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Lasers and Confocal

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Title: Lasers and Confocal


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/ele
    ctroncycle/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 (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
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/gai
    nbandwidth/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/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

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/dio
    delasers/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 La
ck 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 ? 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
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