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.