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 today’s Power point slides and Internet Explorer
I. Lecture: Intro to Confocal, optics
II. Paper discussion: Zucker 2006
III. TBA: Collect Z-series of Artemia samples
IV. Assignment due Jan. 29
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TBA times with Dr. Hertzler: Spring 2007
Time Tuesday Wednesday Thursday Friday
8
9 SEM Cell Biology TEM Cell Biology
10 Group 1 Office Group 2
11 Amy, Lauren, Rachel
Hours Andrea, Emily, Molly
12
1 403 Group 3 Lab meeting
2 students Becky, Ellen, Katie
Group 4
3 UCC Amanda, Brittaney, Joe
4 Faculty Meeting
Seminar
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Outline: Understanding Microscopy
A. Introduction to Confocal Microscopy1. Confocal versus conventional (widefield) fluorescence2. Optical sectioning3. Imaging modes and applications4. Advantages, limitations of confocal
B. Essential Optics1. Wave/particle nature of Light2. Diffraction3. Numerical aperture4. Lateral resolution5. 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
Laser
Scan Head
Microscope
Controllerbox 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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Human brain slice Rabbit muscle fibers Sunflower pollen grain
Widefield
Confocal
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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
1. XY fluorescence imaging
a) Singleb) Doublec) Single or Double +
transmitted (not confocal)
d) 3-channel (need 3 lasers)
2. XYZ imaging, 3-D reconstruction
3. 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)
Red light
650 nm
long wavelength
low frequency
low energy
Photon as a wave packet of 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
λ < d λ > d
See: Microscopy primer,
One long continuous wave,unlike light from a lampor the sun.
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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/diffraction/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/imageformation/airydiskbasics/index.html– How does resolution vary with wavelength and
numerical aperture?
• http://micro.magnet.fsu.edu/primer/java/imageformation/airyna/index.html– What is the effect of higher NA?
• http://micro.magnet.fsu.edu/primer/java/imageformation/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 < 1.00
• For oil objective, max NA = (1.515)(1.00) = 1.5.– In practice, it is 1.4.– All oil objectives have NA > 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.
objobjlateral
condobjlateral
NANA
λ.r
NANA
λ.r
61.0
2
221
thenNANA If
aperture. numerical isNA h, wavelengtis Where
221
condobj
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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 = 0.4λ/NA so best resolution is closer to 0.15 μm).
μm 0.2nm 2621.4
nm 5200.61r
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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 don’t 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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No Zoom 2X Zoom
Zooming for maximum XY resolution
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Java Tutorial: 3D Airy disk is the Point Spread Function
• http://micro.magnet.fsu.edu/primer/java/imageformation/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_lrg/support/presentation04.shtml
•
μm 2nm 2165
0.5625
nm 1218
(0.75)
nm)(1.5) 1.4(580
)(
4.1
:lensNA 0.75 40XFor
2
2
axial
axial
objaxial
r
r
NA
nr
μm 0.6nm 621
1.96
nm 1218
(1.4)
nm)(1.47) 2(580
)(
2
:lensNA 1.4 60XFor
2
2
axial
axial
objaxial
r
r
NA
nr
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Ideal step sizes
Ideal step size(higher Z resolution, e.g. NA=1.4)
Ideal step size(lower Z resolution, e.g. NA=0.7)
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Z axis under- and over-sampling
UndersampledToo few sections for full Z
resolutionBut: full Z resolution may
not be needed.
Oversampled:Overlapping sections add no
additional information since full Z resolution is realized;
just makes a bigger file.
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XY and Z resolutions (μm)
10X
0.4 NA
20X
0.7 NA
40X
0.75 NA
60X
1.4 NA
rlateralfluorescein
488/5180.790 0.451 0.421 0.226
rlateralrhodamine
543/5800.885 0.505 0.471 0.253
raxial
step
fluorescein
488/5186.80 2.22
1.1
1.93
1
0.555
0.275
raxial
step
rhodamine
543/5807.61 2.49
1.25
2.17
1
0.621
0.3
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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: