Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan...
Transcript of Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan...
![Page 1: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/1.jpg)
Advanced fluorescence microscopy techniques
Ferhan AyaydinBRC, CELLULAR IMAGING (CI) LABORATORY
10 May 2017
„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001
![Page 2: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/2.jpg)
Lecture Outline
History of microscopesFluorescence and Fluorochromes
Fluorescence MicroscopeConfocal Microscope
Applications of Confocal Microscope
Ferhan Ayaydin
![Page 3: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/3.jpg)
History of Microscopy
"Emeralds are usually concave so that they may concentrate the visual rays.The Emperor Nero used to watch in an Emerald the gladiatorial combats."
Pliny the Elder 23-79 A.D
![Page 4: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/4.jpg)
The first known compound microscope, made by Zacharias and Hans Janssen in the 1590's.
Antoni van Leeuwenhoek was an amateur Dutch scientist who was granted for his discoveries in microscopy and high quality, but crude optical microscopes.
Jeroen Rouwkema, Wikipedia
Public domain, Wikipedia
History of Microscopy
![Page 5: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/5.jpg)
The Robert Hooke’s design was a functional improvement over the traditional motif, and even included a lighting apparatus to aid in specimen illumination
Hooke's microscope, from an engraving in Micrographia.
Public domain, Wikipedia Public domain, Wikipedia
History of Microscopy
![Page 6: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/6.jpg)
History of Microscopy
18th Century MicroscopesEdal Anton Lefterov, Wikipedia
![Page 7: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/7.jpg)
19th Century
Hannes Grobe, Wikipedia
History of Microscopy
![Page 8: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/8.jpg)
20th Century MicroscopesPublic domain, Wikipedia Public domain, Wikipedia
History of Microscopy
![Page 9: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/9.jpg)
21st Century Microscopes
Ferhan Ayaydin
History of Microscopy
![Page 10: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/10.jpg)
fluorescent mineralsHannes Grobe, Wikipedia
![Page 11: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/11.jpg)
UV light
white light
Fluorescence and Fluorophores
Didier Descouens, Wikipedia
the term fluorescence comes from the mineral “fluorite”
TransControl, Wikipedia
![Page 12: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/12.jpg)
Fluorescence occurs when a molecule relaxes to its ground state following
excitation
Excitation: S0+hv S1
Emission: S1S0+hv
Stoke’s shift
Jablonski diagram of absorbance, non-radiative decay, and fluorescence
Public domain, Wikipedia
![Page 13: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/13.jpg)
Detection of proteins by Immunofluorescence
Common FluorochromesFITCRhodamineTexas RedCyanine dyes
Novel AlexaFluor dyeswide spectrum, stablebrighter and bleach resistant
Jakodak, Wikipedia
Maria Francia & Boris Striepen, University of Georgia, Wikipedia
![Page 14: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/14.jpg)
Staining Organelles with FluorochromesNucleusDAPIHoechst dyesEthidium BromidePropidium IodideAcridine Orange
MitochondriaMitotrackerMitofluor dyesNonyl acridine orange
Golgi/ERER trackerfluorescent Ceramidefluorescent Sphingomyosin
LysozmeLysotrackerGerry Shaw, Wikipedia
![Page 15: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/15.jpg)
What if my research project requires a fluorescent dye that….
...specifically stains lung cancer cells?
Public domain, Wikipedia
![Page 16: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/16.jpg)
Novel fluorescent chemical discovery through combinatorial chemistry
Ferhan Ayaydin
![Page 17: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/17.jpg)
Discovery of novel live cell permeable fluorescent chemicals
oil bodies
mitochondria
membrane
mitochondria
14585 compounds > microarray scanner > confocal microscopyFerhan Ayaydin and Soujanya Kuntam
![Page 18: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/18.jpg)
Novel dyes to stain plant oil bodies in live cells
B2
C6
Ferhan Ayaydin and Soujanya Kuntam
![Page 19: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/19.jpg)
Green Fluorescent Protein :GFP
GFP is a small protein (27 kD) and the DNA sequences coding for GFP can be manipulated by recombinant DNA technology to create gene fusion
promoter | GFP | your favorite protein
promoter | your favorite protein | GFP
Sierra Blakely, Wikipedia
![Page 20: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/20.jpg)
• The GFP chromophore consists of a cyclic tripeptide derived from Ser-Tyr-Gly at positions 65–67 in the protein and is only fluorescent when embedded within the fully folded, complete GFP molecule.
• EGFP: Ser65 to Thr mutation (UV to blue excitation)
• Nascent GFP is not fluorescent, since chromophore formation occurs post-translationally. The chromophore is formed by a cyclization reaction and an oxidation step at Tyr66 that requires molecular oxygen
The Mechanism of Glow
Jim G, Wikipedia
![Page 21: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/21.jpg)
YELLOW Fluorescent Protein (YFP)(Thr 203 to Tyr)
Citrine variant is very bright relative to EYFP and has been demonstrated to be much more resistant to photobleaching, acidic pH, and other environmental effects
Another derivative, named Venus, is the fastest maturing and one of the brightest yellow variant
CYAN Fluorescent Protein (CFP)(Tyr66 to Tryptophan)
BLUE Fluorescent Protein (BFP)(Tyr66 to His)
Fluorescent Protein Color Variants
RED Fluorescent Protein (RFP)Susano-o, Wikipedia
![Page 22: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/22.jpg)
Artwork by Nathan Shaner, photography by Paul Steinbach, created in the lab of Roger Tsien in 2006, Wikipedia
A San Diego beach scene drawn with an eight color palette of bacterial colonies expressing fluorescent proteins derived from GFP and the red-fluorescent coral protein dsRed. The colors include BFP, mTFP1, Emerald, Citrine, mOrange, mApple, mCherry and mGrape.
![Page 23: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/23.jpg)
The biggest advantage of using GFP is...
Ferhan Ayaydin
![Page 24: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/24.jpg)
Nuno Nogueira, Wikipedia
upright inverted
Fluorescence Microscope
Masur, Wikipedia
![Page 25: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/25.jpg)
Henry Mühlpfordt, Krzysztof Blachnicki, Wikipedia
Fluorescence Microscope
![Page 26: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/26.jpg)
Mercury Arc Lamp
UV IR
DETECTOR
filter block
![Page 27: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/27.jpg)
dichoric mirror
Filter sets used in fluorescence microscopy
filter cube
Ferhan Ayaydin
excitation filter
emission filter
![Page 28: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/28.jpg)
Confocal laser scanning microscopy
Danh, Wikipedia
![Page 29: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/29.jpg)
Optical sectioning with confocal microscope
Ferhan Ayaydin
![Page 30: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/30.jpg)
Advanced Applications of Confocal Microscopy
Protein Dynamics and Interaction
A) Bleaching techniquesFRAP, iFRAP, FLIP
B) Photoconversion techniquesPA-GFP, Kaede/Kikume, PS-CFP, EosFP, DRONPA
C) Protein-protein InteractionsFRET, BiFC
![Page 31: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/31.jpg)
FRAP: Fluorescence Recovery After Photobleaching
Selective Laser bleachingwith Laser Scanning Confocal Microscope
Fluorescence recovery
A) Bleaching techniques: FRAP, iFRAP, FLIP
Ferhan Ayaydin
![Page 32: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/32.jpg)
Ferhan Ayaydin
FRAP: Protein Mobility Comparison
protein X protein Y
A) Bleaching techniques FRAP, iFRAP, FLIP
![Page 33: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/33.jpg)
FRAP: Kinetics of Fluorescence Recovery
A) Bleaching techniques FRAP, iFRAP, FLIP
immobile fraction
mobile fractionshalf recovery: t1/2
![Page 34: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/34.jpg)
Public Domain , Wikipedia
A) Bleaching techniques FRAP, iFRAP, FLIP
Diffusion-limited fluorescence recovery
Reaction-limited recovery
![Page 35: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/35.jpg)
Ferhan Ayaydin
A) Bleaching techniquesFRAP, iFRAP, FLIP
iFRAP: Inverse FRAP
prebleach bleach recovery(outside of region of interest)
dissociation parameters of molecules can be measured
Region of interest(ROI) bleaching laser
![Page 36: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/36.jpg)
A) Bleaching techniquesFRAP, iFRAP, FLIP
Ferhan Ayaydin
![Page 37: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/37.jpg)
A) Bleaching techniquesFRAP, iFRAP, FLIP
Ferhan Ayaydin
![Page 38: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/38.jpg)
A) Bleaching techniquesFRAP, iFRAP, FLIP
Ferhan Ayaydin
![Page 39: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/39.jpg)
Ferhan Ayaydin
![Page 40: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/40.jpg)
B) Photoconversion techniquesPA-GFP, Kaede/Kikume, PS-CFP, EosFP, DRONPA
Wikipedia
![Page 41: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/41.jpg)
C) Protein-protein InteractionsFRET, BiFC
Ferhan Ayaydin
![Page 42: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/42.jpg)
Ferhan Ayaydin
C) Protein-protein InteractionsFRET, BiFC
![Page 43: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/43.jpg)
Ferhan Ayaydin
C) Protein-protein InteractionsFRET, BiFC
![Page 44: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/44.jpg)
C) Protein-protein InteractionsFRET, BiFC
BiFC: Bimolecular Fluorescence Complementation
Public domain,Wikipedia
![Page 45: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/45.jpg)
Discussion:
What happens if we use half GFP, half CFP and half YFP?
Multicolor Complementation Is that possible?
C) Protein-protein InteractionsFRET, BiFC
![Page 46: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/46.jpg)
Public domain, Wikipedia
![Page 47: Advanced fluorescence microscopy techniques · Advanced fluorescence microscopy techniques Ferhan Ayaydin BRC, CELLULAR IMAGING (CI) LABORATORY 10 May 2017 „Practice-oriented, student-friendly](https://reader031.fdocuments.in/reader031/viewer/2022021812/5e5ab78f4dcbab45120976d3/html5/thumbnails/47.jpg)
Thank you for your attention!
This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practice-
oriented, student-friendly modernization of the biomedical education for strengthening the international
competitiveness of the rural Hungarian universities " TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project.