Transcript Confocal Hardware

Announcements
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Artemia reports due today: please put on front desk.
Paper today: Wongprasert et al. 2003 (Katie leads)
Paper for next week: Jud et al. 2007 (Molly leads)
Start computers, open lecture PP for simulations again.
Revised Fluoview manual (PDF file) is available on
Fluoview computer.
• TBA and assignment this week: Multi-channel imaging,
including laser transmitted DIC
– Bright field contrast techniques
– Collect images of kidney slices and submit report (Fig. 1A:
channel 1, 1B: channel 2, 1C: merge of channels 1 and 2; 1D:
channel 3 (DIC image).
– Using references, describe some structures in your images in
your figure legend.
Microcopy
Facility
Assistant
Johns Hopkins
University
General
Description:
Qualifications:
https://hrnt.jhu.
edu/jhujobs/job
_view.cfm?view
_req_id=26703)
A microscopy specialist is sought to provide user support in Johns Hopkins
School of Medicine Microscope Facility. This facility provides light, fluorescence
and electron microscopy services to more than 200 users throughout Johns
Hopkins. In addition to assisting with routine maintenance of the facility equipment
and supplies, the candidate will help assist in training and supervising new users,
as well as help users troubleshoot experiments. The candidate will also be trained
to help users analyze images and data. Because of the diversity of equipment
within the facility, we will provide initial training as necessary. Consequently, the
candidate must display resourceful independence, a willingness to learn and have
strong analytical skills.
The primary duties of the candidate, when trained, will be to provide training and
supervision for new users when they use one of many confocal and wide-field light
microscopes (Zeiss, Olympus, and Nikon). Basic knowledge of cell biology is
critical for communicating with users. Helping users with software and specimen
preparation and interpretation may be needed as part of user services on projects.
Regular duties include cleaning these microscope work areas and maintaining
relevant supplies. Secondary duties will include collecting and maintaining a
library of protocols, manuals and tutorials for users. With all duties, timely recordkeeping (electronic & written) are required.
BS/BA, or equivalent, in biological sciences, chemistry, or related field required.
Comparable laboratory experience may be substituted for some education. At least
one year of relevant work experience required. Previous experience with
fluorescence microscope imaging, including basic image processing, is required.
Experience with specimen preparation for light microscopy (immunostaining,
histology/pathology), confocal microscopy, or interpretation of cellular physiology
in images is useful, but not required. Strong organizational skills coupled with
strong interpersonal and communication skills (both oral and written) are essential.
NOTE: The successful candidate(s) for this position will be subject to a preemployment background check
Paper Discussion Schedule
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Today, Jan. 22 (Hertzler): Zucker 2006
Jan. 29 (Katie): Wongprasert et al. 2003
Feb. 5 (Molly): Jud et al. 2007
Feb. 12 (Becky): Anders 1988
Feb. 19 (Rachel): Tan et al. 2005
Feb. 26 (Ellen):
March 12 (Emily)
March 19 (Amy)
March 26 (Amanda)
April 2 (Andrea)
April 9 (Brittaney)
April 16 (Lauren)
April 23 (Joe and Molly):
Outline: Contrast Enhancement,
Confocal Hardware
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B.
C.
Resolution and sampling frequency: XY and Z
Kohler Illumination and microscope setup
Contrast Enhancement
1.
2.
3.
4.
5.
D.
Components of LSCM
1.
2.
3.
E.
Brightfield
Interference of light
Phase contrast
Polarization
DIC
Scan Head
Lasers
Light Detectors
Week 4 TBA
Nyquist Sampling Theorem: XY
• Hibbs, p. 126: “When a
continuous, analogue image is
digitised, the information content
of the signal will be retained only
if the diameter of the area
represented by each pixel is 2.3x
smaller than the optical
resolution limit of the
microscope.”
– So an objective with a theoretical
resolution of 0.2 μm requires a
pixel size of 0.08 μm.
• How do you determine the pixel
size (sampling frequency)?
– Measure it with your scale bar at
different zoom factors:
From: Pawley, 2006. Handbook of Biological
Confocal Microscopy. Springer: New York.
Nyquist sampling of an image of two points
separated by the Rayleigh resolution
(Pawley 2006)
Sampling interval
= d/2.3
From: Pawley, 2006. Handbook of Biological Confocal Microscopy. Springer: New York.
5. Axial Resolution (Z or raxial)
• Minimum distance between the 3D diffraction patterns (PSFs) of two
points along the Z axis that can still be seen as two.
raxial
2

2
( NAobj )
where λ is the wavelengt h and
η is the refractive index of the medium
• From: Pawley, 2006. Handbook of Biological Confocal Microscopy.
Springer: New York.
5. Axial Resolution (Z or raxial)
• So with η = 1.5 for methyl salicylate:
For 40X 0.75 NA lens :
raxial
2 n

( NAobj ) 2
2(580 nm)(1.5) 1740 nm

2
(0.75)
0.5625
 3093 nm  3 μm
For 60X 1.4 NA lens :
raxial
2 n

( NAobj ) 2
2(580 nm)(1.5) 1740 nm

2
(1.4)
1.96
 888 nm  0.9 μm
raxial 
raxial 
raxial
raxial
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)
Undersampled
(lower Z resolution, e.g. NA=0.7)
XY and Z resolutions (μm), XY Zoom and
Z step sizes (1024 X 1024 box size)
Dye
λex/λem
10X
0.4 NA
20X
0.7 NA
40X
60X
0.75 NA 1.4 NA
fluorescein
488/518
0.790
4X
0.451
3X
0.421
1.5X
0.226
none
rhodamine
543/580
0.885
4X
0.505
3X
0.471
1.5X
0.253
none
raxial
Step+
fluorescein
488/518
9.71
3.2
3.17
1.1
2.76
0.9
0.793
0.26
raxial
Step+
rhodamine
543/580
10.9
3.7
3.55
1.2
3.09
1.0
0.888
0.30
rlateral
Zoom*
rlateral
Zoom*
*Nyquist sample frequency of 2.3
+Nyquist
sample frequency of 3.0
B. Kohler Illumination
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Purpose: Bright, even illumination
without illuminating unnecessary areas
or excess flare.
Steps
1. Focus on the sample.
2. Close field diaphragm until it can be
seen, focus and center the condenser.
3. Open field diaphragm until it disappears
from view.
Upright Scope
Epiillumination
Source
Brightfield
Source
Olympus BX50 Upright ‘scope
Inverted Microscope
Brightfield
Source
Epiillumination
Source
C. Enhancing contrast in LM
Fibroblast in Culture: Four Types of Light Microscopy
Bright-Field
Differential Interference Contrast
Phase-Contrast
Dark-Field
1. Bright-field microscopy
• Is the simplest, but object must be colored to be seen.
Histological staining usually requires killing the sample.
• Staining utilizes absorption; e.g. red stain absorbs
green and blue light, passing only red light. The
specimen is now an amplitude object, where contrast is
seen by reducing the amplitude of certain wavelengths of
light.
• Microscopy of living cells, which are usually transparent,
are limited by contrast, or the difference between light
and dark.
• How can we see them without staining them?
– By exploiting the fact that samples are phase objects, which
slow light down relative to other parts of specimen or to
background.
2. Interference
3. Phase-Contrast Microscopy
• Annular Ring in Phase Condenser
focuses cone of light onto sample.
– Specimen light is shifted -1/4
wavelength
• In Phase ring of Objective:
– Direct light (background) passes
through thin, dark part.
– Diffracted light (specimen) passes
through thick, light part, shifted -1/4
wavelength.
• Specimen light shifted by ½
wavelength total.
• Rings must be aligned to get
phase effect.
Alignment of phase rings
• Jave tutorial:
http://micro.magnet.fsu.edu/primer/java/ph
asecontrast/phasemicroscope/index.html
ALIGNED
Phase Contrast Microscope
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Surround wave (red) is undiffracted light that passes around and through the
sample.
Diffracted wave (blue) interacts with sample, is retarded by ¼ wavelength relative
to S wave.
Particle wave (green) results from interference between S and D waves.
Amplitude difference between S and P determines the level of contrast
PC scope shifts diffracted beam from specimen an additional ¼ wavelength to ½ λ,
creating maximal destructive interference between S and D.
Causes decrease in amplitude (brightness) in P, which can be seen against
brighter background.
Object dimmer
Background bright
λ/2
Object brighter
Background dimmer
Limitations of Phase Contrast
• Phase images are usually surrounded by halos around
the outlines of details. Such halos are optical artifacts,
which sometimes obscure the boundaries of details.
• The phase annuli do limit the working numerical aperture
of the optical system to a certain degree, thus reducing
resolution.
– 20X PlanApo 0.7 NA compared with 20X Phase 0.4 NA.
• Phase contrast does not work well with thick specimens
because shifts in phase occur from areas slightly below
or slightly above the plane that is in focus. Such phase
shifts confuse the image and distort image detail.
4. Polarization Microscopy
• Useful for crystalline
materials or oriented
structures in biological
materials, e.g.
– Mitotic spindle fibers
– Microfilament bundles
– Striated muscle fibers
• These structures are said
to be birefringent (having
double refraction),
meaning that they have
at least two refractive
indices.
Birefringent skeleton in sea urchin larva
Polarization of Light
AKA
Analyzer
Note: laser light is already polarized
Polarizing Sunglasses
• Human eye can’t
detect difference in
randomly oriented
versus polarized light.
• When polarizing
sunglasses filter out
parallel waves, eye
detects less glare,
lower amplitude.
Isotropic versus anisotropic materials
Light slowed equally vibrating
in any direction.
Light slowed less when vibrating N-S
n1
n1
n1
n1
Isotropic:
Glass, salt
n2
Light slowed more when vibrating E-W
Anisotropic (birefringent – with two
Refractive indices):
Sugar, muscle, gout crystals
Birefringence
• First clue to explanation of
polarization came from
observation of calcite crystals
by Erasmus Bartholin in 1669.
• http://www.microscopy.fsu.edu/
primer/java/polarizedlight/icela
ndspar/index.html
• One of the light rays emerging
from a birefringent crystal is
termed the ordinary ray, while
the other is called the
extraordinary ray.
Incident ray oblique to optical axis of crystal
Split, both
Polarized;
perpendicular
Incident light perpendicular to optical axis of specimen: same trajectory,
different path length causes interference when recombined.
Birefringent samples oriented 45o to
crossed polarizers are maximally bright
• Java tutorial:
http://micro.magnet.fsu.edu/primer/java/polarizedlight/cry
stal/index.html
5. Differential Interference
Contrast
• Also called Nomarski optics; uses plane polarized light.
• Similar to Phase Contrast in that light from low contrast sample is
caused to interfere destructively to produce amplitude changes.
• Produces contrast where changes in thickness, slope, or refractive
index occur in cell, especially along edges, to give a pseudo three
dimensional appearance.
Red blood cells
Cheek cells
Filamentous alga
DIC images have no halos.
DIC produces superior axial resolution, optical sectioning.
DIC Pathway:
Components
• Light from lamp passes through
Polarizer, is separated into O
and E waves by Wollaston
Prism (specific to each lens),
then to Condenser.
• Phase specimen creates
different optical path lengths for
O and E, shifting their phase.
• After passing through specimen,
light passes through Objective,
and is recombined, resulting in
interference, by second
Wollaston Prism, then to
Analyzer (second Polarizer) to
Eyepiece.
Effect of Bias Retardation in
Analyzer
• Controls how O and E waves are
recombined.
• Affects brightness, contrast, and color
(optical staining) of specimen.
• Java tutorial:
http://micro.magnet.fsu.edu/primer/java/dic
/lightpaths/index.html
Advantages, Disadvantages of DIC
• Advantages:
– Uses full NA of the lens, achieving optimal
resolution and some optical sectioning ability.
– Provides optical color staining.
– No phase halos as with phase contrast.
• Main Disadvantage:
– Tissue culture plastic or birefringent sample
features can produce confusing effects.
D. Confocal Hardware
1. Fluoview 300 Scan Head
Anatomy
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1
10
4
3
9
5
7
8 Beam splitter
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2. Lasers available for Olympus
Fluoview Confocal Microscopes
• Blue argon-ion (488 nanometer) laser (WE HAVE)
• Multi-line argon-ion (457, 488, and 514 nanometers)
laser
• Green helium-neon (543 nanometer) laser (WE HAVE)
• Red helium-neon (633 nanometer) laser (WE MAY GET)
• Yellow krypton-ion (568 nanometer) laser
• Blue-violet helium-cadmium (442 nanometer) laser
• Violet and blue-violet diode (405 and 440 nanometer)
lasers
• Ultraviolet argon-ion (351 nanometer) laser
• Infrared (750 nanometer) laser
Adjusting Offset and PMT/Gain to
maximize range of grey levels collected
Pixel intensity
256 (4047)
0
Figure 5(a) illustrates the raw confocal image along with the signal from the
photomultiplier. After first applying a negative offset voltage to the
photomultiplier, the signal and image appear in Figure 5(b). Note that as the
signal is shifted to lower intensity values, the image becomes darker (upper
frame in Figure 5(b)). When the gain is next adjusted to the full intensity
range (Figure 5(c)), the image exhibits a significant amount of detail with good
contrast and high resolution.
Note on Building stereo images
• Stereo Factor: Sets the deviation
between the left and right eyes
when building a pair of stereo 3D
images or a 3D image to be
viewed through color (red/green)
eyeglasses.
– You can change this.
• Z Stretch Factor: Provides each
section with a feeling of
thickness. Usually this value
does not need to be changed.
Week 4 TBA
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Kohler illumination
Phase contrast
Polarization
DIC
Laser transmitted DIC
Assignment: Collect multi-channel
fluorescence and laser transmitted
images of prepared kidney slide, save,
submit report as before.