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
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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
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9. Types of lasers
Argon ion laser – ionize argon gas to produce excited state
Continuous wave emission
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Argon ion lasers can produce approximately 10 wavelengths in the ultraviolet region and up to 25 in the visible region, ranging from 275 to nanometers and to 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
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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