Announcements, Agenda Week 3

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Announcements, Agenda Week 3

• Reading for today Ch. 1, 2 in Hibbs, Zucker 2006
• Start up your computers you will need them for
  some in-class exercises.
• Open todays Power point slides and Internet
  Explorer

• Lecture Intro to Confocal, optics
• Paper discussion Zucker 2006
• TBA Collect Z-series of Artemia samples
• Assignment due Jan. 29

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TBA times with Dr. Hertzler Spring 2007
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Outline Understanding Microscopy

• Introduction to Confocal Microscopy
• Confocal versus conventional (widefield)
  fluorescence
• Optical sectioning
• Imaging modes and applications
• Advantages, limitations of confocal
• Essential Optics
• Wave/particle nature of Light
• Diffraction
• Numerical aperture
• Lateral resolution
• Axial resolution
• Useful resource Molecular Expression Microscopy
  Primer
• http//micro.magnet.fsu.edu/primer/index.html

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Laser Scanning Confocal Microscope Components
Scan Head
Microscope
Controller box
Laser
Computer, display
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1. Conventional versus confocal fluorescence
Conventional epifluorescence
Confocal epifluorescence
Sea urchin eggs (100 µm diameter) stained with
antibody to tubulin.
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Widefield
Confocal
Sunflower pollen grain
Human brain slice
Rabbit muscle fibers
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Wide-field fluorescence dichroic (dichromatic)
mirror
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Confocal Light Path

• Confocal means having the same focus.
• Basis of optical sectioning coherent light
  emitted by the laser system (excitation source)
  passes through a pinhole aperture that is
  situated in a conjugate plane (confocal) with a
  scanning point on the specimen and a second
  pinhole aperture positioned in front of the
  detector (a photomultiplier tube).

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2. Optical slicing
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3. Imaging Capabilities

• XY fluorescence imaging
• Single
• Double
• Single or Double transmitted (not confocal)
• 3-channel (need 3 lasers)
• XYZ imaging, 3-D reconstruction
• Time-lapse
• Including 4D

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Applications

• Immunolabelling
• Organelle ID
• Protein trafficking
• Locating genes on chromosomes
• Analysis of molecular mobility
• Multiple labeling
• Live cell imaging
• Transmission imaging
• Measurement of subcellular functions and ion
  concentrations

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4. Advantages, limitations of confocal microscopy

• Optical sectioning ability
• Can image cells/tissues internally
• 3D reconstruction
• Improved spatial relationships of structures
• Excellent resolution
• Close to theoretical limit of LM 0.2 µm
• Improved multiple labeling
• Since specific wavelengths of light used by
  lasers
• Very high sensitivity
• Capable of collecting single fluorescent molecule
• Easy manipulation and merging of images
• Since they are digital
• Computer controlled
• Complex settings can be programmed and recalled.

• Expensive to buy and maintain.
• 250,000
• Difficult to operate.
• Fixed material easy, live difficult.
• Fluorescent tag usually required.
• May be bulky or toxic
• Objects smaller than 0.2 not resolved
• Need to use EM.
• Damaging high intensity laser
• Need to minimize exposure, especially in live
  cells.
• Digital images are easily mishandled.
• Honesty in imaging very important.

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B. Basic Optics1. The nature of light

• Light behaves as both a particle and a wave.
• Can bounce (reflect) and bend (diffract or
  refract)
• Has wave properties
• Amplitude
• Wavelength visible is between 400-700 nm
• White light carries all visible wavelengths
• Frequency
• Direction of travel
• Direction of vibration

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• Relation between Wavelength, Frequency, Energy

Blue light 488 nm short wavelength high
frequency high energy (2 times the red)
Photon as a wave packet of energy
Red light 650 nm long wavelength low
frequency low energy
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Light-Matter Interactions

• Absorption
• Reflection
• Refraction bending of light as it passes, at an
  angle, from one material to another
• Diffraction bending of light as it passes an
  edge
• Fluorescence spontaneous emission of light after
  excitation
• Polarization
• Dispersion

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2. Diffraction Bending of light as it passes an
edge
One long continuous wave, unlike light from a
lamp or the sun.
? lt d
? gt d
See Microscopy primer,
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Diffraction Pattern from SlitResults from
Interference
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Java Tutorial Diffraction Patterns

• http//micro.magnet.fsu.edu/primer/java/diffractio
  n/basicdiffraction/index.html
• How does the width of the central maximum vary
  with the wavelength?

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Diffraction Through a Circular Aperture creates
an Airy Disk

• The radius of the Airy disk is the distance r
  from the center to the first dark ring, given by
  the resolution equation.

Increasing resolution of lens
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Resolution and Airy disk patterns
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Java Tutorial Airy Pattern Basics

• http//micro.magnet.fsu.edu/primer/java/imageforma
  tion/airydiskbasics/index.html
• How does resolution vary with wavelength and
  numerical aperture?
• http//micro.magnet.fsu.edu/primer/java/imageforma
  tion/airyna/index.html
• What is the effect of higher NA?
• http//micro.magnet.fsu.edu/primer/java/imageforma
  tion/rayleighdisks/index.html
• What is the Rayleigh criterion?

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3. Numerical aperture (NA)
NA n sin ? where n refractive index and ?
the collecting angle. nair 1.00 and noil
1.515.
W.D.
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Maximum theoretical NA

• Maximum collecting angle is 90o
• sin 90o 1.00.
• For dry objective, max. NA (1.00)(1.00) 1.0
• In practice, it is 0.95.
• All dry objectives have NA lt 1.00
• For oil objective, max NA (1.515)(1.00) 1.5.
• In practice, it is 1.4.
• All oil objectives have NA gt 1.00

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4. Lateral Resolution (XY or rlateral)

• The smallest distance two objects can be imaged
  as two. Depends on wavelength and NA.

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Optimal Resolution for LM

• Visible light ranges from 400-700 nm
• Best NA lens is 1.4
• Calculate best theoretical resolution using 520
  nm emission of fluorescein
• (Footnote for confocal, the resolution equation
  is slightly better rlateral 0.4?/NA so best
  resolution is closer to 0.15 µm).

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XY under- and over-sampling

• Optimal zoom settings (for full xy resolution)
  for 512 X 512 pixel box are given for various
  lenses on p. 126.
• You dont need to operate at these settings
  unless you want to push the resolution limit.
• Rules of thumb for 1024 X 1024 box
• 60X 1.4 NA 4X max zoom
• 40X 0.75 NA 5X max zoom
• 20X 0.7 NA 6X max zoom
• Zooming higher than this creates empty
  magnification.

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Zooming for maximum XY resolution
2X Zoom
No Zoom
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Java Tutorial 3D Airy disk is the Point Spread
Function

• http//micro.magnet.fsu.edu/primer/java/imageforma
  tion/depthoffield/index.html

This Z step will not resolve the objects in Z
axis.
This Z step will resolve the objects in Z axis.
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5. Axial Resolution (Z or raxial)

• Minimum distance between the 3D diffraction
  patterns of two points along the Z axis that can
  still be seen as two.
• Depends on wavelength and NAobj as follows
• Rule of thumb step size ½ Z resolution. See
  also http//www2.bitplane.com/sampling/index.cfm
  and http//www.cemedigital.com/clients/brand_aic_l
  rg/support/presentation04.shtml

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Ideal step sizes
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Z axis under- and over-sampling
Oversampled Overlapping sections add no
additional information since full Z resolution is
realized just makes a bigger file.
Undersampled Too few sections for full Z
resolution But full Z resolution may not be
needed.
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XY and Z resolutions (µm)
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The bottom line on optimal step size

• The Nyquist Sampling Theorem states that the
  pixel size should be 2.3X smaller than the
  resolution limit of the microscope (p. 126).
• So 1.4 NA objective with rlateral 0.2 µm
  requires xy pixel size of 0.08 µm, optimal zoom
  of 3.7X at 512 X 512.
• Step size should be 3X xy pixel size 0.24 µm
  for 1.4 NA objective with raxial 0.6 µm

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Week 3 TBA

• Assignment (each person)
• Collect Z-series of one of your Artemia samples,
  using the 20X lens and a step size of 1 or 2 um.
• Display the sections in tile mode.
• Save (as a normal TIFFs) extended focus images in
  black and white, showing (a) every section of the
  Z-series, (b) the top 1/3, (c) the middle 1/3,
  and d) the bottom 1/3.
• Always include a scale bar on your images.
• Save in the BIO553 file on the imaging computer.
• Turn in a description of your images using the
  form available on Blackboard.

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Paper discussion

• Today, Jan. 22 Zucker 2006 (Hertzler)
• Jan. 29 (Hertzler)
• Feb. 5
• Feb. 12
• Feb. 19
• Feb. 26