Total Internal Reflection Fluorescence Microscopy

Transcription

1 Total Internal Reflection Microscopy Nicole O Neil Indiana University October 24, 2005 Agenda Why use TIRFM? Theory behind TIR Snell s Law Instrumentation Evanescent Wave Excitation of Fluorophores Advantages/Disadvantages Applications Why would you use TIRFM? In cellular and molecular biology, a large number of events occur in the cell membrane or surface cell adhesion binding of cells by hormones and secretion of neurotransmitters membrane dynamics Problem with conventional fluorescence microscopes Fluorophores exist in equilibrium Background fluorescence overwhelms detector What is a Fluorophore? A component of a molecule which causes it to be fluorescent. A functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. Fluorophores typically used: Green fluorescent protein (GFP) and its derivatives 1

2 Theory behind TIR Instrumentation Snell s Law n 1 x sinθ c = n 2 n 1 = refractive index of air n 2 = refractive index of water θ c = critical angle When the angle of incidence is greater than the critical angle, all light undergoes reflection Blue line: angle of incidence Green line: angle of reflection As light reflects from the interface, an electromagnetic field is produced called an Evanescent Field EF can only be produced by TIR if: Medium of illumination incidence is greater than specimen medium Angle of incidence is greater than critical angle Evanescent Field is capable of exciting fluorophores Range of excitation is limited because Evanescent Field decays exponentially nm Only able to penetrate few hundred nm into cell membrane 2

3 2 basic approaches: Prism method Easy to adjust θ c Restricted access to sample Geometric constraints on specimen manipulation Objective lens method (mainstream) Diificult to adjust θ c Greater access to sample Greater flexibility in specimen manipulation Intermediate dielectric layers Used when boundary between glass coverslip and aqueous buffer is not clearly defined Use of thin metallic coating creates a resonance effect- Surface Plasmon Resonance Used to quench fluorescence within 10 nm of surface Advantages Higher resolution than Widefield Fig. a: TIRFM Fig. c: Widefield Greater Signal to Noise Ratio Fig. b: TIRFM Fig. d: Widefield Fluorescent microspheres in solution Widefield vs. TIRF 3

4 Advantages Cont. TIRFM can be applied to ordinary research-grade laboratory microscopes. Not as much specimen needed as confocal methods. 10 nm sections needed for TIRFM 600 nm sections needed for confocal methods Living cells survive longer Disadvantages Can only analyze small samples nm sections Limited to specimen regions having an appropriate refractive indices Specimen must contain fluorphores Applications of TIRFM Single molecules Molecules in solution in Brownian motion Imaging of minute structures Endocytosis or exocytosis occuring in cell membrane Single protein trafficking between cells Measure fluorophore concentrations Record binding/unbinding equilibria and kinetic rates Applications Cont. Miami University of Ohio Total Internal Reflectance Infrared Microspectroscopy as a detection technique for HPLC Coupled the ideas of IR and TIRFM TIRFM allows detection of small molecules IR allows characterization of the molecules 4

5 Applications Cont. Institute for Microsystem Technology; Freiburg, Germany Real-time detection of nucleic acid interactions by TIRFM Hybridization progress of molecules from the liquid phase in a flow cell to immobilized oligonucleotides on a transducer can be observed in real-time Kinetic analysis Melting curves Detection of single-point mutations on DNA Conclusion TIRFM is useful in looking at very minute structures Cell membrane Small molecules TIRFM is able to produce a higher resolution picture Use of evanescent waves Small range of excitation References Lehr, G.P.; Reimann, M.; Brandenburg, A.; Sulz, B.; Klapproth, H. Real- Time Detection of Nucleic Acid Interaction by Total Internal Reflection. Analytical Chemistry. 2003, 75, Nikon MicrscopyU. TIRF Microscopy: Introduction and Applications. 28 Sept <>. Olympus Microscopy Resource Center. Total Internal Reflection Microscopy: Introduction and Theoretical Aspects. 28 Sept < tml>. Patterson, Brian M.; Danielson, Neil D.; Sommer, Andre J. Attenuated Total Internal Reflectance Infrared Microspectroscopy as a Detection Technique for High-Performance Liquid Chromatography. Analytical Chemistry. 2003, 75, Skoog, D.A.; Holler, F.J.; Nieman, T.A. Principles of Instrumental Analysis. Brooks/Cole Publishing Co., 5th edition, Questions? 5