Transcript Slide 1

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 Zseries of Artemia
samples
IV. Assignment due
Jan. 29
TBA times with Dr. Hertzler: Spring 2007
Time
Tuesday
Wednesday
Thursday
Friday
9
SEM
Cell Biology
TEM
Cell Biology
10
Group 1
Office
Group 2
11
Amy, Lauren,
Rachel
Hours
Andrea,
Emily, Molly
1
403
Group 3
Lab meeting
2
students
Becky, Ellen,
Katie
Group 4
3
UCC
8
12
4
Faculty
Meeting
Amanda,
Brittaney, Joe
Seminar
Outline: Understanding Microscopy
A. Introduction to Confocal Microscopy
1. Confocal versus conventional (widefield) fluorescence
2. Optical sectioning
3. Imaging modes and applications
4. Advantages, limitations of confocal
B. Essential Optics
1. Wave/particle nature of Light
2. Diffraction
3. Numerical aperture
4. Lateral resolution
5. Axial resolution
Useful resource: Molecular Expression Microscopy Primer:
• http://micro.magnet.fsu.edu/primer/index.html
Laser Scanning Confocal
Microscope Components
Scan Head
Microscope
Controller
box
Computer, display
Laser
1. Conventional versus confocal
fluorescence
Conventional epifluorescence
Confocal epifluorescence
Sea urchin eggs (100 μm diameter)
stained with antibody to tubulin.
Widefield
Confocal
Human brain slice
Rabbit muscle fibers Sunflower pollen grain
Wide-field fluorescence: dichroic
(dichromatic) mirror
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).
2. Optical slicing
3. Imaging Capabilities
1. XY fluorescence
imaging
a) Single
b) Double
c) Single or Double +
transmitted (not
confocal)
d) 3-channel (need 3
lasers)
2. XYZ imaging, 3-D
reconstruction
3. Time-lapse
•
Including 4D
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
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.
B. Basic Optics
1. 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
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
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
2. Diffraction:
Bending of light as it passes an edge
One long continuous wave
unlike light from a lamp
or the sun.
λ<d
See: Microscopy primer,
λ>d
Diffraction Pattern from Slit
Results from Interference
Java Tutorial: Diffraction Patterns
• http://micro.magnet.fsu.edu/primer/java/diff
raction/basicdiffraction/index.html
• How does the width of the central
maximum vary with the wavelength?
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
Resolution and Airy disk patterns
Java Tutorial: Airy Pattern Basics
• http://micro.magnet.fsu.edu/primer/java/imagefor
mation/airydiskbasics/index.html
– How does resolution vary with wavelength and
numerical aperture?
• http://micro.magnet.fsu.edu/primer/java/imagefor
mation/airyna/index.html
– What is the effect of higher NA?
• http://micro.magnet.fsu.edu/primer/java/imagefor
mation/rayleighdisks/index.html
– What is the Rayleigh criterion?
3. Numerical aperture (NA)
NA = n sin 
where n = refractive index and  = the collecting angle.
nair = 1.00 and noil = 1.515.
W.D.
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
4. Lateral Resolution (XY or rlateral)
• The smallest distance two objects can be
imaged as two. Depends on wavelength and NA.
1.22 λ
rlateral 
NA obj  NA cond
Where  is wavelengt
If NA
h, NA is numerical
obj
rlateral 
 NA
1.22 λ
2 NA obj
cond

then
0 . 61 
NA obj
aperture.
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:
r
0.61  520 nm
 2 26 nm  0.2 μm
1.4
• (Footnote: for confocal, the resolution equation
is slightly better: rlateral = 0.4λ/NA so best
resolution is closer to 0.15 μm).
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.
Zooming for maximum XY
resolution
No Zoom
2X Zoom
Java Tutorial: 3D Airy disk is the
Point Spread Function
• http://micro.magnet.fsu.edu/primer/java/im
ageformation/depthoffield/index.html
This Z step will
not resolve the
objects in Z axis.
This Z step will
resolve the
objects in Z axis.
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:
For 40X 0.75 NA lens :
raxial 
raxial 
For 60X 1.4 NA lens :
1 .4  n
( NA obj )
raxial 
2
1.4(580 nm)(1.5)
(0.75)
2
raxial  2165 nm  2 μm

1218 nm
0.5625
raxial 
2n
( NA obj )
2
2(580 nm)(1.47)
(1.4)
2

1218 nm
1.96
raxial  621 nm  0.6 μm
• 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/presentati
on04.shtml
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)
Z axis under- and over-sampling
Undersampled
Too few sections for full Z
resolution
But: 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.
XY and Z resolutions (μm)
10X
0.4 NA
20X
0.7 NA
40X
60X
0.75 NA 1.4 NA
rlateral
fluorescein
488/518
0.790
0.451
0.421
0.226
rlateral
rhodamine
543/580
0.885
0.505
0.471
0.253
raxial
step
fluorescein
488/518
6.80
2.22
1.1
1.93
1
0.555
0.275
raxial
step
rhodamine
543/580
7.61
2.49
1.25
2.17
1
0.621
0.3
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
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 Zseries, (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.
Paper discussion
•
•
•
•
•
•
Today, Jan. 22: Zucker 2006 (Hertzler)
Jan. 29: (Hertzler)
Feb. 5:
Feb. 12:
Feb. 19:
Feb. 26: