Stephen Dubsky 2012 1

Stephen Dubsky
Stephen.Dubsky@monash.edu
 Resolution

Types of Microscopes
Brightfield
Darkfield
Fluorescence

Application example zebrafish heart

Stephen Dubsky 2012 Micrographia Robert Hooke (1665) 4

 Many biologically important processes and
structures exist on very small scales
Cells are typically around 1 10 um in size

melanogaster
(fly) wing
using 100x
bright field
microscopy

African Water
Mongoose Skin
Fibroblast Cells:
Fluorescence
microscopy

mysis zooplankton,
dark field microscopy 6
 Simple setup

Trans-illumination

Measures the absorption of light by the

sample

Brightfield illumination, which yields dark

objects on a bright background, is the
simplest technique for optical microscopy.
In brightfield illumination, the light source is
positioned below the sample. Light then
propagates through the sample, and is
observed by the objective lens and sensor,
which are positioned above the sample.
The darker the sample, the denser the
specimen, as denser samples absorb more
light. The simplicity of brightfield
illumination is the main reason this
technique is so popular in optical
microscopy.

Image Appearance

A typical brightfield illumination image has a

dark sample with a white background. The
darker the regions on a sample, the more
absorption of light that has occurred. For
example, plant cells would appear darkest
at the nucleus and central region where
cellular matter is most dense, and lighter in
the cytoplasm void of ribosomes, the
endoplasmic reticulum, and other
Light source (halogen
intracellular components. Animal cells are
more dicult to image with this technique
without staining of the sample, which
ultimately kills live cells.

lamp)
Condenser focuses

Figure 1: Brightfield Illumination Image of

Tissue Paper

Technical Details

There are four key components in the light

path for brightfield illumination.

1. Light Source: trans-

illumination on sample
illumination below sample that propagates
through to condenser and objective lens.
Typically a broadband source such as a
quartz halogen bulb.

Objective lens
2. Condenser Lens: collects
trans-illuminated light and focuses to
sample.

3. Objective Lens: collects

Ocular lens
light which propagates through sample and
enhances details by a factor of
magnification.

4. Eyepiece/Camera: views
or records the image.

There are some limitations to the brightfield

illumination technique, which include very
low contrast for cellular or biological
samples, low optical resolution due to
limitations of light, and the requirement for
stained samples prior to imaging or
viewing. However, simplicity of the http://www.rahulgladwin.com/
brightfield technique is a huge benefit when
first imaging an unknown sample.
Additional optical microscopy applications
include darkfield illumination, phase 8
contrast,
 Light source (halogen
lamp)
Condenser focuses
illumination on sample
Objective lens
Ocular lens

fluorescence, and dierential interference

contrast.

This is the halogen light source

http://www.rahulgladwin.com/

Requires absorbing material for contrast

Often poor contrast for biological samples,

such as cells

Used often for histology (fix, section, stain)

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Histological section of lung tissue taken with Mosquito larvae (http://sci-toys.com/)
microscope. Nuclei stain blue and cell cytoplasm
is pink. (Source: http://www.istockphoto.com/)

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Images the light scattered by a specimin

Can improve contrast over bright-field,

particularly for unstained living organisms

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Darkfield illumination is a technique in
optical microscopy that eliminates
scattered light from the sample image. This
yields an image with a dark background
around the specimen, and is essentially the
complete opposite of the brightfield
illumination technique. The primary imaging
goal of the darkfield illumination technique
is to enhance the contrast of an unstained
sample, which is incredibly powerful, yet
simple, for live cellular analysis or samples
that have not gone through the staining
process.

Image Appearance

A typical darkfield illumination image has a

white/bright specimen with a dark
background and environment filling the
image. This is the exact opposite of a
brightfield illumination image, and is useful
for unstained specimens or images that
require increased contrast. The advantage
with using darkfield illumination is that
unstained specimens can remain alive and
vital, whereas their brightfield counterparts
must be treated and are no longer active.
Also, it is possible to acquire more
qualitative results with this technique
through live cellular analysis. For additional
information on the brightfield technique,
please read Optical Microscopy
Application: Brightfield Illumination.

Figure 1: Darkfield Illumination Image of

Tissue Paper

Figure 2: Optical Path for Darkfield

Illumination

Technical Details

The light path of the darkfield illumination

technique is typically applied to an upright
microscope, as seen in Figure 2. The light
path consists of three key components.

1. Light Source: enters the

microscope and hits the dark field patch
stop, which is a disc used to block light
from entering the condenser and leaves a
circular ring of illumination.

2. Condenser Lens: collects

outer ring of illumination and focuses it on
the sample.

3. Objective Lens: light hits

the sample, and is transmitted or scattered
from it. Scattered light enters the objective
lens, whereas transmitted light is not
collected by the lens. The direct illumination
block assists in this step.

Additional optical microscopy

Stephen Dubsky 2012 12

applications include brightfield illumination,

phase contrast, fluorescence, and
dierential interference contrast.

Stephen Dubsky 2012 12

Brightfield Darkfield
13

Good for use with unstained biological

samples

As only scattered light is imaged, samples

must be very strongly illuminated

This can lead to damage of the specimen

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Micrasterias rotata (algae) undergoing cell
division (200x)
http://www.nikonsmallworld.com/ mysis zooplankton,
dark field microscopy 15

Flower stem of a spiny sow thistle 150x

Stephen Dubsky 2012 16

 Uses chromatic filters to image fluoresced
light

Allows selective imaging of fluorescently

labelled proteins

Very powerful method for biological studies

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Photon absorbed
Electron exited
Electron relaxes to
lower energy state
(energy lost)
Lower energy light
emitted

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 19

Sample

Microscope
Objective

= 560nm
= 532nm
Laser Dichroic Mirror
Beam
Lens
Expander

http://www.physics.emory.edu/faculty/ CCD
weeks//confocal/

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Fluorescence microscopy is an optical
microscopy technique that utilizes
fluorescence, which is induced using
fluorophores, as opposed to absorption,
scatter, or reflection. A fluorophore is a type
of fluorescent dye used to mark proteins,
tissues, and cells with a fluorescent label
for examination by fluorescence
microscopy. A fluorophore works by
absorbing energy of a specific wavelength
region, commonly referred to as the
excitation range, and re-emitting that
energy in another specific wavelength
region, commonly referred to as the
emission range.

Fluorescence microscope systems can

range from very simple, such as an
epifluorescent microscope, to extremely
complex, such as confocal or multiphoton
systems. Whether simple or complex,
fluorescence microscopes share the same
basic concept: excitation energy is used to Sample
illuminate a sample which then emits a type
of wavelength energy, albeit weak, that is
quantifiable. The excitation and emission
wavelengths do not share the same center
wavelength, and this allows specialized
optical filters to increase overall contrast
Microscope
and signal.

Image Appearance

Objective
Figure 1 shows a real-world fluorescence
sample. The sample is excited by a shorter
wavelength source (350 - 500nm), and then = 560nm
emits a fluorescent wavelength that is = 532nm
longer than the excitation wavelength
(500nm +). Images such as this cannot be
captured if not for the advanced optical
Laser Dichroic Mirror
filtering techniques in fluorescence
microscopy which allow narrow bandwidths
of light to propagate through to the sensor Beam
Lens
resulting in very crisp, high contrast images.
Fluorescent proteins in specimens cause Expander
the unique emission colors. These proteins,
such as GFP, are often derived from marine
life.

Figure 1: Fluorescence Image of

Microspheres

CCD
Technical Details

In general, the technical details of a

fluorescence microscope mirror any
standard brightfield illumination microscope
or darkfield illumination microscope. The
innovation comes from the intricate filtering
techniques that are used to selectively
utilize narrow bands of light. The three
critical filters needed for a precision
fluorescence microscope are the
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Sample

excitation, dichroic, and emission filters. For

more in-depth information, please read
Fluorophores and Filters in Fluorescence Microscope
Microscopy.

1. Excitation Filter: placed Objective

within the illumination path of a
fluorescence microscope. It filters out all
wavelengths of the light source except for
= 560nm
the excitation range of the fluorophore or
specimen under inspection.

= 532nm
Laser
2. Dichroic Filter: placed
between the excitation filter and emission Dichroic Mirror
filter at a 45 angle. It reflects the excitation
signal towards the fluorophore under
inspection and transmits the emission Beam
signal towards the detector.
Lens
3. Emission Filter: placed
within the imaging path of a fluorescence
Expander
microscope. It filters out the entire
excitation range of the fluorophore under
inspection and transmits the emission
range of the fluorophore.

CCD
Figure 2: Basic Optical Filtering
Arrangement for Fluorescence Microscopy

Additional optical microscopy applications

include brightfield illumination, darkfield
illumination, phase contrast, and dierential
interference contrast.

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 Nucleic acid stains (DAPI) Bind to DNA
By having a confocal pinhole, the
microscope is really ecient at rejecting out
Label the nuclei of cells
of focus fluorescent light. The practical
eect of this is that your image comes from
a thin section of your sample (you have a
small depth of field). By scanning many thin
sections through your sample, you can
build up a very clean three-dimensional
image of the sample. Some examples of
Labelled-phalloidin
this are given at this page, looking at
emulsions.

Also, a similar eect happens with points of

light in the focal plane, but not at the focal
Stains actin fibres in mammalian cells
point -- emitted light from these areas is
blocked by the pinhole screen. So a
confocal microscope has slightly better
resolution horizontally, as well as vertically.
In practice, the best horizontal resolution of
a confocal microscope is about 0.2
microns, and the best vertical resolution is
Genetic modification
about 0.5 microns. I wrote a brief
discussion here of the dierence between
resolution and magnification.
Make a cell or protein of interest fluoresce
Eg. GFP green fluorescent protein

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Photobleaching
Chemical damage to fluorescent molecules from the
excited electrons
Limits fluorescence exposure time

Fluorescence results in limited light, need large

NA, sensitive cameras etc. for dynamic imaging
Only fluorescently labelled structures are
imaged
Sometimes combined with a brightfield image to get
the full picture

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Imagine we have some lenses inside the
microscope, that focus light from the focal
point of one lens to another point. This is
represented by the blue rays of light in the
above picture. The red rays of light
represent light from another point in the
sample, which is not at the focal point of
the lens, but which nonetheless get imaged
by the lenses of the microscope. (Note that
the red and blue rays in the picture are
meant to distinguish the two sets of rays,
but they aren't meant to be dierent
wavelengths of light.) The image of the red
point is not at the same location as the
image of the blue point. (You may
remember this from introductory optics,
perhaps you have seen a formula such as
1/s + 1/s' = 1/f for locating the image
formed by a lens. Points don't need to be at
Beam splitter
the focal point of the lens in order for the
lens to form an image.)

So, we want to just look at the blue point,

that is, the point directly at the focus of the
lens. If we put a screen with a pinhole at the

Light source
other side of the lens system, at the image
of the blue point, then all of the light from
the original blue point will pass through this
pinhole. However, most of the light from the
red point is still out of focus at this screen,
and gets blocked by the pinhole.

This solves one of the problems of regular

fluorescence microscopy. Normally, the
sample is completely illuminated by the
Aperture
excitation light, so all of the sample is
fluorescing at the same time. Of course, the
highest intensity of the excitation light is at
the focal point of the lens, but nonetheless,
the other parts of the sample do get some

Focal plane
of this light and they do fluoresce. This
contributes to a background haze in the
resulting image. Adding a pinhole/screen
combination solves this problem. Because
the focal point of the objective lens of the
microscope forms an image where the
pinhole is, these two points are known as
Aperture
"conjugate points" (or alternatively, the
sample plane and the pinhole/screen are Light detector
conjugate planes). The pinhole is conjugate
to the focal point of the lens, thus it is a
confocal pinhole.

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Improves resolution (particularly in z

direction) by removing out-of-focus
structures
Scanning procedure reduces temporal
resolution, but fast confocal is now becoming
widespread for live imaging

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Endothelial cells
Cavefish
Nucleus: Blue DAPI
Neuron cells express GFP
Microtubules: Green (FITC)
Actin: Red (labelled phalloidin)
A fibroblast: is a type of cell that synthesizes the extracellular matrix and 25
collagen, the structural framework (stroma) for animal tissues, and plays a

To look at small things, we need high

magnification

This has consequences for our imaging

system

Our application determines the required

resolution of the system

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 It is important to understand the difference
between resolution, magnification and pixel size

Resolution is defined by smallest object feature

that can distinguished

It is the minimum distance between

distinguishable objects in an image

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A fibroblast: is a type of cell that synthesizes the extracellular matrix and

collagen,[1] the structural framework (stroma) for animal tissues, and plays a
critical role in wound healing.
Actin (green): muscle contraction, cell motility, cell division and cytokinesis,
vesicle and organelle movement, cell signaling, and the establishment and
maintenance of cell junctions and cell shape.

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http://www.photonics.com/
 What contributes to the resolution of the
system?

Sensor Resolution
Magnification and native pixel size
Optical resolution
Numerical aperture, diffraction limit

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The effective pixel size sets a lower limit on

the smallest structure that can be resolved

The smallest resolvable feature scale is equal

to 2 times the effective pixel size (2 x native
pixel size / M)

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 Minimum sampling rate

required to avoid aliasing,

equal to 2x the highest

frequency

contained within the signal

Harry Nyquist (1889-1976)

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Two different sinusoids that fit the same set of samples. The sampling rate is
not sufficient to resolve the red sinusoid and avoid aliasing

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 The digital image is a signal of discrete
measurements that are regularly spaced

A signal (object feature) must be larger than 2

pixels in order to resolve it with the sensor

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The smallest resolvable feature in terms of

sensor resolution is equal to 2 times the
effective pixel size (2 x native pixel size / M)

36 px disc, 12 px hole 9 px disc, 3 px hole 3 px disc, 1.5 px hole

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 Sensor resolution need high magnification
Need short object distance
M = f ( f - S1 )-1
Image plane

Object plane No image

Object plane
formed!

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Sensor resolution need high magnification

Need short object distance
M = f ( f - S1 )-1

Practical limit on
magnification

No image
Object plane
formed!

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 In theory, the resolution will be dependent on
the diffraction limit of the imaging system

This diffraction limit is dependent on the

numerical aperture (NA)

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Diffraction is the bending of light around the

edge of an object

Similar but distinct to refraction

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 Each point on the light wavefront behaves like a point source, and the
superposition of the resulting waves becomes the new wavefront
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Lenses can be modeled as a pinhole

Large aperture = less diffraction
Short aperture = more diffraction

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 phase shift = 0 phase shift = pi (180 degrees)

Combined

Wave 1

Wave 2

Constructive intereference Destructive intereference

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The point spread function (PSF) describes the

response of an imaging system to a point
source or point object

Airy pattern
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 We want a high NA for high resolution imaging

If airy disc is large, objects are more Increasing NA

difficult to distinguish

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Diameter of the airy disc:

Light Wavelength
Airy disc diameter
d =
Lens
2 ( n sin )
Refractive index Light gathering angle

Light
gathered d =
2 NA
Sample
Numerical Aperture

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 d =
2 ( n sin )
d =
2 NA
In practice maximum optical resolution
achievable is ~/2 (~250nm)
We can improve our resolution
Increasing NA (oil immersion lens) Electron micrograph of antenna
Decreasing wavelength surface detail of a wasp
electrons: wavelengths ~100,000 times
shorter
X-rays: ~1000 times shorter
More complex light methods (super-
resolution)
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1951 USAF Resolution test 48

 Stephen Dubsky 2013 49

Modulation transfer function(MTF)

How does the system respond to a modulated
signal (line pairs)
Modulation = (Imax Imin) / (Imax + Imin)
MTF(f) = Mod. of image / mod. of object
MTF(f) = Mi / M0

Relates to point spread function

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 51

Take an image of a resolution target

Target contains modulations (line-pairs) at various scales
Measure peak to trough intensity of line-pairs
Plot modulation transfer vs. spatial frequency

Characteristic resolution

Arbritrary threshold value 0.2

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A 21day old zebrafish. Their optical clarity and relatively easy maintenance make them a
favorite for geneticists and developmental biologists.

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Excellent model for embryonic heart

development

Transparency allows easy microscopic

investigation

Easy genetic manipulation allows for

investigation of congenital heart disease

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Courtesy of Dr. Robert Bryson Richardson
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Courtesy: Aidan Jamison 57

Courtesy: Aidan Jamison 58

Stephen Dubsky 2012 59