Structured illumination microscopy and reflection confocal

1. Structured illumination microscopy and reflection confocal

2. Structured illumination • Sample image through a grating 3X, moving the grating each time by 1/3 pixel • Only in-focus data will have sharp fringes • Out-of-focus data will be relatively invariant • I = ((I1-I2)2+(I1-I3)2+(I2-I3)2)1/2 - throws out all the out-of-focus information • Imperial College Powerpoint

3. Structured illumination illuminators • Zeiss Apotome • Whole microscope is approx $100K • Grabs images 1/second, processes in real time • Thales Optem Optigrid • Can be added onto existing scopes • Relative cheap ($20K) • OptiGrid

4. Mathematical foundations of structured illumination • From a mathematical sense, we can say that we are sampling 3-dimensional image space with a vertical grid spacing of 1/3 pixel • This should give us a vertical resolution comparable to X and Y • That is far better than confocal, where Z resolution is limited to 0.8 µ

5. Extended resolution • Standing wave fluorescence microscopy • Principle: Perform fluorescence microscopy with a laser as illumination source • Light reflected from coverslip sets up a standing wave • Set intensity threshold such that standing wave only excites fluorophores at its peak

6. Standing Wave Fluorescence

7. Problems • Works well for very thin specimens • For, thicker specimens, however, you get excitation of fluorophores in other parts of the cell by adjacent standing waves • It is possible to mathematicvally deconvolve, but a pain!

8. 4Pi confocal microscopy • Instead of using reflection, split laser beam to have two interfering beams from opposite sides of the specimen • Significantly, the confocal aperture ensures that you are only getting fluorescence from one standing wave

9. Leica TCS 4Pi

10. Actin filaments in fibroblast by 4Pi Gustafsson, Curr. Opin. Struct. Biol. 9:627

11. Resolution of techniques Gustafsson, Curr. Opin. Struct. Biol. 9:627

12. STED • Depends on phenomenon of Stimulated Emission Depletion • If we raise a fluorophore to an excited state, hitting it with the emitted wavelenth can revert it to the ground state • STED microscopy shines a short λ spot in field center, and surrounds it with a doughnut of longer λ; STED reduces the size of the spot

13. STED Stephan Hell Resolution can be 2X confocal resolution Hell’s group has demonstrated 50 nm resolution Leica is marketing a STED microscope But: Microscope is very elaborate and expensive Pumps a lot of energy into the specimen: photobleaching and phototoxicity

14. Reflection confocal • Instead of setting the emission wavelength longer than excitation wavelength, set them to the same. • Gives image contrast based on reflected (or really, backscattered) light • Problem – hard to interpret

15. Reflection-enhanced backscatter confocal microscopy • Cells plated on a specular reflective substrate look like they were imaged in DIC when viewed by reflection confocal

16. Mode of image formation in REBCM In other words, REBCM is an interferometric technique that measures height above the coverslip

17. Note fringes along filopodia

18. REBCM can show individual microtubules

19. REBCM offers increased resolution 100 nm fluorescent beads are resolved in REBCM, but not in fluorescence