Cell Biology Application of Microscopy
Class 4: Optical Aberrations and Objective Lens
Arlene Wechezak,
Algae and diatoms (10X)
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Numerical Aperture
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Numerical Aperture
The light gathering ability of an objective
NA = n • sin(µ)
n = refractive index
µ = ½ angular aperture
NA is directly related to resolution, brightness, working distance
http://micro.magnet.fsu.edu/primer/index.html
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Numerical Aperture and Working Distance
NA is inversely proportional to working distance
http://micro.magnet.fsu.edu/primer/index.html
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Numerical Aperture and Image Brightness
Image brightness is proportional to NA
Higher NA is better at gathering light
F (transmitted) = 104 NA2/M2
Separate objective/condenser
F (reflected) = 104 (NA2/M)2
Objective acts as condenser
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Transmitted Light (Diascopic Illumination)
Transmitted light microscopy is the type of microscopy where the light is transmitted
from a source on the opposite side of the specimen from the objective. Usually the light
is passed through a condenser to focus it on the specimen.
http://micro.magnet.fsu.edu/primer/index.html
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Reflected Light (Episcopic Illumination)
Reflected light microscopy is often referred to as incident light, episcopic illumination, or
metallurgical microscopy, and is the method of choice for fluorescence and for imaging
specimens that remain opaque even when ground to a thickness of 30 micrometers.
http://micro.magnet.fsu.edu/primer/index.html
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Reflected Light (Episcopic Illumination)
(a) Surface of an integrated circuit (reflected DIC). (b)The jewel bearing of a watch
mechanism (reflected brightfield). (C) Surface structure of a superconducting wire
(reflected darkfield). (d) Magnetic thin film (reflected polarized light).
http://micro.magnet.fsu.edu/primer/index.html
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Numerical Aperture and Image Brightness
F (trans) = 104 NA2/M2
F (refl) = 104 (NA2/M)2
Separate objective/condenser
Objective acts as condenser
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NA and Resolution
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Numerical Aperture and Resolution
Resolved
Not resolved
Resolution (r) = 0.61 • λ/NA
= 0.61 • λ/[(NAobjective + NAcondenser)/2]
http://micro.magnet.fsu.edu/primer/index.html
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Numerical Aperture and Resolution
Low NA
http://micro.magnet.fsu.edu/primer/index.html
High NA
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Microscope Magnification
http://micro.magnet.fsu.edu/primer/index.html
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Magnification
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Magnification vs Resolution
200x, 0.4 NA
200x, 0.1 NA
“Empty magnification” is a situation where the image is magnified,
but no additional detail is resolved. http://www.microscopyu.com/articles/formulas/formulasmagrange.html
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Range of Useful Magnification
500 ~ 1000x NA of Objective
http://micro.magnet.fsu.edu/primer/index.html
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Magnification vs Resolution
Dry – up to 1.0 NA
http://micro.magnet.fsu.edu/primer/index.html
Oil – up to 1.51 NA (theoretical)
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Aberration
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Aberrations in Lens
•  On-axis aberration
Chromatic
Spherical
•  Off-axis aberration
Coma
Astigmatism
Field curvature
•  Geometrical distortion
Barrel distortion
Pincushion distortion
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On-axis: Chromatic Aberration
Blue halo around edge
shows the chromatic aberration
•
Wavelengths are not refracted equally; colored halos form around object
•
Can be corrected by using multiple lenses with different color-dispersion
http://micro.magnet.fsu.edu/primer/java/aberrations/chromatic/index.html
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On-axis: Spherical Aberration
•
The best focus, in an imperfectly or non-corrected lens, will be somewhere
between the focal planes of the peripheral and axial rays, an area known as the
“disc of least confusion”.
•
Can be introduced by improper microscope tube lens, coverslip thickness
(deviate from 0.17 mm), or immersion oil.
•
Can be corrected by using lens elements of different shapes to bring the more
central and more peripheral rays to common focus.
http://micro.magnet.fsu.edu/primer/java/aberrations/spherical/index.html
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On-axis: Spherical Aberration
•
Spherical aberrations are not only influenced by the optical properties
of an objective but also by the properties of the cover glass and
mounting medium.
•
In practice, there are mainly two factors that considerably intensify
spherical aberration:
1.  The difference in the refractive indices of the immersion medium and
mounting medium. The more the refractive index of the immersion
medium deviates from the refractive index of the mounting medium, the
more marked the spherical aberration.
2.  The distance between the cover glass and the specimen structure to be
examined. In general, spherical aberration intensifies as the sample depth
increases. Ideally, therefore, the specimen should be positioned directly
under the cover glass.
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Coverslips Thickness
The standard thickness of the coverslips is 0.17 mm (#1.5!!), all of the
unadjustable objectives are spherically corrected to this thickness. #1 (0.15 mm)
coverslips are too thin and #2 (0.22 mm) are too thick.
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On-axis: Spherical Aberration
http://micro.magnet.fsu.edu/primer/index.html
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Coverslips Correction Collar
Compensation for cover glass thickness can be accomplished by adjusting the
mechanical tube length of the microscope, or by the utilization of specialized
correction collars that change the spacing between critical lens elements inside the
objective barrel.
http://micro.magnet.fsu.edu/primer/java/aberrations/slipcorrection/index.html
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Off-axis: Field Curvature
•
•
•
The effect of field curvature means that a flat
structure is imaged on a curved surface.
This artifact is the natural result of using
lenses that have curved surfaces.
Can be corrected using flat-field objectives
(Plan or Plano).
http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield/index.html
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Off-axis: Field Curvature
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Geometrical Distortion
Image distortion is an aberration commonly observed in stereomicroscopy,
and is manifested by changes in the shape of an image rather than the
sharpness or color spectrum.
http://micro.magnet.fsu.edu/primer/java/aberrations/distortion/index.html
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Objective Lens
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Objectives
This is a 250x long working distance “apochromat”, which contains 14 optical elements that
are cemented together into three groups of lens doublets, a lens triplet group, three individual
internal single-element lenses, a hemispherical front lens, and a meniscus second lens.
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Objectives
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Objectives
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Infinity Optical System
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Objective Corrections
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Achromats
•  Least expensive and least corrected.
•  Only blue (486 nm) and red (656 nm) colors are
corrected for chromatic aberration.
•  Only green color (546 nm) is corrected for
spherical aberration.
•  Not corrected for field curvature.
•  Plan Achromats – corrected for field curvature.
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Fluorites (Semi-Apochromats)
•  Named for the mineral fluoride was originally used
in their construction.
•  Chromatically corrected for 2-3 colors.
•  Spherically corrected for 2-3 colors.
•  Not corrected for field curvature.
•  Higher numerical aperture (NA).
•  Moderately expensive.
•  Plan Fluorite – corrected for field curvature.
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Apochromats
•  Most correction and most expensive.
•  Chromatically corrected across the entire visible
light spectrum.
•  Spherically corrected across the entire visible
light spectrum.
•  Highest numerical aperture (NA).
•  Usually available as Plan Apochromats –
corrected for field curvature.
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Objective Corrections
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Working and Parfocal Distance
Working distance is a trade-off
with numerical aperture (NA)!
There are specially designed
long-working distance objectives
for tissue culture
Covslip surface
Parfocal objective lenses are lenses that stay in focus when switched
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Field of View
The diameter of the field in an optical microscope is expressed by the field-ofview number, or simply the field number, which is the diameter of the view field
in millimeters measured at the intermediate image plane.
Field Size = Field Number (fn) ÷ Objective Magnification (M)
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Understand Your Objectives
What’s the magnification?
What’s the immersion medium?
What’s the NA?
What’s the coverslip thickness?
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Contrast Microscopy Objectives
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UV Light Objectives
Fluorescence objectives are designed with quartz and non-fluorescent glasses
that have high transmission from the ultraviolet (down to 340 nm) through the
infrared regions of the electromagnetic radiation spectrum.
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http://www.microscopyu.com/articles/fluorescence/filtercubes/ultraviolet/uv2a/uv2aindex.html
Reflected Light Objectives
Objectives designed to be used with reflected (episcopic) illumination have a
special construction consisting of a 360-degree hollow chamber surrounding the
centrally located lens elements. The outer system functions as a "condenser" and
the inner system as a typical objective.
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Adjustable NA Objectives
Specimens with unusually high fluorescence or very bright darkfield specimens often
induce image flare by light emitted from areas outside the focal plane. To
compensate for this artifact, manufacturers offer high NA objectives that are
equipped with an internal iris diaphragm to increase image contrast during imaging.
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Water Immersion Objectives
The dipping cone is tapered at a 43-degree angle to allow a steep inclination of
approach providing easy access to the sample by the manipulator or microelectrodes
while keeping the specimen and objective immersed in solution. These objectives
also have long working distances.
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Distortion in Aqueous Media
distorted
undistorted
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Condenser
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Condenser
Immersion oil is needed for high NA (>1.0) condensers!!
http://micro.magnet.fsu.edu/primer/java/kohler/condenseraperture/index.html
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Condenser Aberration Correction
X = corrected
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Immersion Medium Affects NA
NA = n • sin(µ)
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Homogenous Immersion System
Homogeneous Immersion System is the ideal situation to achieve maximum
numerical aperture and resolution in an optical microscope. In this case, the
refractive index and dispersion of the objective front lens, immersion oil, substage
condenser front lens, and the mounting medium are equal or near equal.
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Immersion Medium
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Key Immersion Medium Features
1.  Match the refractive index of the lens
2.  Non-drying
3.  Inert to optical surface coatings
4.  Non-toxic (polychlorinated biphenyls)
5.  Easy to remove
6.  Non-fluorescent
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Common Immersion Media
Refractive index for glass ~1.52
IR compatible
UV compatible
A primary problem with common immersion media is their inherently high
absorption characteristics in the UV or IR region of the spectrum.
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