission light can then be filtered from the excitation light to reveal the location of the fluorophores.
  • Fluorescence microscopy 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.
  • The image produced is based on the second light source or the emission wavelength of the fluorescent species rather than from the light originally used to illuminate, and excite, the sample.

Fluorescence Microscope

  • A fluorescence microscope uses a mercury or xenon lamp to produce ultraviolet light.
  • The light comes into the microscope and hits a Dichroic mirror - A mirror that reflects one range of wavelengths and allows another range to pass through. The dichroic mirror reflects the ultraviolet light up to the specimen.
  • The ultraviolet light excites fluorescence within molecules in the specimen. The objective lens collects the fluorescent-wavelength light produced. This fluorescent light passes through the dichroic mirror and a barrier filter (that eliminates wavelengths other than fluorescent), making it to the eyepiece to form the image
Simple illustration of the wide-field fluorescence microscopy.
Schematic illustration of the wide-field fluorescence microscopy.

Working

  • 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.
Filter cube of a Fluorescence Microscope

Parts of Fluorescence Microscope

Fluorescence Microscope parts with mechanism.

Typical components of a fluorescence microscope are:

Fluorescent dyes (Fluorophore)

  • A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation.
  • Fluorophores typically contain several combined aromatic groups, or plane or cyclic molecules with several π bonds.
  • Many fluorescent stains have been designed for a range of biological molecules.
Fluorophore - A fluorescent chemical compound
A detailed Fluorophore

A light source

A Xenon Arc lamp
Filter cube consists of: (a)The excitation filter; (b) The Dichroic Mirror; (c) The emission filter

The excitation filter

  • The exciter is typically a bandpass filter that passes only the wavelengths absorbed by the fluorophore, thus minimizing the excitation of other sources of fluorescence and blocking excitation light in the fluorescence emission band.
Excitation filter

The dichroic mirror

  • A dichroic filter or thin-film filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors.
Dichroic mirror

The emission filter

  • The emitter is typically a bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band – especially the excitation light.
  • By blocking unwanted excitation energy (including UV and IR) or sample and system autofluorescence, optical filters ensure the darkest background.
Emission Filter

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 labeling with fluorescent stains or,
  • In the case of biological samples, expression of a fluorescent protein.

Alternatively the intrinsic fluorescence of a sample (i.e., autofluorescence) can be used. In the life sciences fluorescence microscopy is a powerful tool which allows the specific and sensitive staining of a specimen in order to detect the distribution of proteins or other molecules of interest. As a result, there is a diverse range of techniques for fluorescent staining of biological samples.

A sample of herring sperm stained with SYBR green in a cuvette illuminated by blue light in an epifluorescence microscope. The SYBR green in the sample binds to the herring sperm DNA and, once bound, fluoresces giving off green light when illuminated by blue light.

Biological Fluorescent Stains

  • Many fluorescent stains have been designed for a range of biological molecules.
  • Some of these are small molecules which are intrinsically fluorescent and bind a biological molecule of interest. Major examples of these are nucleic acid stains such as DAPI and Hoechst (excited by UV wavelength light).
  • DAPI (4',6-diamidino-2-2-phenylindole) is a fluorescent stain that binds strongly to A-T rich regions in DNA.
  • Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA.
  • A major example of fluorescent stain is phalloidin which is used to stain actin fibres in mammalian cells.
  • There are many fluorescent molecules called fluorophores or fluorochromes such as fluorescein, Alexa Fluors or DyLight 488, which can be chemically linked to a different molecule which binds the target of interest within the sample.

Immunofluorescence

  • Immunofluorescence is a technique which uses the highly specific binding of an antibody to its antigen in order to label specific proteins or other molecules within the cell.
  • A sample is treated with a primary antibody specific for the molecule of interest.
  • A fluorophore can be directly conjugated to the primary antibody.
Two-color immunofluorescence image of human keratynocytes stained with an anti-TOM20 (mitochondria, in magenta) and anti-HSP60 (membrane, magenta).

Epifluorescence Microscopy

  • Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in life sciences.
  • The excitory light is passed from above (or, for inverted microscope, from below), through the objective lens and then onto the specimen instead of passing it first through the specimen.
  • The fluorescence in the specimen then gives rise to the emitted light which is focused to the detector by the same objective lens that is used for excitation.
Epifluorescence microscope scheme

Applications of Fluorescence Microscopy

  • To identify structures in fixed and live biological samples.
  • Fluorescence microscopy is a common tool for today’s life science research because it allows the use of multi-color staining, labeling of structures within cells, and the measurement of the physiological state of a cell.
  • Diagnostic of diseases.

Advantages of Fluorescence Microscope

  1. Fluorescence microscopy is the most popular method for studying the dynamic behavior exhibited in live-cell imaging.
  2. This stems from its ability to isolate individual proteins with a high degree of specificity amidst non-fluorescing material.
  3. The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer.
  4. Different molecules can now be stained with different colors, allowing multiple types of the molecule to be tracked simultaneously.
  5. These factors combine to give fluorescence microscopy a clear advantage over other optical imaging techniques, for both in vitro and in vivo imaging.

Fluorescence Microscopy gallery

A z-projection of an osteosarcoma cell, stained with phalloidin to visualise actin filaments. The image was taken on a confocal microscope, and the subsequent deconvolution was done using an experimentally derived point spread function.
Endothelial cells under the microscope. Nuclei are stained blue with DAPI, microtubules are marked green by an antibody bound to FITC and actin filaments are labeled red with phalloidin bound to TRITC. Bovine pulmonary artery endothelial (BPAE) cells.
3D dual-color super-resolution microscopy with Her2 and Her3 in breast cells, standard dyes: Alexa 488, Alexa 568. LIMON microscopy.
Super-resolution microscopy: Co-localization microscopy (2CLM) with GFP and RFP fusion proteins (nucleus of a bone cancer cell) 120.000 localized molecules in a wide-field area (470 µm2) measured with a Vertico-SMI/SPDMphymod microscope.
Yeast cell membrane visualized by some membrane proteins fused with RFP and GFP fluorescent markers. Imposition of light from both of markers results in yellow color.
Human lymphocyte nucleus stained with DAPI with chromosome 13 (green) and 21 (red) centromere probes hybridized []Fluorescent in situ hybridization (FISH)]
Fluorescence microscopy images of sun flares pathology in a blood cell showing the affected areas in red.