FLUORESCENT SENSING IN BIOLOGICAL SYSTEMS

Summary

Fluorescence is a phenomenon where a substance (the fluorophore) absorbs light of a certain wavelength and enters a higher-energy state. After a very short time, the fluorophore returns to the ground state and emits light of a different wavelength. A prominent example where fluorescence is used as a diagnostic tool is opthalmology. Blood vessels in the retina can be made visible by injecting the patient with a solution that contains fluorescein. After a short time, the blood carries the fluorescein to the eye, and the blood vessels of the retina become prominently visible under ultraviolet light. Fluorescent molecules can be combined with physiologically active compounds, and sites where the fluorescently labeled compound accumulates become visible under fluorescent microscopy. A popular example is green fluorescent protein (GFP), which can be linked to gene expression and allows to study cell signaling and gene-related processes. In a third example, fluorophores can be designed to become sensitive to their environment. Examples include fluorophores that change their fluorescent behavior in the presence of calcium ions, in acidic fluids, in polar environments, or in viscous fluids. Since fluorescence measurement is highly quantitative, fluorophores with sensitivity for environmental properties can act as high-speed, versatile, and accurate sensors. The UGA Biophotonics lab, headed by Dr. Mark Haidekker, investigates novel applications of spectroscopic and laser-optical imaging techniques with a focus on fluorescent sensing. These techniques include the measurement of microviscosity and the visualization of microviscosity and microflow patterns in fluidic systems and in cells.

Situation

The research is primarily driven by biomedical challenges. One example concerns the cardiovascular system in patients with severe hemorrhagic shock. A new hypothesis states that tissue hypooxygenation associated with blood volume restitution is related to a hypoviscosity-induced failure of the capillary beds rather than th lack of red blood cells. Under this hypothesis, rapid volume restitution with high-viscosity plasma expanders would be necessitated. However, physiological blood viscosity must not be exceeded. This is a difficult task since neither the remaining blood volume nor the effects of hemodilution by interstitial fluid in hemorrhagic patients is known. Conventional mechanical viscometers cannot meet the demand for continuous, real-time viscosity monitoring, and completely new techniques need to be developed. This is a situation where fluorescent {\em molecular rotors}, i.e., a group of viscosity-sensitive fluorophores, promise an innovative solution. The viscosity sensitivity of molecular rotors can be used in a variety of fields, including cell research, monitoring of polymerization processes, and measurement and imaging of flow in microfluidic systems. A completely different challenge was presented by a collaborator, Dr. Michael Toews (Tifton campus). Stink bugs (Heteroptera: Pentatomidae) negatively impact the cotton industry. Cotton bolls become damaged and the lint unusable after the boll has been fed upon by stink bugs. State-of-the-art prevention of stink bug damage involves scouting for stink bug damage by manually harvesting and breaking open bolls in the field. Based on the occurrence of lint stains, the optimum time for insecticide application can be determined, albeit with high manual effort. Dr. Haidekker discovered that damaged bolls exhibit a strong green fluorescence near the stained lint. This fluorescence is not observed in bolls that have not been affected by stink bugs. It is now the goal of the biophotonics lab to develop practical methods to detect stink bug damage with fluorescent sensing methods, thus eliminating the need for breaking open the bolls.

Response

Several new techniques were developed and presented. To address the question of continuous, real-time blood plasma viscosity measurement, a revolutionary new method was devised using fluorescent molecular rotors. These molecules act as bio-mechanosensors in almost any liquid environment and report changes of viscosity through changes in fluorescent emission intensity and lifetime. By chemical synthesis, it is possible to target different sites with molecular rotors. Hydrophilic derivatives can be used in blood plasma or other aqueous systems, whereas lipophilic derivatives can be integrated into the cell membrane. These membrane-bound molecular rotors are an interesting new research tool in cell physiology research. We found that molecular rotors have comparable reporting accuracy to FRAP (fluorescence recovery after photobleaching, an established technique to measure membrane viscosity) but dramatically reduce experimental complexity and time. In the 2009-2010 academic year, the focus of the Biophotonics lab was to advance understanding and application of molecular rotors. The main area of interest is the newly discovered shear-stress sensitivity of a subgroup of molecular rotors which can be used for ultrahigh-sensitivity flow measurement, flow pattern imaging, closed-loop flow control, and microfluidics-related applications. We have established a method to image flow patterns (M.Sc. Thesis of Adnan Mustafic), but the mechanism of shear sensitivity is still unknown. More systematic experiments are required to further understand the mechanism. In response to the newly discovered stink bug-related fluorescence, the fluorescence was characterized and unambiguously linked to stink bug damage (manuscript submitted). The present focus is the development of imaging systems and methods to detect stink bug-related fluorescence without opening the cotton boll. Initial results show that the characteristic fluorescence emission can be detected on the outer wall of the boll, particularly near the puncture sites. The long-term vision is the development of a hand-held device that can rapidly quantify stink bug damage in the field.

Impact

High level of interest from the national scientific community as ascertained through peer-reviewed publications, patents and one grant: Book: M.A. Haidekker, Advanced Biomedical Image Analysis. John Wiley \& Sons, 2011. ISBN 978-0-470-62458-6. Book chapter: Haidekker MA, Nipper ME, Mustafic A, Lichlyter D, Dakanali M, Theodorakis EA. Dyes with segmental mobility: molecular rotors. In: Demchenko AP (editor). Advanced Fluorescence Reporters in Chemistry and Biology I. Fundamentals and Molecular Design. Springer Series on Fluorescence, Vol. 8. Wolfbeis O (series editor). Springer-Verlag, 2010, pp. 267-308. ISBN 978-3642047008. Invited review: Haidekker MA, Theodorakis EA. Environment-Sensitive Behavior of Fluorescent Molecular Rotors. Journal of Biological Engineering 2010; 4: 11. Peer-reviewed article: Mustafic A, Huang H-M, Theodorakis EA, Haidekker MA. Imaging of Flow Patterns with Fluorescent Molecular Rotors. J Fluorescence 2010; 20(5): 1087-1098. Peer-reviewed article: Sutharsan J, Lichlyter D, Wright NE, Dakanali M, Haidekker MA, Theodorakis EA. Molecular rotors: synthesis and evaluation as viscosity sensors. Tetrahedron 2010; 66: 2582-2588. Peer-reviewed article: Sutharsan J, Dakanali M, Capule CC, Haidekker MA, Yang J, Theodorakis EA. Rational Design of Amyloid Binding Agents Based on the Molecular Rotor Motif. Chem Med Chem 2010; 5: 56-60. Patent: Haidekker MA. Supported molecular biofluid viscosity sensors for in vitro and in vivo use Issued 03/02/2010 Patent: Haidekker MA, Toews M. Noninvasive methods and apparatus for detecting insect-induced damage in a plant. UGARF No.: PR09.025; PCT Application.

State Issue

Agricultural Profitability and Sustainability

Details

• Year: 2010
• Geographic Scope: National
• County: Clarke
• Program Areas:
  • Agriculture & Natural Resources