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Final Exam: May 4th, 9-11am DCL 3211
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Last Class: Genetic Engineering 1. Restriction nucleases 2
Last Class: Genetic Engineering 1. Restriction nucleases 2. DNA labeling 3. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 4. Molecular cloning, DNA replication by vector 5. Gene sequencing 6. polymerase chain reaction 7. Monitoring Gene expression 8. the application of genetic engineering: Detect proteins and protein-protein interactions, library screening, gene mutation
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Visualizing Cells
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Resolving Power
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Light Microscope
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Interference between light waves
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Resolution Calculation
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Two ways to get contrast
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Four Types of light microscopy
Bright field, phase contrast, differential interference contrast, Dark-field microscopy
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Fluorescence Microscope
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Fluorescent Dyes
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Blue: DNA; Green: microtubules; Red: centrimere
Fluorescent image Blue: DNA; Green: microtubules; Red: centrimere
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Immunofluorescence
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Immunostaining
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Confocal Fluorescence Microscopy
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The difference between conventional and confocal microscopes
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3D reconstruction from confocal images
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Transmission Electron microscopy (TEM, resolution 0.002 nm)
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A root-tip cell under electromicroscopy
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The scanning electron microscope (SEM)
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Stereocillia from a hair cell
TEM DIC SEM
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Summary of Visualizing Cells
Transmitted lights Fluorescence Electron microscopy
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Fluorescence Proteins and Live Cell Imaging
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A Cell and A City
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Track Molecular Motions
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Jellyfish and GFP Osamu Shimomura discovered GFP in 1962
Shimomura O, et al, 1962. J. Cell. Comp. Physiol.
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Dr. Douglas Prasher Prasher DC, et al Gene
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GFP and its labeling strategy
Recombinant Gene Target Molecule GFP Transcription Translation Recombinant Protein GFP Target Molecule 510 nm 488 nm Wang et al. Annual Review in Biomedical Engineering, 2008
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Martin Chalfie Chalfie M, et al. 1994. Science
Inouye S, Tsuji FI FEBS Lett.
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Passive Applications of GFP
GFP-microtubules
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The Discovery of DsRed (discosoma, coral reef from Indo-pacific)
Sergey A. Lukyanov The Discovery of DsRed (discosoma, coral reef from Indo-pacific) Matz MV, et al Nature Biotech.
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Roger Y. Tsien Tsien RY. 1998, Ann Rev Biochem.
Tsien RY. 2005, FEBS Letters Giepmans, BN. et al Science
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Multiple color visualization
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Photoactivatable Fluorescence Proteins
Lukyanov, KA. et al Nature Rev Mol Cell Biology
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Photoactivatable Fluorescence Proteins
UV UV PA-FP PA-FP PS-FP PS-FP C UV Blue Dronpa Dronpa Wang et al. Annual Review in Biomedical Engineering, 2008
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Photoactivatable Proteins Dronpa
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Circularly Permutated Proteins
cpFP B N C Inserted Domain Stimulator Domains for interaction A FP 144 145 cpFP C 1-144 Breakage Site N Wang et al. Annual Review in Biomedical Engineering, 2008
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Calcium Oscillation in Heart
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Technologies utilizing FPs
Fluorescence Lifetime Microscopy (FLIM) Chromophore Assisted Laser Inactivation (CALI) Fluorescence Resonance Energy Transfer (FRET) Applications of FRET Biosensors
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Fluorescence Lifetime Microscopy (FLIM)
A Excitation Emission Frequency Domain Fluorescence Intensity Time B Fluorescence Intensity Time Excitation Emission Time Domain Wang et al. Annual Review in Biomedical Engineering, 2008
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Chromophore Assisted Laser Inactivation (CALI)
FP FP ROS Target Molecule Wang et al. Annual Review in Biomedical Engineering, 2008
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Spy on their Actions! FRET
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The Principle of Fluorescence Resonance Energy Transfer (FRET)
When the fluorophores are far apart: No FRET Excitation Emission When fluorophores are close: FRET occurs Excitation Emission FRET
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The General Design of FRET-based Fluorescent Probes A
527 nm 433 nm 476 nm EYFP ECFP EYFP ECFP 433 nm B 527 nm 433 nm 476 nm EYFP ECFP EYFP ECFP 433 nm C 433 nm 476 nm 433 nm FRET 527 nm EYFP ECFP ECFP EYFP Wang et al. Annual Review in Biomedical Engineering, 2008
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FRET-Based Biosensors
Ras and Rap1 Calcium Miyawaki, et al 1997, Nature Mochizuki, et al 2001, Nature Tyrosine Kinase Abl Ting, et al 2001, PNAS
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Why Src? The first protein tyrosine kinase discovered.
Src plays a significant role in: Cell polarity Adhesion Focal adhesion dynamics Lamellipodia formation Migration Mechanotransduction Cancer development
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Design Strategy ECFP(1-227) SH2(from c-Src) Substrate EYFP Linker
433 nm Weak FRET 490 nm 433 nm Strong FRET 527 nm Src Activation Phosphatase
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The Src kinase induces a FRET response of the Src reporter
Emission spectra of the Src reporter -Src CFP YFP +Src Emission Intensity Arbitrary Units Wavelength (nm)
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EGF induced FRET responses in HeLa Cells
Ratio (CFP/YFP) 0.4 0.3
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The Src reporter with CFP and YFP monomers
ECFP(1-227) SH2(from c-Src) Substrate EYFP Linker A206K 0.5 0.35 A206K Zacharias, D. A. et al, Science, 2002
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Construction of membrane-tethered Src reporter
MGCIKSKRKDNLNDDE mCFP SH2 substrate mYFP Plasma Membrane mCFP mYFP GC Zacharias, D. A. et al, Science, 2002 0.5 0.3
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Application of Mechanical Stimulation by Using Laser Tweezers
Physical Principle of Laser Tweezers F1 F F2
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Polystyrene beads were coated with fibronectin and positioned on cells
Optic Lens Light Fibronectin Bead Cell Body (with Src reporters) F Integrins Actin
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Polystyrene beads were coated with fibronectin and positioned on cells
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Pulling Polylysine-coated beads
did not have significant effects on FRET 0.55 0.35 FRET
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Pulling Fibronectin-coated Beads induced a directed and long-range Src activation
0.44 0.22 Overlay Force
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Pulling Fibronectin-coated Beads induced a directed propagation of Src activation
0.52 0.25
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Cytochalasin D Treated
The directed and long-range activation of Src is dependent on cytoskeleton-integrity 0.44 0.25 Nocodazole Treated 0.45 0.25 Cytochalasin D Treated
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Summary FRET-based biosensors can allow the detection of various biochemical signals with high tempo-spatial resolution in live cells, including the signal transduction in response to mechanical stimulation.
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