IMAGING OPTICS

FUNDAMENTALS
FOR LIFE SCIENCES

Gregory Hollows
Edmund Optics
 IMAGE QUALITY

Resolution Contrast
 HOW DOES DIFFRACTION AND F/#
AFFECT PERFORMANCE?
Not even a perfectly designed and manufactured lens can accurately
reproduce an object’s detail and contrast.
Diffraction will limit the performance of an ideal lens.
The size of the aperture will affect the diffraction limit of a lens.
The smallest achievable spot of a lens = 2.44 x wavelength of light x (F/#)
F/# describes the light gathering ability of an imaging lens (lower F/#
lenses collect more light).
As lens aperture decreases, F/# increases.
 HOW DOES DIFFRACTION AND F/#
AFFECT PERFORMANCE?
The smallest achievable spot of a lens = 2.44 x wavelength of light x (F/#)
 HOW DOES DIFFRACTION AND F/#
AFFECT PERFORMANCE?
The smallest achievable spot of a lens = 2.44 x wavelength of light x (F/#)
9 micron pixels 4.5 micron pixels

~f/8

~f/4

~f/2

2.2 micron pixels

 HOW DOES DIFFRACTION AND F/#
AFFECT PERFORMANCE?
Not even a perfectly designed and manufactured lens can accurately
reproduce an object’s detail and contrast.
Diffraction will limit the performance of an ideal lens.
The size of the aperture will affect the diffraction limit of a lens.
The smallest achievable spot of a lens = 2.44 x wavelength of light x (F/#)
F/# describes the light gathering ability of an imaging lens (lower F/#
lenses collect more light).
As lens aperture decreases, F/# increases.
 HOW DOES CONTRAST DEPEND
ON FREQUENCY?
Suppose two dots are placed close to each other and imaged through a lens.
The two spots will blur slightly.
Moving the spots closer causes the blur to overlap and contrast is decreased.
When the spots are close enough that the contrast becomes limiting, the spacing is
our resolution.
At each spacing of the spots we obtain a specific contrast.
We can plot this information in the form of a Modulation Transfer Function (MTF).
 HOW DOES DIFFRACTION AND F/#
AFFECT PERFORMANCE?
Not even a perfectly designed and manufactured lens can accurately
reproduce an object’s detail and contrast.
Diffraction will limit the performance of an ideal lens.
The size of the aperture will affect the diffraction limit of a lens.
The smallest achievable spot of a lens = 2.44 x wavelength of light x (F/#)
F/# describes the light gathering ability of an imaging lens (lower F/#
lenses collect more light).
As lens aperture decreases, F/# increases.
MODULATION TRANSFER
FUNCTION (MTF) CURVE
DOES INCREASING THE F/#, DECREASING
NAHURT PERFORMANCE?
HOW IS MTF AFFECTED BY
WAVELENGTH?

660nm Light 470nm Light

3b
HOW WAVELENGTH AFFECTS
RESOLUTION
HOW WAVELENGTH AFFECTS
RESOLUTION
HOW WAVELENGTH AFFECTS
RESOLUTION
HOW WAVELENGTH AFFECTS
RESOLUTION
HOW WAVELENGTH AFFECTS
RESOLUTION
 HOW IS MTF AFFECTED BY
WAVELENGTH?

Chromatic
Aberration

Chromatic aberrations can be both on axis and off axis

Lateral Color Axial Color

 COLOR BALANCING IN LENSES

Achromatic Design v. Apochromatic Design

 IMAGE QUALITY

Depth of Field Depth of Focus

HOW CAN APERTURES BE USED TO
IMPROVE DEPTH OF FIELD?
HOW CAN APERTURES BE USED TO
IMPROVE DEPTH OF FIELD?
DOF TO RESOLUTION
COMPARISON
TIP AND TILT IN SENSOR OR
OBJECT
GLOSSARY & IMPORTANT TERMS

• Parfocal Length
Distance between the surface of the specimen and the objective mounting position when in focus

• Infinity Corrected Optical System

An optical system in which the image is formed by an objective and a tube lens with an Infinity
Space between them, into which optical accessories can be inserted

• Finite Conjugate Optical System

An optical system in which the image is formed only by an objective 24
 FINITE CONJUGATE DESIGN
• Light from a source is focused (not from infinity)
• Characterized by DIN or JIS standards
• Utilized when cost and ease of design are concerns
• Offer little to no filtering or in-line illumination
• No tube lens required for focus
• Account for majority of basic microscope systems where only simple
magnification and lighting is required

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INFINITE CONJUGATE DESIGN
• Offer longer working distances
• Allows for larger samples, elaborate mechanics, and room to operate (dyes,
reagents, catalysts)

• Allow for addition of in-line components

• Filters, beamsplitters, and mechanics

• Light rays focused with assistance of secondary/tube

lenses
–Set at specific, long distance from objective (~160-200mm)

• Enable in-line illumination

–Improved lighting and convenient for space constraints

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 BUILDING A CUSTOM SYSTEM FROM
OFF THE SHELF COMPONENTS

• Simplified view of an infinity corrected system

• The more advanced design offers some distinct advantanges

 BUILDING A CUSTOM SYSTEM FROM
OFF THE SHELF COMPONENTS

• The final system can end up

looking something like this
• The beamsplitter allows for
inline illumination to be
introduced into the system
• Notice that theoretically there
has been no change to the
imaging capabilities of the
system
 BUILDING A CUSTOM SYSTEM FROM
OFF THE SHELF COMPONENTS
• Ultimately the system can be come
even more complex by following the
same logic
• In this systems a second
beamsplitter is introduced leading to
another tube (focusing) lens and
second camera
• Now with two tube lenses (with
different focal lengths) in the system
it is possible to get two different
magnifications simaltanously from
one system
 BUILDING A CUSTOM SYSTEM FROM
OFF THE SHELF COMPONENTS
• The results can be
seen in the images to
the right
• This can allow for
image processing on
two images at the
same time
• Additionally this
saves time when
compared to a
system that requires
you to zoom
 BUILDING A CUSTOM SYSTEM FROM
OFF THE SHELF COMPONENTS
• Since in this design there is collimated space between
the lenses it is possibly to add components between
them to enhance the systems abilities
• This includes:
• Beamsplitters
• Filters
• Colored Glass
• Thin Film
• Interference Filters
• Prisms
• Other optical components
Gregory Hollows
Director, Imaging Business Unit
Edmund Optics
Barrington, New Jersey
USA
Phone: (856) 547-3488
Email: ghollows@edmundoptics.com
www.edmundoptics.com
GLOSSARY & IMPORTANT TERMS

• Focal length (F)

Distance between a principal point and a focal point. F1 is the focal length of an objective. F2 is the focal length of a tube lens.
For infinity-corrected systems, magnification is determined by the ratio of the focal length of the tube lens to that of the
objective.
Magnification of Objective = Focal length of tube lens / Focal length of objective

• Field Number and Field of View (FOV)

The field number of an eyepiece (expressed in mm) is determined by the field stop diameter of the eyepiece. FOV is the area
of the specimen that is observable, and is determined by the field number of the eyepiece and magnification of the objective.
FOV = Field number of eyepiece / magnification of objective

• Depth of Field (DOF)

Vertical distance in the specimen, measured from above and below the exact plane of focus, which still yields an acceptable
image. The larger the NA, the smaller the depth of field.
± DOF = λ / (2*(NA)2) - standard wavelength of 550nm

• Aperture Diaphragm
Adjusts the amount of light passing through, and is related to the brightness and resolving power of an optical system. This
diaphragm is especially useful in width dimension measurement of cylindrical objects with contour illumination, and provides
the highest degree of correct measurement/observation by suppressing diffraction in an optimal aperture.
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GLOSSARY & IMPORTANT TERMS

• Oil Immersion
Medium used on objectives with an NA high than 0.95
Examples: air, water, glycerin, paraffin oil, synthetic oil, anisole (Refraction index between 1.01-1.65)

• Field Stop
Used for blocking out unwanted light and preventing it from degrading the image

• Vignetting
This unwanted effect is the reduction of an image’s brightness or saturation at the periphery compared to the image
center. May be caused by external (lens hood) or internal features (dimensions of a multi-element objective).

• Double Image
An image degrading phenomenon in which an image appears as if it is a double image due to redundant light
projection and optical interference within the optical system.

• Flare
Lens flare is typically seen as several starbursts, rings, or circles in a row across the image or view, and is caused
by unwanted image formation mechanisms, such as internal reflection and scattering of light.

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 SPECIFYING AND CHOOSING
OBJECTIVES
• Objective Specifications
• Objective Designs
• Finite Conjugate
• Infinite Conjugate
• Lens Configurations
• Industry Standards
• Mounting Threads
• Tube Lengths
• Choosing the Right Objective

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OBJECTIVE SPECIFICATIONS

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 OBJECTIVE LENS
CONFIGURATIONS
• Achromatic ~ 3-5 lens elements
• Fluorite ~ 5-9 lens elements
• Apochromatic ~ 9-18 lens elements

Achromatic – corrected for chromatic aberration at the red and blue wavelengths only
Apochromatic – corrected for chromatic aberration at the red, blue, and yellow
wavelengths
Fluorite – to be used in low light level detection, specifically fluorescence emission
Plan – objective lens that produces a flat (planar) image by correcting the spherical 37
aberration/curvature of the field of an achromatic/apochromatic lens
 INDUSTRY STANDARDS
Royal Microscopy Society (RMS) - 0.8” x 36TPI, Whitworth
• Society Thread
• ~200mm tube length

Deutsches Insititut fur Normung (DIN) - 0.7965”, 36TPI, 55` Whitworth

• 45mm standard objectives
• 160mm tube length
• Object to image distance 195mm, fix object distance at 45mm, and
remaining 150mm for internal real image position (10mm from end
of tube)

Japanese Industrial Standards (JIS) - 0.7965”, 36TPI, 55` Whitworth

• 36mm standard objectives
• 170mm tube length
• Rare cases have slight variation on Parfocal Distance and Tube Length

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 INDUSTRY STANDARDS

Typical Objective Manufacturer Specifications

• Olympus Standards - RMS thread type, 180mm

tube lens focal length, 45mm parfocal distance
• Nikon Standards - M25 thread type, 200mm tube
lens focal length, 60mm parfocal distance
• Zeiss Standards - RMS thread type, 165mm tube
lens focal length, 45mm parfocal distance
• Mitutoyo Standards - 26mm x 0.706mm pitch (36
TPI), 200mm tube lens focal length, 95mm parfocal 39

distance
SELECTING THE RIGHT OBJECTIVE
“For any conventional optical microscope configuration,
the objective is the most critical component of the
system in determining the information content of the
image.”
Microscopy Techniques -Olympus Microscopy Resource Center

• Brightfield
• Darkfield
• Phase Contrast
• Differential Interference
Contrast (DIC)
• Fluorescence
• Confocal
• Multiphoton
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 BRIGHTFIELD & DARKFIELD
Brightfield Illumination
• Sample contrast comes from
absorbance of light in the sample
• The most basic technique in light
microscopy
• Typical appearance is a bright
background with dark objects
• Zero sample preparation

Darkfield Illumination
• Sample contrast comes from light
scattered by the sample
• Technique used to enhance
contrast in unstained samples
• Typical appearance is a dark
background with bright objects
• Zero sample preparation, but also
lacks overall intensity and
resolution Images from Wiki – tissue paper, 1.6um/pixel
BRIGHTFIELD & DARKFIELD

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 BRIGHTFIELD OBJECTIVES

• Almost any objective will work for this

microscopy technique!
• The illumination source, detector, and sample
heavily influence objective choice
• The lowest cost, most basic objective would
work (<$100), but your image and system
could suffer

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 DARKFIELD OBJECTIVES

• Depending on the light source and

illumination method, a specific objective
type might be necessary
• Darkfield attachments and darkfield ring
lights exist, which allow for the use of
standard brightfield objectives
• However, specialized reflected light
darkfield objectives exist which redirect
light in an oblique fashion via reflector
lenses working in unison with basic Olympus Resource Center

objective design

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 PHASE CONTRAST
• Displays proportional differences in
optical density
• Shows differences in refractive index as
contrast difference
• Nucleus appears darker than
surrounding ECM
• Typically a grey background with light
and dark features - these features
represent change in optical density

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 PHASE OBJECTIVES
• Specific phase contrast objectives exist
• Almost any objective can be used here as well,
but more intricate mechanical designs/systems
needed at image and object space
• Mechanical Apertures
• Phase Plates
• Phase plates and apertures control phase
shifts and light. Image is made up of a
combination of scattered light and background
light
• These specialized phase objectives can be
intricate in design, difficult to use, and
expensive to acquire

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 FLUORESCENCE
• Technique that utilizes
fluorescence, as opposed to
scatter, dispersion, or reflection
• Fluorescence describes light
emission that continues only
during the absorption of excitation
light
• Requires the use of filters, to
separate excitation energy from
emission energy

Green Fluorescent Protein – image courtesy of Nexcelom Bioscience

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FLUORESCENCE OBJECTIVES

• More intricate objective assemblies required

• Low, or weak emission levels are typical
• This requires objectives with extremely high
transmission and high numerical apertures
• Often, wavelength correction is required so an
apochromatic objective becomes necessary
• High numerical apertures result in short working
distances

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 CONFOCAL MICROSCOPY
• Confocal microscopy shares a
common optical pathway with
fluorescence microscopy
• Differences are…
• Addition of a pinhole aperture between light source
and excitation filter
• Addition of pinhole aperture between detector and
emission filter

• These apertures block out-of-focus

light rays

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 CONFOCAL OBJECTIVES
• Almost always requires planar, apochromatic,
immersion objective designs
• Correction collar to compensate for aberrations from
cover glass changes or depth changes in the sample
• Extremely high numerical apertures required
(20x/0.75, 40x/0.9, 60x/1.2-1.25)

Olympus Resource Center 50

FILTERING
GLOSSARY & IMPORTANT TERMS
• Angle of Incidence (AOI) – the angle at which a filter is tilted relative to the primary ray. For
example, an AOI of 0 means the primary ray is perpendicular to the plane of the filter
• Bandpass filter – a filter than transmits a band of wavelengths while blocking the wavelengths
on either side of the passband
• Blocking – the level of rejection of light outside the passband
• Center Wavelength (CWL) – center of the passband for a bandpass filter. Another common
definition is the average of the 50% of peak of each edge of a bandpass filter
• Cone angle – the range of angles of light incident on the filter about the AOI
• Cone angle average – average of transmission or reflection across the cone angle
• Dichroic – a filter that transmits one band and reflects a different band
• Edge filter – a filter that transmits a band of wavelengths and blocks only low side (long wave
pass) or high side (shortwave pass) of wavelengths
• Fused Silica – substrate material composed of amorphous silicon dioxide
• Long Wave Pass (LWP) – an edge-type filter that transmits longer wavelengths and blocks
shorter wavelengths
• Notch Filter – a filter that blocks a narrow range while transmitting wavelengths on either side of
the blocked wavelength range

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GLOSSARY & IMPORTANT TERMS
• Optical Density (OD) – the log (base 10) of transmittance (T)
• Passband – the range of wavelengths that are desired to be transmitted
• Ripple – variations in transmission in the passband
• Shortwave Pass (SWP) – an edge-type filter that transmits the shorter wavelengths
and blocks higher wavelengths
• Spectral Slope – rate of change of the spectrum from a passband to a blocking
band
• Transmitted Wavefront Error (TWE) – distortion of the wavefront of a plane wave
as it transmits through a filter, generally given in units of the HeNe wavelength
(633nm)

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TYPES OF FILTERS

Colored Glass
 TYPES OF FILTERS

Shortpass and Longpass

TYPES OF FILTERS

Bandpass and Notch

 TRADITIONAL VS HARD COATED
• Traditional filters – multiple layers, heavier, and
larger in size. Decrease in transmission due to
number of layers
• Hard Coated filters – single layer, advanced coating
decreases overall size, weight, and results in
improved blocking and transmission

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 MULTILAYER COATINGS
• Coated filers have an angular dependence to them
• This can create blue shift in the coating
transmission

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 FABRICATION TECHNIQUES
• Optical filters can be broken down into two main categories
• Absorptive – light is blocked based on the absorption properties of the glass
substrate used
• Dichroic – unwanted light is reflected, and specific ranges are transmitted
• Absorptive filters are not angularly sensitive, while dichroic filters are heavily
depending on angle of incidence
• Dichroic filters are desirable as they can separate light into two sources. Dichroics
utilize multiple layers to exploit the interference nature of light waves

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 TYPES OF POPULAR FILTERS
• Bandpass Filters – narrowband to broadband, these filters block the
surrounding wavelengths and are typically very sensitive to angles.
Hard sputtered filters are ideal to maximize transmission and blocking
• Edge filters – longpass or shortpass filters, these filters have a
designated cut-on or cut-off wavelength that allows transmission
before or after the designated wavelength. A longpass and shortpass
set can create custom bandpass filters
• Notch Filters – designed to block a pre-selected bandwidth while
transmitting all other wavelengths within the design range of the filter.
Typically used to remove a single laser wavelength, or narrow band,
from an optical system
• Dichroic Filters – coated with a thin film that can vary transmission
and reflection properties. These filters are typically utilized at 45
degree AOI, but can be varied for a specific wavelength parameter.
• Neutral Density Filters – designed to reduce, or attenuate, light in an
optical system. Either absorptive or reflective, they are specified by
optical density on the logarithmic scale

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SYSTEM LAYOUTS AND
FILTERING
COST SENSITIVITY
Attribute Filter Price Impact

Low Medium High

Size (area) X

Flatness X X

TWE X

Wavelength Tolerance X

Spectral Steepness X

Blocking X

Blocking Range X

Substrate Material X

Custom Sizing X

Center Wavelength X

FWHM X
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SWP/LWP X