1. By: Mosa
Charles
MOLECULAR PROBES
FLUORESCENCE

2.  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

3.  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 specimen
 Very sensitive and selective
 Generally poly-aromatic hydrocarbons or heterocyclic molecules.
FLUORESCENT PROBE
Detection of target proteins. Cells stained with multiple fluorescent
probes

4.  Fluorescence is the result of a three-stage process that occur
within molecules known as fluorophores or fluorescent dyes.
 The Process
1. 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

5. MECHANISM OF FLUORESENSCE
Jablonski energy diagram of fluoresence

6. FLUORESCENCE SPECTRA
• Excitation and emission spectra of a fluorophore and the correlation between
excitation amplitude and emission intensity.

7. 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.

8.  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

9.  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

10. Basic requirements of instrumentation
Excitation light source such lasers, or lamps
A fluorophore
Filters to isolate specific wavelengths
Detector to record output
Instruments
Fluorescent microscopes
Fluorescence scanners
Spectrofluorometers and microplate readers
Flow cytometers
FLUORESENCE DETECTION

11.  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

12.  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

13. TPOFF DIAGRAM

14. RESULTS
SINGLE AND DOUBLE PHOTON
COMPARISON
Cell targeting comparison

15. RESULTS

16. RESULTS
Fluorescence of in vivo targeted tumor cells.

17. 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.
F
The Molecular Probes® Handbook—A Guide to Fluorescent
Probes and Labeling Technologies
http://www.lifetechnologies.com/us/en/home/references/mole
cular-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.