1. Lasers and Confocal

2. Laser • Acronym: Light Amplification by Stimulated Emission of Radiation • Ordinary light emission: Comes from spontaneous decay of excited state to ground levels • Stimulated emission: molecule remains in excited state until stimulated to emit by incoming light that is insufficient to raise it to the next higher excited state

3. Simulation • http://micro.magnet.fsu.edu/primer/java/lasers/electroncycle/index.html

4. Design of a laser • Medium (such as ruby crystal) that has reflective mirrors at both ends • Mechanism to pump energy (stimulated absorption) in (flashtube, accelerating coils, pump laser) so that we get a population inversion: circumstyance in which there are more (atoms, molecules) in the excited state than the ground state • Under these circumstances, additional light is more likely to generate stimulated emission than stimulated absorption • At that point, further pulses give stimulated emission.

5. Design of a laser (cont’d) • This phenomenon of stimulated emission gives rise to a standing wave • That standing wave can generate constructive interference to escape from the end of the crystal Different lasers with different pumps

6. Ruby laser • Ruby laser • Length of cavity, index of refraction of material determines wavelength • Note that emission is: • Phase coherent • Nearly monochromatic

7. Cavity resonance modes and gain bandwidth • Multimode lases are not monochromatic • Wavelengths of light are extremely small compared to size of cavity • Laser modes are distibuted over a narrow range of frequencies, termed gain bandwidth

8. Varying cavity modes can affect gain bandwidth • http://micro.magnet.fsu.edu/primer/java/lasers/gainbandwidth/index.html

9. Types of lasers • Argon ion laser – ionize argon gas to produce excited state • Continuous wave emission • http://micro.magnet.fsu.edu/primer/java/lasers/gainbandwidth/index.html • Argon ion lasers can produce approximately 10 wavelengths in the ultraviolet region and up to 25 in the visible region, ranging from 275 to 363.8 nanometers and 408.9 to 686.1 nanometers, respectively. In the visible light spectral region • Typically most power at 458, 488, 514 are in visible range

10. Ion laser spectra

11. Semiconductor diode laser • Electrical pumping • Wide variety of wavelengths

12. Beam shaping in diode lasers • http://micro.magnet.fsu.edu/primer/java/lasers/diodelasers/index.html Ti-sapphire mode-locked lasers

13. Ti-sapphire lasers • Wavelength adjustable by changing cavity length • Modelocking ensures better monochromacity • Tunable over a broad range using prism to spread spectrum and slit to select wavelength

14. Laser illuminators for widefield fluorescence • Because lasers are phase coherent, you set up standing wavers between optical components • Results in fringes when you try to image • Solution: optical fiber mode scramblers

15. Optical fibers total internal reflection Scramblers work by curving optical fibers to remove phase coherence: Advantages of laser sources for widefield fluorescence: - Monochromacity - Intense illumination in a small spot

16. Confocal laser scan microscopy • Instead of defocussing source over the image plane, focus it to a point • Scan that point over the specimen to buld up an image

17. Advantage: Out of focus loght may be rejected by a paired emission aperture

18. Result: Optical sections

19. Pollen grain optical sections

20. Reconstruction of optical stacks

21. Confocal technologie • Specimen scan confocal • Use a Piezo device to scan specimen as you build up images • Advantage: can be used in transmission • Major disadvantages: • specimen size limitation • Shear on specimen

22. Laser scan confocal microscope Advantages: Flexibility Ease of use Disadvantages Speed Monochromacity Cannot be used for transmitted-light confocal

23. Spinning disc confocal Advantages: White light Speed Disadvantages Lack of sensitivity

24. Intermediate techniques • Slit scan confocal – Use a cylindrical lens to spread beam into a fan bean • Scan that beam across specimen • Instead of pinhole, use a slit to reject out-of-focus information, and use a line detector • Real time speed • However, resolution, contrast, and optical sectioning are nonisotropic

25. Confocal caveats • The meaning of optical sections: no sharply defined boundaries; Gaussian intensity distribution • Means that very bright objects can “spill over” • Importance of setting black level and gain • In X and Y, maximum resolution is ~0.1 µm; in Z, approximately 0.8 µm. Problems for colocalization

26. The problem of chromatic aberration • Lenses that have chromatic abberration bring different wavelengths to focus at different points • Even apchromats are only corrected at blue, green and red; we often use purple (DAPI) or near infrared (Cy5) dyes

27. Problems (continued) • Spherical aberration • As we focus into a specimen, we are focusing though aqueous medium. • If we are using an oil immersion lens, we will get spherical aberration, because η is wrong • One solution: High NA water-immersion objectives • Signal-to-noise: much worse for confocal than deconvolved widefield • Fluorophore overlap: rhodamine, for example, is excited by 488, as well as 514 • Detection: turn of 514 excitation • Fix • 1. Use other dyes • 2. Sequential scanning • Multispectral analysis to deconvolve overlapping fluorophores