Confocal Microscope NEU260

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Confocal Microscope NEU260
Confocal Microscope Basic
Hiroyuki Hakozaki National Center for Microscopy
and Imaging Research University of California,
San Diego
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Fluorescence Microscope

• Fluorescence
• Fluorescence Microscope

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Fluorescence Microscope
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Confocal Microscope

• Optical Sectioning

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Detail Optics of Confocal Microscope
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Theoretical Lateral Resolution

• Lateral resolution in fluorescence microscope
• Lateral resolution in confocal microscope
• Pinhole size is bigger than 1AU
• Pinhole size is smaller than 0.25AU

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Lateral Resolution in expample

• Example
• Objective Lens NA1.4
• Excitation wavelength ?ex488nm
• Emission wavelength ?em520nm
• Lateral Resolution of Fluorescence Microscope
• 189nm
• Confocal Microscope
• Big pinhole size
• 178nm
• Small pinhole size
• 132nm

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Difference between Axial Resolution and Optical
Slice Thickness

• Axial Resolution
• How point fluorescence image will spread in Z
  direction.
• Optical Slice Thickness
• How thick optical sectioning created by pinhole.

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Theoretical Axial Resolution

• What does axial resolution mean?
• You can resolve two points when they are
  separated more than axial resolution.
• Axial Reslution
• Pinhole size is bigger than 1AU
• Pinhole size is smaller than 0.25AU

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Theoretical Optical Slice Thickness

• Optical Slice Thickness
• Pinhole size is bigger than 1AU
• Pinhole size is smaller than 0.25AU

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Axial Resolution and Optical Slice Thickness in
example

• Optical Slice Thickness (NA1.3, n1.52, l496nm)

• Axial Resolution (PH0, n1.52,
  l496nm)

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Spherical aberration

• What is spherical aberration
• Focus in oil vs. in water

• Axial size conversion and resolution change in
  each Z position

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Digital Sampling

• To get resolution that optics support, sampling
  rate (Pixel Size) need to be more than twice of
  resolution.

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Spinning disk confocal

• Optics
• Light source can be same with normal fluorescence
  microscope
• Detector need to be 2D detector like CCD
• Use disk to scan area
• faster scan speed

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Spinning disk confocal

• Multi point scanning
• More duration time than single point scanning
  High sensitivity
• Poor Z resolution
• Micro Lens Array to increase excitation light
  power

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References

• James B. Pawley
• Handbook of Biological Confocal Microscopy
  (Second Edition)
• Zeiss Stefan Wilhelm, Bernhard Grobler, Martin
  Gluch, Hartmut Heinz
• Confocal Laser Scanning Microscopy
• Nikon
• www.microscopyu.com
• Yokogawa
• www.yokogawa.com/scanner/index.htm