Lasers and Confocal

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

Simulation http://micro.magnet.fsu.edu/primer/java/las ers/electroncycle/index.html

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.

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

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

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

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

Types of lasers Argon ion laser – ionize argon gas to produce excited state Continuous wave emission http://micro.magnet.fsu.edu/primer/java/lasers/gainband width/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

Ion laser spectra

Semiconductor diode laser Electrical pumping Wide variety of wavelengths

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

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

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

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

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

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

Result: Optical sections

Pollen grain optical sections

Reconstruction of optical stacks

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

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

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

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

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

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

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