1. New TURF for TIRF Joel Schwartz Stowers Institute for Medical Research Imaging Center

2. 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..

3. 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..

4. Index of refraction “bends” light Some refractive indices to know: water1.33 air1.0003 glass1.517 coverglass1.523 immersion oil1.516 cell cytosol1.38 mountvariable

5. At a specific critical angle [θ critical = 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. The basics of imaging cells by TIRF microscopy

6. http://micro.magnet.fsu.edu/primer/java/tirf/penetration/index.html

7. 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) Evanescent wave penetration λ 0 = 488; n 2 =1.52; n 1 =1.38 d empirical = 190 nm λ 0 = 647; n 2 =1.52; n 1 =1.38 d empirical = 238 nm λ 0 = 488; n 2 =1.78; n 1 =1.38 d empirical = 142 nm The evanescent wave penetration (d) : d = λ 0 /4π (n 2 2 sin 2 θ-n 1 2 ) -1/2

8. TIRF selectively illuminates the cellular membrane

9. 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..

10. Prism-based TIRF limit access to sample Axelrod et al. Traffic 2001

11. Prism systems can be placed under a culture dish

12. Prism-based TIR on an upright microscope Axelrod et al. Traffic 2001 Trapezoid TIR prism on condenser and the position of the beam is adjusted by moving external lens.

13. TIRF is commonly done inside the objective

14. The objective influences penetration depth 100X 1.65 NA objective: θ c = sin -1 (n 1 /n 2 ) = 50.83º [calculated using n 2 = 1.78 (RI coverglass and immersion liquid) and n 1 = 1.38] Maximum Angle θ m from the optical axis that TIR will occur is: NA = n 2 sin θ m 60X 1.45 NA θ m = 72.54º 100X 1.65 NA θ m = 67.97º TIRF objectives are now starting to come with compensation collars for varying temperature and cover slip thickness 100X 1.45 NA objective: θ c = sin -1 (n 1 /n 2 ) = 65.22º [calculated using n 2 = 1.52 (RI coverglass and immersion liquid) and n 1 = 1.38]

15. 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

16. 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..

18. TIRF illumination enhances contrast

19. YFP on the Membrane

20. TIRF is more sensitive to Z-axial drift Hogan, Biophotnics International May 2006 48-51

21. TIRF measures endocytosis of clathrin coated vesicles

22. Color Coded Motion Red Green Blue RGB

23. Clathrin coated pits are move in and out of the membrane

24. Membrane-localized fluorophores are difficult to separate from mitochondria

25. TIRF selectively visualizes the membrane localized fluorophores

26. Total Internal Reflection Fluorescence (TIRF) Microscopy is used to reduce background 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. Applications of TIR microscopy

27. 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..

28. The new rig 405, 440, 491, 561, 638 AOTF operated Environmental Chamber Back-thinned EM-CCD Axiocam HS BAD IDEA!

29. High tech TIRF calibration device

30. FRET measures protein proximity

31. 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

32. 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). We idealized the system to excite CFP for FRET measurements

33. The new TIRF scope is capable of specific membrane photoactivation

34. 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..

35. Dickinson et al. Biotechniques. 31:1272 2001. Spectral images separate overlaping spectra

36. The system is a linear transfer function similar to CT scanning and reconstruction CTIS provides space and color information without any moving parts NOT YET AVAILABLE

37. CGH Disperser Spectral Imaging: CTIS Space and Color in a Single Shot

38. Ford et al., Optics Express’01 (9) 444-453. Raw Data on CCD The CTIS images are deconvolved to generate the actual image Color projection of final data stack

39. 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

41. http://micro.magnet.fsu.edu/primer/java/tirf/evaintensity/index.html