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Fluorescence microscopy
- 1. PRESENTED BY: JYOTSNAVERMA RAHUL VERMA SRISHTI SHARMA
- 3. A processby which a photon is absorbed at one wavelength and released at a different wavelength or energy. JABLONSKI DIAGRAM
- 8. A fluorescencemicroscope 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.
- 10. Fluorescence microscopyrequires intense, near- monochromatic, illumination which some widespread light sources, like halogen lamps cannot provide. Four main types of light source are used, including xenon arc lamps or mercury-vapor lamps . Lasers are most widely used for more complex fluorescence microscopy techniques like confocal microscopy and total internal reflection fluorescence microscopy while xenon lamps, and mercury lamps, and LEDs with a dichroic excitation filter are commonly used for widefield epifluorescence microscopes.
- 13. Epifluorescence microscopyis 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 throgh 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.
- 14. A fluorophore(or fluorochrome, similarly to a chromophore) 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. Types: internal: are part of molecule .e.g.Trp amino acid in proteins. external: molecules that lack internal fluoroscense are linked with other small fluorescent molecules externally
- 15. Many fluorescentstains 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 like DAPI and Hoechst (excited by UV wavelength light). DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to A-T rich regions in DNA. Hoechst stains are part of a family of bluefluorescent dyes used to stain DNA.
- 16. A majorexample 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.
- 17. 1. Immunology: Anantibody is first prepared by having a fluorescent chemical group attached, and the sites (e.g., on a microscopic specimen) where the antibody has bound can be seen, and even quantified, by the fluorescence. 2. Cell and molecular biology: • detection of colocalization using fluorescence-labelled. • Imaging structural components of small specimens, such as cells. • Conducting viability studies on cell populations (are they alive or dead?) • Viewing specific cells within a larger population with techniques such as FISH. Detection and determination of the proteins localization in cell and tissue 3. Diagnostic of diseases
- 19. The greenfluorescent protein (GFP) is a protein composed of 238 amino acid residues (27kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The GFP,first isolated from the jellyfish Aequorea victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression.
- 20. GFP hasa beta barrel structure consisting of eleven β-strands, with an alpha helix that runs through the center of the barrel, containing the covalently bonded chromoph ore 4-(p- hydroxybenzyliden e)imidazolidin-5- one (HBI) running through the center.
- 21. The chromophoreis located in the middle of the beta-barrel, it is occasionally referred to as the “light in the can.” Five shorter alpha helices form caps on the ends of the structure. The beta barrel structure is a nearly perfect cylinder, 42Å long and 24Å in diameter,creating what is referred to as a "β- can" formation, which is unique to the GFP- like family. HBI, the spontaneously modified form of the tripeptide Ser65–Tyr66–Gly67, is nonfluorescent in the absence of the properly folded GFP scaffold.
- 22. Biological marker: Fusion of GFP to a protein does not alter the function ,mobility abilities or location of the protein. This feature has enabled researchers to use GFP in living systems, and it has led to GFP’s widespread use in cell dynamics and development studies. Reporter Gene: - The first applications of GFP were as a reporter gene -Gene expression level in living cells.
- 23. By joining theGFP gene to the gene of the protein of interest so that when the protein is made it will have GFP hanging off it. Since GFP fluoresces, one can shine light at the cell and wait for the distinctive green fluorescence associated with GFP to appear.
- 24. GFP hasthe ability to exhibit intrinsic fluorescence through three amino acids that cyclise (Ser65-Tyr66-Gly67) and then undergo an oxidation step during a complex maturation process. The 3 amino acids forming the chormophore in GFP undergoes oxidation and cyclization. The formed active chromophore contains conjugated double bonds. These double bonds store and release the energy from electrons.
- 25. Topology ofFolding The above diagram illustrates the topology of the folding pattern. Beta-strands are shown in green, alpha-helices in red and connecting loops in black. The numbers of the residues at the beginning and end of the secondary structure elements are given as well.
- 27. It isRoger Tsien who is responsible for much of our understanding of how GFP works and for developing new techniques and mutants of GFP. His group has developed mutants that start fluorescing faster than wild type GFP, that are brighter and have different colors (see below, the E stands for enhanced versions of GFP, m are monomeric proteins and tdTomato is a head-to- tail dimer).
- 29. The DsRedis a protein tetramer having the property of emitting a fluorescence color red . From a coral (Discosoma ), this protein is intrinsically fluorescent. Its gene can be fused in vitro to the gene of a protein that one wishes to study. The recombinant gene is then reintroduced into cells or an embryo , which will then synthesize the then fluorescent fusion protein. It can then be observed using a fluorescence microscope, for example. This method makes it possible to study proteins in their natural environment: the living cell. DsRed has excitation maxima at 558 nm (green light) and emission maxima wavelength is 583nm.
- 30. The chromophoreis formed by a cyclization reaction from amino acids Gln66 , Tyr67 , and Gly68 . The Tyr67 amide nitrogen binds to the Gln66 backbone carbonyl (with elimination of a water molecule) generating the imidazolone ring. Oxidation step 1:generates a green fluorescing structure. Second oxidation step:involving the amide nitrogen of Phe65 enlarges the resonant pi electon cloud to the red fluorescing chromophore.
- 31. One ofits crippling defects is the slow processing of the protein as one of the intermediate forms has a green fluorescence similar to that of the green fluorescent protein (GFP). Furthermore, DsRed is an obligate tetramer and can form large protein aggregates in living cells. In contrast to the jellyfish fluorescent proteins, which have been successfully used to tag hundreds of proteins, DsRed conjugates have proven much less successful and are often toxic.
- 32. But wild-typeDsRed has several drawbacks, including slow chromophore maturation and poor solubility. To overcome the slow maturation, we used random and directed mutagenesis to create DsRed variants that mature 10–15 times faster than the wild-type protein. An asparagine-to-glutamine substitution at position 42 greatly accelerates the maturation of DsRed, but also increases the level of green emission. Additional amino acid substitutions suppress this green emission while further accelerating the maturation. To enhance the solubility of DsRed, we reduced the net charge near the N terminus of the protein. The optimized DsRed variants yield bright fluorescence even in rapidly growing organisms such as yeast.
- 33. There arenow different variants of DsRed that have been obtained by modifying it by genetic engineering. These mutation steps made it possible to obtain dimeric, monomeric forms, to increase the brightness or to change the range of wavelengths involved in the excitation and emission spectra. Variants obtained by mutagenesis: dimer2 : dimeric variant mRFP1 : monomeric variant , slow maturation dTomato : dimeric variant tdTomato : tetrameric variant mOrange : monomeric variant, orange fluorescence mBanana : monomeric variant, orange fluorescence mHoneydew : monomeric variant, green-orange fluorescence mStrawberry : monomeric variant, red fluorescence between DsRed and mRFP. mCherry : monomeric variant, mRFP-like fluorescence with faster maturation and better stability
- 34. http://www.chm.bris.ac.uk/motm/GFP/GFPh.htm https://www.conncoll.edu/ccacad/zimmer/GFP- ww/GFP-1.htm https://www.microscopyu.com/techniques/fluores cence/introduction-to-fluorescence-microscopy https://www.conncoll.edu/ccacad/zimmer/GFP- ww/GFP-1.htm