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By: Mosa Charles
MOLECULAR PROBESFLUORESCENCE
What is it? Detector molecules that investigate, or analyze other
molecules, macromolecules, molecular aggregates or organisms.
Major driving force of molecular imaging.What can be explored?
Small molecules Peptides Proteins Apatamers
DNA or RNA with special ability to bind to other molecules. Nanoparticles
MOLECULAR “SPECIFIC” PROBES
What is it? They allow the detection of components of complex
bimolecular assemblies such as live cells. They are designed to respond to a specific stimulus and
localize in specific region of a biological specimenVery sensitive and selective Generally poly-aromatic hydrocarbons or heterocyclic
molecules.
FLUORESCENT PROBE
Detection of target proteins.
Cells stained with multiple fluorescent probes
Fluorescence is the result of a three-stage process that occur within molecules known as fluorophores or fluorescent dyes.
The Process1. Excitation
Energy from an external source 2. Excited-State Lifetime
This is a very short time. Fluorophore undergoes conformational changes Two important things happen as result
3. Fluorescence Emission The fluorophore returns to ground state. The photon is emitted
FLUORESCENCE
MECHANISM OF FLUORESENSCE
Jablonski energy diagram of fluoresence
FLUORESCENCE SPECTRA• Excitation and emission spectra of a fluorophore and the correlation
between excitation amplitude and emission intensity.
STOKES SHIFT
Fluorophores with greater stokes shift show clear distinction between excitation and emission light in a sample.
Fluorophores with smaller Stokes shift has a smaller difference between excitation and emission wavelengths.
Early fluorescence Employed fluorophores that only emitted light on the visible
range 390nm to 700nm
New Technology Fluorophores can now detect beyond the visible spectrum
UV and IR ranges
ELECTROMAGNETIC SPECTRUM
Molar Extinction Coefficient (ε) The quantity of light that can be absorbed by a given
wavelength. Measured in M-1cm-1
Quantum Yield Number of photons emitted divided by the number of
photons absorbed. Provides the efficiency of the fluor.
FLUOROPHORE BRIGHTNESS
Basic requirements of instrumentationExcitation light source such lasers, or lampsA fluorophoreFilters to isolate specific wavelengths Detector to record output
Instruments Fluorescent microscopesFluorescence scannersSpectrofluorometers and microplate readersFlow cytometers
FLUORESENCE DETECTION
Intensity Same parameters as absorbance
Instrument dependent Reference standards essential to calibration
Applications Cell number Amount of fluorophore localized to cells
Or discrete cellular compartments Rate if gene expression and protein synthesis Rate of cell motility or movement of intracellular components Amount of DNA, RNA or protein in a sample DNA, RNA or protein sequence Enzyme activity Viability
QUANTITATIVE USE
Detection and Analysis of Tumor Fluorescence Using a Two-Photon Optical Fiber Probe.
Purpose In vivo tumor analysis Demonstrate the benefits of TPOFF for in vivo biosensing. Demonstrate the benefits of a single-mode fiber Detection of tumor antibodies and tumor markers.
The Project Tumors developed in Mice Ex vivo detection In vivo detection
RESEARCH ARTICLE
TPOFF DIAGRAM
RESULTSSINGLE AND DOUBLE PHOTON
COMPARISON
Cell targeting comparison
RESULTS
RESULTS
Fluorescence of in vivo targeted tumor cells.
Thomas TP, Myaing MT, Ye JY, Candido K, Kotylar A, Beals J, Cao P, Keszler B, Patri AK, Norris TB, Baker JR, Jr.: Detection and Analysis of Tumor Fluorescence Using a Two–Photon Optical iber Probe. Biophysical Journal, 2004:86(6), 3959–3965. FThe Molecular Probes® Handbook—A Guide to Fluorescent Probes and Labeling Technologies http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook.html
WORKS CITED
Thomas P, Ye JY, Yang C, Myaing M, Majoros IJ, Kotlyar A, Cao Z, Norris TB, Baker JR, Jr.: Tissue distribution and real–time fluorescence measurement of a tumor–targeted nanodevice by a two photon optical fiber fluorescence probe. Proc. of SPIE, 2006:6095, 1–7.