Title: Lasers and Confocal
1Lasers and Confocal
2Laser
- 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
3Simulation
- http//micro.magnet.fsu.edu/primer/java/lasers/ele
ctroncycle/index.html
4Design 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.
5Design of a laser (contd)
- 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
6Ruby laser
- Ruby laser
- Length of cavity, index of refraction of material
determines wavelength
Note that emission is Phase coherent Nearly
monochromatic
7Cavity 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
8Varying cavity modes can affect gain bandwidth
- http//micro.magnet.fsu.edu/primer/java/lasers/gai
nbandwidth/index.html
9Types of lasers
- Argon ion laser ionize argon gas to produce
excited state - Continuous wave emission
- http//micro.magnet.fsu.edu/primer/java/lasers/gai
nbandwidth/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
10Ion laser spectra
11Semiconductor diode laser
- Electrical pumping
- Wide variety of wavelengths
12Beam shaping in diode lasers
- http//micro.magnet.fsu.edu/primer/java/lasers/dio
delasers/index.html
Ti-sapphire mode-locked lasers
13Ti-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
14Laser 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
15Optical 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
16Confocal 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
17Advantage Out of focus loght may be rejected by
a paired emission aperture
18Result Optical sections
19Pollen grain optical sections
20Reconstruction of optical stacks
21Confocal 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
22Laser scan confocal microscope
Advantages Flexibility Ease of
use Disadvantages Speed Monochromacity Cannot
be used for transmitted-light confocal
23Spinning disc confocal
Advantages White light Speed Disadvantages La
ck of sensitivity
24Intermediate 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
25Confocal 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
26The 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
27Problems (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 ? 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