Module 3

Light Optical Microscopy

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Introduction to seeing
Growth of Microscopes & Microscopy
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland manufactured the
first compound microscopes
• 1590 – 1609 - Galileo – one of the earliest microscopists (coined the term
“microscope”
• 1665 - Robert Hooke (1635-1703)- book Micrographia, devised the compound
microscope.
• Ernst Abbe together with Carl Zeiss published a paper in 1877 defining the
physical laws that determined resolving distance of an objective
• Early 20th Century Professor Köhler developed the method of illumination
still called “Köhler Illumination
• 1960: Invention of Laser
• 1990s- Super resolution microscopy : STED and iSCAT to go beyond
diffraction limit of light
 Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of a particular
wavelength of white light produces colored light (Color of objects esp.
Dyes)
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another medium with different optical density. (Class 10 experiment)
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition (Just seen in
previous module)
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow (Used in
Monochromators)
Properties of Light
 Reflection and Refraction
• Snell’s Law: The angle of
Transmitted reflection (Ør) is equal to the
Reflected Beam (refracted)Beam angle of incidence (Øi)
regardless of the surface
material
r t
• The angle of the transmitted
i beam (Øt) is dependent upon
the composition of the
Incident Beam material

n1 sin Øi = n2 sin Øt
The velocity of light in a material
of refractive index n is c/n
He sees the
fish here….

But it is really here!!

 Properties of thin Lenses
f f

p q
1 1 1
+ =
p q f

Resolution (R) = 0.61 x

l q
Magnification =
(lateral)
NA p
(Rayleigh criterion)
 Refraction & Dispersion

Short wavelengths are “bent”

more than long wavelengths

Light is bent and the resultant colors separate (dispersion).

Red is least refracted, violet most refracted.
 Filter
Control

Absorption

No blue/green light
red filter
 Absorption Chart
Color in white light Color of light absorbed
red blue green
blue red green
green red blue
yellow blue
magenta green
cyan red
black red green blue

gray pink green blue

 Color wheel Chart
Color in white light Color of light absorbed
red blue green
blue red green
green red blue
yellow blue
magenta green
cyan red
black red green blue

gray pink green blue

 The light spectrum
Wavelength ---- Frequency
Blue light
Photon as a
488 nm wave packet
short wavelength of energy
high frequency
high energy (2
times the red)
Red light
650 nm
long wavelength
low frequency
low energy
Properties of Lenses
 Magnification
• Definition
• Types of lenses: Simple lens and Compound Lens
• Formula of Magnification: m=h/h’ =-v/u
• An object can be focussed generally no closer than 250
mm from the eye (depending upon how old you are!)
• this is considered to be the normal viewing distance for 1x
magnification
• Young people may be able to focus as close as 125 mm so
they can magnify as much as 2x because the image covers
a larger part of the retina - that is it is “magnified” at the
place where the image is formed
 Magnification
1000mm

The projected image is 28 times larger than we

would see it at 250 mm from our eyes.
35 mm slide
If we used a 10x magnifier we would have a
magnification of 280x, but we would reduce 1000 mm
the field of view by a factor of 10x. M = 35 mm = 28

The field of view (FoV) is the extent of the observable world that is seen at
any given moment.
 Numerical Aperture (NA)
NA=1 - theoretical NA = n(sin )
maximum numerical
aperture of a lens n: refractive index of the
operating with air as imaging medium between
the imaging medium the front lens of objective
and specimen cover glass
Objective lens
Angular aperture
(72 degrees)
 One half of A-A

Specimen
cover glass

NA of an objective is a measure of its ability to gather

light and resolve fine specimen detail at a fixed object
distance.

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Numerical Aperture (NA)

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 Numerical Aperture
NA = n(sin )

Imaging Medium

Air
n=1.0

Immersion oil
n=1.515

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oil immersion objective use in microscope at~0:33
George Biddell Airy and
‘Airy Discs’ (1835)

Ernst Abbe and ‘Abbe’s The diffraction limits of the microscope!

Resolution (d=λ/2 NA), the specimen must be viewed using either
Diffraction Limit’ (1873) shorter wavelength (λ) light or through an imaging medium with a relatively
high refractive index or with optical components which have a high NA (or, indeed, a
combination of all of these factors) ???

John William Strutt and

‘The Rayleigh Criterion’ (1896)

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 Abbe, Zeiss & Schott
Ernst Abbe together with Carl Zeiss published a paper in 1877
defining the physical laws that determined resolving distance of
an objective. Known as Abbe’s Law
“minimum resolving distance (d) is related to the
wavelength of light (lambda) divided by the Numeric
Aperture, which is proportional to the angle of the
light cone (theta) formed by a point on the object, to
the objective”.

Abbe and Zeiss developed oil immersion systems by making oils that matched the refractive index of
glass. Thus they were able to make the a Numeric Aperture (N.A.) to the maximum of 1.4 allowing
light microscopes to resolve two points distanced only 0.2 microns apart (the theoretical maximum
resolution of visible light microscopes).
Dr Otto Schott formulated glass lenses that color-corrected objectives and produced the first
“apochromatic” objectives in 1886.
 Point Spread Function
Even a “point” forms a finite spot on detector.
No matter how small an emitting light, it always forms a finite-sized spot,
PSF ~ l/2NA
You can “never” get better than l/2NA ~ 500 nm/2* 1.4 ~ 175 nm

Resolution x,y = l/2*NA

Resolution z = 2l/NA2
 Resolution: The Abbe/ Rayleigh criteria
How well can you resolve two nearby (point) objects?

Light
always
spreads
out to ~
l /2NA
The resolution is limited to how well you can separate two overlapping PSFs.
Rayleigh Criteria ~ ~ l/2NA ~ 200-250 nm
Can resolve things to l/2N.A.

Resolution x,y = l/2*NA

Abbe’s Diffraction limit
Resolution z = 2l/NA2

The Rayleigh Criterion: Resolution = 1.22 λ/NAobj+NAcond

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[Link] at~5:35-6:00
 Microscopy as a compromise
• Magnification
• Resolution
• Brightness
• Contrast

Image brightness is governed by the Contrast is defined as the difference in light

light-gathering power of the objective, intensity between the image and the adjacent
which is a function of numerical background relative to the overall background
aperture intensity.

Image Brightness ∝ (NA/M)2 Percent Contrast (C) = ((I(s) - I(b)) x 100)/I(b)

where NA is the objective numerical aperture Where I(b) is the intensity of the background and I(s) is the
and M is the magnification specimen intensity.
 Köhler illumination (invented 1893):
Makes illumination uniform
(used with sources like light bulbs; irrelevant for lasers)

B. Köhler
(old technique) Illumintion:
A. Critical Conjugate planes are
Illumination. the illuminating bulb
Conjugate planes filament and
are the illuminating Condenser
bulb filament and diaphragm. Second
sample plane (O). conjugate planes are
When adjusted the Field diaphragm
correctly, the image and the sample plane.
of the filament is When adjusted
seen coincident with correctly, the image of
the sample image. A the field diaphragm
diffusing glass filter The trick is to make sure that you are not and the sample are
(d) is used to blur imaging the light source. The filament is out of coincident. The
the filament image. the plane of focus, and thus uniformly diffuse. filament is out of the
FD: Field diaphragm: CD: Condenser diaphragm plane of focus, and
[Link] thus uniformly diffuse.
 Köhler Rays

Kohler Illumination gives

the most uniform
illumination
Each part of the light
source diverges to whole
specimen
Each part of the specimen
gets light that converges
from the whole light
source

Arrows mark
conjugate planes
 Köhler Illumination
condenser
eyepiece
Field iris Specimen Field stop
retina

Conjugate planes for image-forming rays

Specimen Field stop

Field iris

Conjugate planes for illuminating rays

 Types of Microscopes
• Stereo Microscope.
Upright or
• Compound Microscope. Inverted
• Inverted Microscope.
✓ Köhler Illumination
• Metallurgical Microscope. ✓ Fluorescence
• Polarizing Microscope. Illumination

Limited by diffraction of visible light and lens aberrations

 Illumination Techniques

Transmitted Light Reflected (Incident) Light

• Bright-field • Bright-field
• Oblique • Oblique
• Darkfield • Darkfield
• Phase Contrast • Polarized Light
• Polarized Light • DIC (Differential Interference
• DIC (Differential Interference Contrast)
Contrast) • Fluorescence (Epi)
How can we use the properties of light to create
contrast?

Absorption
Scattering
Refraction
Phase
Polarization
 Contrasting techniques

• Brightfield
• Darkfield
• Phase Contrast
• Polarization Contrast
• Differential Interference Contrast (DIC)
 Brightfield
Principle: Light is transmitted through the sample and absorbed by it.

Application: Only useful for specimens that can be contrasted via dyes. Very little contrast in
unstained specimens. With a bright background, the human eye requires local intensity fluctuations
of at least 10 to 20% to be able to recognize objects.

Cross section of sunflower root Piece of artificially grown skin

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([Link]/.../dt/PI_BioTechnica2001.[Link] )

→ all our microscopes can be used for brightfield

Sample Preparation

DO IT YOURSELF
 Maximizes detectability
Darkfield Cost in resolution
Principle: The illuminating rays of light are directed through the sample from the side by putting a dark
disk into the condenser that hinders the main light beam to enter the objective. Only light that is scattered
by structures in the sample enters the objective.

Application: People use it a lot to look at Diatoms and other unstained/colourless specimens

Darkfield

Symbiotic Diatom colony

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→ we do not have microscopes set up for darkfield

Dark field illumination is the elimination of the 0 order
Diffraction - Change of Wavelength
(Undeviated light that is not diffracted)

Short wavelength Long wavelength

10x 40x 63x

-1 0 +1
-2 +2 +3
+4

+5

Blue “light”
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 Specimens for DF microscopy
• Containing refractive objects scattered
about with empty space between them.
• No dark field occurs if objects are too
crowded or if a thick solid specimen turns
light into the microscope.
• Very thin histological sections can be used
if unstained or if only certain components
are stained, as in silver stains.
• Biological fluids from animals and plants,
cell cultures, microbes, foods, fibers,
crystals, colloids, and sub-microscopic
particles are all suitable for dark field
microscopy.
 Rheinberg Illumination

• Special variant of Dark

field illumination

• The Good: Striking

contrast
• The Bad: “dark field” like
resolution

• (good for seeing things,

not as good for measuring)
DO IT YOURSELF
 Rheinberg Illumination

• Which filter was

used to take the
picture of the tick?

DO IT YOURSELF
What if you can’t see something by bright-field?
Light has a phase, (plus an amplitude)
You may be able to see a phase change.
 Enhancing Contrast in Optical Microscopy
Investigations dealing with inherently low-contrast specimens, such as unstained
bacteria, thin tissue slices, and adherent live cells, rely on specialized contrast-
enhancing techniques to assist with imaging these virtually transparent samples.
Use dyes
(w color contrast) Two Phase techniques

Polarized light
requires
birefringence
(usually not present
to a significant
degree in animal
cells) to generate
contrast. Muscle cells
are birefringent.
 Fluorescence Microscopy and
Confocal Microscopy

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James Pawley, Ed. “Handbook of Biological Confocal Microscopy, 3rd ed.”
Murray JM et al., J. Microsc. 2007, 228: p.390-405
Slides : [Link]
Handbook of Biological Confocal Microscopy. Ed. J. Pawley, Plenum Press
Fundamentals of Light Microscopy and Electronic Imaging. D. B. Murphy, Wiley-Liss Inc.
[Link]/science-lab/introduction-to-widefield-microscopy
 Fluorescence Microscopy (Wide field)
An optical microscope that uses fluorescence instead
of, or in addition to, scattering, reflection, and
absorption, to study the properties of organic or
inorganic substances

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applications-advantages-limitations/
What is Fluorescence ?
 Optical Sectioning and 3D reconstruction

x y
 Optical Sectioning and 3D reconstruction

x y
 Optical Sectioning and 3D reconstruction

x y
 Fluorescence Illumination of a single point
Camera Fluorescence is emitted along
entire illuminated cone

Tube lens
Excitation light

Emission light

Objective lens

Sample

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 Widefield Point
Illumination Illumination

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[Link]/services/confocal
-and-fluorescence-
microscopy

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[Link]/science-
lab/introduction-to-widefield-
microscopy/
The Pinhole
Effect

Conjugate plane

y
z
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x y x
The Pinhole
Effect

Pinhole

52
 Fluorescence Microscopy
(Wide field vs Confocal)

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 Light sources

• Excitation light must be focused to a

diffraction limited spot

• Could be done with an arc lamp Excitation light

and pinhole – but very inefficient

• Enter the laser:

• Perfectly collimated and Objective lens
high power
Sample
 Being confocal…
Scan excitation spot point-by-
Detector point to build up image

Pinhole

Tube lens
Excitation light

Emission light

Objective lens

Sample
Should I Scan?

Changing entrance angle

of illumination moves
illumination spot on sample

Objective lens
The emission spot moves,
Sample so we have to make sure
pinhole is coincident with it
 Confocal optical path
Scanning mirrors Dichroic Pinhole

x
y Detector

Relay lens Laser

Objective
 Sampling
Scanning involves digitisation in x, y, z, intensity, and t

Resolution is affected by sampling during the digitisation

process

0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 22 45 66 11 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 65 12 0 0 0 0
0 0 0 0 0 0 0 0 99 0 0 0 0

pixels 0 0 0 0 0 0 0 7 0 0 0 0 0
0 0 6 5 0 0 0 2 8 21 5 2 0
0 0 0 3 0 0 0 0 0 0 0 0 0

(voxels) 0 0 0 0 0 0 0 0 0 0 0 0 0

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 Pixel under/sampling
Specimen

Large pixels

Small pixels, lucky alignment

Small pixels, unlucky alignment

Very small pixels

512x512 —250 kbyte (1 channel)

1024x1024 —3 Mbyte (3 channel)
2048x2048
More the pixels:
smoother looking image - more xy information
more light exposure of specimen
larger file size
slower imaging (less temporal resolution) 59
 4 principal factors for image quality

• Spatial resolution
Ultimately set by the optics, but can be limited by digitisation (therefore affected by
image size and zoom). Affected by pinhole: super-resolution (1.4x) is possible at small
pinholes
• Intensity resolution
Ultimately set by detector, but limited by digitisation and low photon sampling. Aim to
fill whole dynamic range with image information.
• Signal-to-noise ratio
Degree of visibility of image over background noise, given variability in system.
• Temporal resolution
Depends on raster scan rate (+averaging). 512x512 at 2/s.

Imaging depends on compromising between these factors,

e.g. you might want to optimise resolution of light intensity at expense of spatial or
temporal resolution.
60
 A Possible Solution: Spinning Disk Confocal

• Image with many pinholes at once, so fast

• Use CCD as detector, so much higher QE
 Pros/Cons of spinning disk
• Fast – multiple points are illuminated at once
• Photon efficient – high QE of CCD
• Gentler on live samples – usually lower laser
power

• Fixed pinhole – except in swept-field

• Small field of view (usually)
• Crosstalk through adjacent pinholes limits sample
thickness
 Which imaging technique should I use?
Total Internal Reflection Fluorescence (TIRF) Microscopy DO IT YOURSELF
1-5 mm TIRF (for samples at the coverslip)

Wide-field (+deconvolution)

Fast
1-20 mm
Sample Thickness

Spinning Disk Confocal

Sensitivity
10-100 mm Line-scanning confocal

>20 mm Point scanning Confocal

Slow
>50-100 mm 2-photon confocal

DO IT YOURSELF
Is Confocal Microscopy Better?
1. More Color Possibilities
Because the images are detected
by a computer rather than by eye,
it is possible to detect more color
differences.
 Insulin-Cy5 CRLR-FITC

Glucagon-Rhodamine Overlay
Is Confocal Microscopy Better?
1. More Color Possibilities
2. Less Cross Talk
Is Confocal Microscopy Better?
2. Less Cross Talk
In most applications, fluorochromes have overlapping emission spectra. Hence, the
emission signals cannot be separated completely into different detection channels
resulting in “bleed through” or cross talk.

However, if the fluorochromes have distinct excitation spectra, the fluorochromes

can be excited sequentially using one excitation wavelength at a time. This is only
possible with confocal systems that offer the multitracking feature.
Is Confocal Microscopy Better?
1. More Color Possibilities
2. Less Cross Talk
3. Optical Sectioning of
Objects Without Physical
Contact
Is Confocal Microscopy Better?
3. Optical Sectioning of Objects Without Physical Contact

Zebra fish embryo wholemount

Neurons (green)
Cell adhesion molecule (red)

Monika Marks, Martin Bastmeyer

University of Konstanz
Is Confocal Microscopy Better?
1. More Color Possibilities
2. Less Cross Talk
3. Optical Sectioning of
Objects Without Physical
Contact
4. Three-Dimensional
Reconstruction of
Specimen
Is Confocal Microscopy Better?
4. Three-Dimensional Reconstruction of Specimen

3D shadow projection
Tight junctions (red)
Cytoskeletal structures (green)

Prof. Wunderli-Allenpach
ETH, Zurich
Is Confocal Microscopy Better?
1. More Color Possibilities
2. Less Cross Talk
3. Optical Sectioning of
Objects Without Physical
Contact
4. Three-Dimensional
Reconstruction of
Specimen
5. Improved Resolution
Is Confocal Microscopy Better?
5. Improved Resolution

Rat Cerebellum
Astrocytes (green)
Mn dismutase (red)

Jorg Lindeman
University of Magdeburg
Confocal Microscopy
• Confocal can be
used with any
instrument / imaging
mode
• BF, DF, PC, DIC,
etc.
• Raman Spectroscopy
End