1. Functional cellular imaging by light microscopy MICROSCOPIES

2. Why use Light? Good (enough) resolution: Spatial – classically a few hundred nanometers; now tens of nm Temporal - <millisecond Compatible with live cells, tissues, organisms Many probes available for imaging: Fluorescent antibodies, GFP, indicators for Ca 2+ membrane potential, etc. Relatively inexpensive and simple (vs. E.M., PET, FMRI etc)

3. Three decades of building microscope systems 19772008

4. On the importance of looking “You can observe a lot just by watching.” Yogi Berra Former Yankee Catcher and Great American Sage

5. And sometimes you can see beautiful things ! Chaotic and spiral calcium waves in Xenopus oocyte: Jim Lechleiter, U. Texas

6. Major types of light microscopy; 1. Transmitted (reflected) light. Poor contrast; paucity of specific labels/functional probes; poor depth resolution. 2. Fluorescence. High contrast (black background); numerous fluorescent dyes, proteins and functional probes; permits 3D imaging (confocal, 2-photon)

7. A fluorescence microscope

8. But – conventional fluorescence imaging provides little depth discrimination, so images are terrible because of out-of-focus fluorescence e.g. Pollen grain imaged by conventional epifluorescence microscopy

9. One solution – Confocal microscopy Out-of-focus light is rejected by blocking it with a pinhole aperture

10. Confocal sections through pollen grain at 1 um intervals

11. 3-D reconstruction of pollen grain

12. Another way to avoid out-of-focus fluorescence and achieve 3D imaging – Two-photon microscopy Especially good for looking deep into tissues (e.g. brain) without damaging cells

13. Practical theory of 2-photon microscopy 1.Near simultaneous absorption of the energy of two infrared photons results in excitation of a fluorochrome that would normally be excited by a single photon of twice the energy. 2.The probability of excitation depends on the square of the infrared intensity and decreases rapidly with distance from the focal volume.

14. Advantages of 2-photon microscopy 1.Increased penetration of infrared light allows deeper imaging. 2.No out-of-focus fluorescence. 3.Photo-damage and bleaching are confined to diffraction- limited spot. 4.Multiple fluorochrome excitation allows simultaneous, diffraction-limited, co-localization. 5.Imaging of UV-excited compounds with conventional optics.

16. Two-photon imaging of exocytosis in pancreatic acinar cells

17. Exocytic events evoked by addition of acetylcholine

18. C 25  m 5  m 15  m DC EL AL M PF TZ 100  m Single-cell imaging in intact lymph node Miller et al., 2002. Science 296: 1869-1873

19. Another solution – Total Internal Reflection (TIRF) Microscopy Excite fluorescence in only a very thin layer right next to a coverglass Good for looking at things happening in or very near the plasma membrane of a cell

20. Total internal reflection microscopy glass air glass

21. Total internal reflection microscopy glass air Evanescent wave

22. Through-the-lens total internal reflection fluorescence microscopy (TIRFM)

23. © Molecular Expressions Microscopy Primer

24. Cultured cells expressing GFP-tagged membrane protein imaged by conventional epifluorescence

25. The same cells viewed by TIRFM

26. TIRFM imaging of single-channel Ca 2+ fluorescence signals (SCCaFTs): Ca 2+ entry through plasma membrane channels expressed in Xenopus oocytes

27. Imaging single channel events with high time resolution: SCCaFTs recorded at 500 frames s -1

28. © Molecular Expressions Microscopy Primer The diffraction limit

29. The position of a single point source (e.g. a fluorescent molecule) can be localized with much higher precision, limited only by the number of photons that can be collected. What we then need is to have only sparse sources at any given time, so as to avoid unresolved overlap Sidling around the diffraction limit

30. Photoactivation Localization Microscopy (PALM) (Betzig et al., Science 2006) Express protein of interest tagged with a photoactivatable fluorescent protein (eg.g. EOS) in cell Stochastically photoactivate a low density of molecules per frame and localize using Gaussian function active state Fluorescence emission Bleached state Activating laser 405 nm Excitation laser 532 nm Repeat thousands of times Non-fluorescent state

31. Photoactivation Localization Microscopy (PALM) Express protein of interest tagged with a photoactivatable fluorescent protein (eg.g. EOS) in cell Stochastically photoactivate a low density of molecules per frame and localize using Gaussian function

32. Imaging actin tagged with EosFP (photoactivatable protein) Eos-actin TIRF Eos-actin PALM

34. Fibroblasts expressing DsRed

35. “Greening” of DsRed

36. “greening” results from enhancement of green fluorescence and reduction of red fluorescence

44. Clustered T cells after activation

45. Evanescent field excites Ca 2+ -dependent fluorescence only in a thin layer next to cell membrane Ca 2+  indicator (fluo-4) in cytosol