Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
New TURF for TIRF Joel Schwartz Stowers Institute for Medical Research Imaging Center • What is TIRF? • Why do we constantly use acronyms to describe everything? • Microscope Configurations – Prism vs Prismless • Biological Applications – Brief Aside • Unique attributes to our system – Calibrated TIRF planes – TIRF-FRET – TIRF-photoactivation • Not ready for prime time players.. • What is TIRF? • Why do we constantly use acronyms to describe everything? • Microscope Configurations – Prism vs Prismless • Biological Applications – Brief Aside • Unique attributes to our system – Calibrated TIRF planes – TIRF-FRET – TIRF-photoactivation • Not ready for prime time players.. Index of refraction “bends” light Some refractive indices to know: water 1.33 air 1.0003 glass 1.517 coverglass 1.523 immersion oil 1.516 cell cytosol 1.38 mount variable The basics of imaging cells by TIRF microscopy At a specific critical angle [θcritical = sin-1(n1/n2)] light is totally reflected from the glass/water interface. The reflection generates a very thin electromagnetic field that has an identical frequency to that of the incident light, providing a means to selectively excite fluorophores within ≤ 100 nm of the coverslip. http://micro.magnet.fsu.edu/primer/java/tirf/penetration/index.html Evanescent wave penetration The evanescent wave penetration (d) : d = λ0/4π (n22 sin2θ-n12)-1/2 note: d is only the depth at which the intensity of the evanescent wave is 37% of the initial intensity. Thus, can empirically determine the experimental depth at which fluorophores are visible using fluorescent beads (Keyel, Watkins, and Traub 2004 JBC) λ0 = 488; n2=1.52; n1=1.38 dempirical = 190 nm λ0 = 647; n2=1.52; n1=1.38 dempirical = 238 nm λ0 = 488; n2=1.78; n1=1.38 dempirical = 142 nm TIRF selectively illuminates the cellular membrane • What is TIRF? • Why do we constantly use acronyms to describe everything? • Microscope Configurations – Prism vs Prismless • Biological Applications – Brief Aside • Unique attributes to our system – Calibrated TIRF planes – TIRF-FRET – TIRF-photoactivation • Not ready for prime time players.. Prism-based TIRF limit access to sample Axelrod et al. Traffic 2001 Prism systems can be placed under a culture dish Prism-based TIR on an upright microscope Trapezoid TIR prism on condenser and the position of the beam is adjusted by moving external lens. Axelrod et al. Traffic 2001 TIRF is commonly done inside the objective The objective influences penetration depth 100X 1.45 NA objective: θc = sin-1(n1/n2) = 65.22º [calculated using n2 = 1.52 (RI coverglass and immersion liquid) and n 1 = 1.38] 100X 1.65 NA objective: θc = sin -1(n1/n2) = 50.83º [calculated using n2 = 1.78 (RI coverglass and immersion liquid) and n 1 = 1.38] Maximum Angle θm from the optical axis that TIR will occur is: NA = n2 sin θm 60X 1.45 NA θm = 72.54º 100X 1.65 NA θm = 67.97º TIRF objectives are now starting to come with compensation collars for varying temperature and cover slip thickness TIRF Comparison Prism Method 1. “Purer” evanescent wave 2. Limited access to sample 3. Few commercial manufactures 4. Open laser systems 5. Typically lower NA objectives Prism-less Method 1. Higher NA will allow confinement closer to surface 2. Not as pure an evanescent wave as prism 3. Commercial system readily available • What is TIRF? • Why do we constantly use acronyms to describe everything? • Microscope Configurations – Prism vs Prismless • Biological Applications – Brief Aside • Unique attributes to our system – Calibrated TIRF planes – TIRF-FRET – TIRF-photoactivation • Not ready for prime time players.. TIRF illumination enhances contrast YFP on the Membrane TIRF is more sensitive to Z-axial drift Hogan, Biophotnics International May 2006 48-51 TIRF measures endocytosis of clathrin coated vesicles Color Coded Motion Red Green Blue RGB Clathrin coated pits are move in and out of the membrane Membrane-localized fluorophores are difficult to separate from mitochondria TIRF selectively visualizes the membrane localized fluorophores Total Internal Reflection Fluorescence (TIRF) Microscopy is used to reduce background Applications of TIR microscopy •Selective visualization of cell/substrate contact regions. •Visualization and spectroscopy of single molecule fluorescence near a surface. •Tracking of secretory granules in intact cells before and during the secretory process. •Micromorphological structures and dynamics on living cells. •Long-term fluorescence movies of cells during development in culture. •Comparison of membrane-proximal ionic transients with simultaneous transients deeper in the cytoplasm. •Measurements of the kinetic rates of binding of extracellular and intracellular proteins to cell surface receptors and artificial membranes. • What is TIRF? • Why do we constantly use acronyms to describe everything? • Microscope Configurations – Prism vs Prismless • Biological Applications – Brief Aside • Unique attributes to our system – Calibrated TIRF planes – TIRF-FRET – TIRF-photoactivation • Not ready for prime time players.. The new rig 405, 440, 491, 561, 638 AOTF operated Axiocam HS Back-thinned EM-CCD BAD IDEA! Environmental Chamber High tech TIRF calibration device Fluorescencce (Epi) 1.0 0.8 Red =120nm Green=88nm 0.6 0.4 0.2 0.0 0 100 200 300 Distance (nm) 400 500 FRET measures protein proximity TIRF enhances signal to noise measurements of membrane associated FRET 1 um z-axial ~ 15 receptors ~ 75 associated proteins 100 nm ~ 15 receptors ~ 15 associated proteins We idealized the system to excite CFP for FRET measurements Excitation of CFP leads to some YFP excitation because YFP is ~5 fold brighter than CFP. CFP emission also bleeds into the YFP channel (i.e. there will always be some “FRET” signal). The new TIRF scope is capable of specific membrane photoactivation • What is TIRF? • Why do we constantly use acronyms to describe everything? • Microscope Configurations – Prism vs Prismless • Biological Applications – Brief Aside • Unique attributes to our system – Calibrated TIRF planes – TIRF-FRET – TIRF-photoactivation • Not ready for prime time players.. Spectral images separate overlaping spectra Dickinson et al. Biotechniques. 31:1272 2001. CTIS provides space and color information without any moving parts NOT YET AVAILABLE The system is a linear transfer function similar to CT scanning and reconstruction Spectral Imaging: CTIS Space and Color in a Single Shot CGH Disperser The CTIS images are deconvolved to generate the actual image Ford et al., Optics Express’01 (9) 444-453. Raw Data on CCD Color projection of final data stack Thank You • Imaging Center – – – – – – Cameron Cooper Paul Kulesa Sarah Smith Danny Stark Jessica Teddy Miranda Smith • Adv. Inst. And Physics – Winfried Wiegraebe – Josef Huff – Amanda Combs http://micro.magnet.fsu.edu/primer/java/tirf/evaintensity/index.html