Biomedical Optics

Microscopy: Bright field, Dark Field, Polarised,

Phase, DIC, Fluorescence
BY: KAMUHANDA SUCCESS
Microscopes

Microscope: Micro = Gk. “small” + skopien = Gk. “to look at”

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Microscopes
"Microscope" was
first coined by
members of the first
"Academia dei
Lincei" a scientific
society which
included Galileo

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History: The First Description of
Microorganisms

• Robert Hooke
• Observed fruiting structures of molds
in 1665 and was the first to describe
microorganisms
• Compound microscopes were mostly
of poor quality and could only magnify
up to 20-30 times.
• Chromatic aberrations
• Chester More Hall, a barrister, 1730s
• Observed that flint glass (newly made
glass) dispersed colors much more
than “crown glass” (older glass)
• Designed a system that used a
concave lens next to a convex lens
which could realign all the colors
→ the first achromatic lens.
• Hooke claimed they were too difficult
to use - his eyesight was poor.
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History: The First Description of
Microorganisms
• Antoni van Leeuwenhoek
• He is incorrectly called "the
inventor of the microscope"
• Created a “simple”
microscope with one ground
lens that could magnify to
about 275x
• Published drawings of
microorganisms in 1676
• The field of microbiology was
unable to develop until
Leeuwenhoek constructed
microscopes that allowed
scientists to see organisms
too small to be seen with the
naked eye.

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Simplest Microscope
• Magnifying Glass
• Single lens magnifier makes
the image appear larger.
• Our brain processes the light
as though coming in a straight
line so the image appears
larger

Example:
250mm
M=
fLens

5x

f=50m

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Compound Microscope
• The compound microscope uses
at least two lens systems
• The condenser
• Provide illumination
• Increase the resolution
• The objective
• Forms an intermediate real image
of the sample at the objective tube
length
• Modern Objective lens
• Multi-element lens
• The number of lenses in a modern
microscope can easily exceed 20.
• The eyepiece
• Forms a virtual image of that
intermediate image to the retina of
the eye
• For a photodetector, use a
projection lens to form a real
image from the intermediate image

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Compound Microscope
• Current microscope
objectives tend to be
infinity corrected
• Parallel rays out of the
objective
• Infinite tube length
• Require an additional
lens in the tube to form
the intermediate image
• Advantages
• Objectives are simpler
• Optical path is parallel
through the
microscope body
• Elements inserted in
the path do not affect
the image Benefits of infinity correction.
(A) Insertion of reflector or filter causes lateral and axial shift.
(B) Two telan lenses generate infinity space to eliminate the shift
(C) Objective directly provides infinity space

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Compound Microscope
Optical image formation: Basic concepts
si
xo

so

1 1 1
= + F=focal length
So=distance of object from principal plane of lens
f s o si Si=distance of image from principal plane of lens

si f
M =− =− M=magnification
so x0 X0=distance of object from focal back focal plane of lens
- sign because of inverted image
f yi f
= =
s0 − f yo si − f
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Compound Microscope
Eyepiece

Tube lens

Objective (Zeiss: f=164.5mm)

250mm fTube 250mm

M =  
fObjective 250mm fEyepiece

fTube 250mm
M = 
f Objective fEyepiece

MCompound Microscope = MObjective  MEyepiec

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Compound Microscope

http://academic.pgcc.edu/~kroberts/Lecture/Chapter%204/04-04_CompoundLM_L.jpg 12
Compound Microscope

Upright microscope Inverted microscope Stereo microscope

.

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Compound Microscope

Split-image comparison of firing pin

Comparison (“CSI”) microscope imprints in coaxial incident light

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Optical Resolution
• Numerical aperture (NA) of
a lens
• A measure of its ability to
gather light and resolve
fine specimen detail at a
fixed object distance
• NA=n*sina
• n=refractive index of
medium
• a=half angle of light
collection cone
• For fixed diameter ⟶ as
magnification increases,
the working distance, i.e.
the distance from the edge
of the objective to the
sample, decreases

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 Optical Resolution
• Resolution of an optical
microscope
• The shortest distance
between two points on a
specimen that can still be
distinguished by the observer
or the camera as separate
entities
• Resolution of an ideal optical
system
• Theoretically d = λ/2
• Limited by the process of
diffraction → formation of an
airy disk pattern when a beam
of light is focused onto a spot

http://www.olympusfluoview.com/java/resolution3d/index.html
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http://www.microscopyu.com/tutorials/java/imageformation/airyna/index.html
Optical Resolution
• Point Spread Function
• Diffracted rays interfere Point source
• Either in a constructive or In a
destructive way →
interference rings.
• The mathematical Objective lens
representation of this
phenomenon is called the Diffracted rays
Converging rays
Point Spread Function (PSF)
• The diffraction pattern of a Image formation by
point source of light. an objective lens
• Intensity profile of a
diffraction spot
• Central spot and surrounding
rings
• The separation distance
between The center of the
spot and the first minimum
depends on the angular
aperture of the lens.

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Optical Resolution
• Airy disk in 2D and 3D
• Axial resolution is inversely proportional to the squared NA

Axial resolution
n
d ax = 4
Lateral resolution

d lat = 1.22
 ( )
NA 2

NA
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Optical Resolution
• Resolution  =4
n
dlat = 1.22 d
(NA + NAcond )
ax
NAobj + NAcond
2
• The shorter the wavelength and the obj
higher the NA the better the
resolution 10 x, 0.3 NA objective at 530 nm light ,
• For standard light microscopy, 10x Eyepiece
diffraction limited resolution is on
the order of 200 nm M = M obj Meyep = 10x10 =100
• How to improve? 530nm
d lat = 1.22 = 1.08m
• Larger NA (lenses, immersion fluid) 0.3 + 0.3
• Shorter λ 530nm
d ax = 4 = 5.89m
• Add a condensor (0.3 + 0.3)2

• D = 1.22 λ / (NAobj. + NAcond.) 60 x, 1.3 N.A. objective at 530 nm light,

• So, for a 1.3 NA lens and 10x Eyepiece
condensor, D drops to ~250 nm M = M obj M eyep = 60x10 = 600
• Examples 530nm
d lat = 1.22 = 248.7nm
• 10 x, 0.3 NA 1.3 +1.3
• 60 x, 1.3 N.A. 530nm
d ax = 4 = 313.6nm
(1.3 +1.3)2

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Objective Specifications

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Objective Specifications
• Oil immersion
• Required for large NA

Images reproduced from: http://micro.magnet.fsu.edu/

Please go to this site and do the tutorials 21
Objective Specifications
• Aberrations
• Spherical aberration Achromats Most common
• Most severe Lowest price
• Immersion fluid Poorly corrected, bad
for demanding
• Field curvature applications.
• Chromatic aberration Fluorites or Semi Plan Mid-grade lenses, better
Apochromats correction, flat field.
• Astigmatism, coma
• http://micro.magnet.fsu.ed Plan Apochromats Best grade, most
expensive (>$3,000 for
u/primer/lightandcolor/opti some) , very well
calaberrations.html corrected.

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Major Light Paths
• Illumination
• Major goal: provide
uniform sample
illumination
• Imaging
• Major goal: reproduce
magnified sample image
with minimal distortion,
high light collection
efficiency, and high
resolution

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Major Light Paths
• Köhler illumination
• Creates an evenly
illuminated field of view
while illuminating the
specimen with a very
wide cone of light
• Two conjugate image
planes are formed
• One contains an imag e
of the specimen
• The other the filament
from the light

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Major Light Paths
• Transillumination

• Condenser aperture: will

affect the numerical aperture
of the condenser
• Field diaphragm: will affect
size of field that is illuminated
at the focal plane

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Major Light Paths
• Epi-illumination

• Aperture diaphragm:
will affect the numerical
aperture of the objective
for illumination
• Field diaphragm: will
affect size of field that is
illuminated at the focal
plane

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0 Units 50 Units 100 Units

50 50 50

C ONTRAST
Brightness of Specimen-Brightness of Background

0.50
50 – 100 / 100 = -
C=
max(Brightness of Specimen,Brightness of Background)

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2
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Contrast
• Ability to tell the difference
between objects and
background
• Specimen properties that produce
• changes in brightness
• color differences
• Arise from
• Light absorption, reflection,
scattering, diffraction
• Spatial variation in refractive index
• Birefringence
• Fluorescence and similar optical
phenomena.
• Can be improved using stains
• The sample has to be sacrificed,
fixed, sectioned, and stained
• Variety of stains →stain different
cells/cellular components with
different colors
• Immunohistochemistry → antibody
based staining

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Contrast
• Contrast can be enhanced
by different
illumination/imaging
techniques
• Brightfield
• Darkfield
• Phase Contrast
• Polarized Light
• DIC (Differential
Interference Contrast)
• Fluorescence (and related
techniques)

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Brightfield Microscopy
• Simplest type of microscopy
• Light floods the objective, making
the field bright
• Objects absorb or deflect light out
of the field, making the objects
dark against the bright
background.
• Contrast provided mainly by
absorption
• Biological specimens are not
highly absorbing naturally
• Use stains, which typically require
fixation, i.e. cells no longer alive

• Used routinely in Blood cells Tissue histology

histopathology and hematology
and basic science studies for
which looking at live specimen
is not crucial

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Darkfield Microscopy
• In Darkfield Illumination
• Light from outside the Field,
does not normally enter the
objective, making the field
dark.
• Light striking objects is
displaced into the objective.
• Objects appear bright against
dark background  easier
to see
• Useful for examining
• Live organisms
• Microorganisms which cannot
be stained by standard
methods
• Treponema pallidum, the
causative agent of syphilis

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Polarized Light Microscopy
• Specimen is placed between
2 crossed polarizers.
• When Polarizers are crossed,
only items that rotate the
plane of polarization reach the Polarizer 2 (Analyzer)
detector.
• Only light produced by
birefringent particles (e.g.
crystals) or coming from the
edges of particles (“edge
birefringence”) is visible.
• Looks sometimes like
Darkfield
• Wave plate adds color
Polarizer 1
• Orientation-specific (linear
Polarization)

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 Polarized Light Microscopy

Background

Brightfield
Birefringent
Material

Polarized Light

Photomicrografy under polarized microscopy.

Parallel collagen fibers between the implant
Pol+ Red I

Color of sample surface (white arrows). Oblique fibers in

and background direction to bone crest (yellow arrows). Bar -
modified by 500µm
wave plate

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Phase Contrast Microscopy
• First microscopic method
which allowed visualization
of live cells in action
• Nobel prize in physics was
awarded to Frits Zernike in
1953 for its discovery
• It enhances contrast in
transparent and colorless
objects by influencing the
optical path of light
• It uses the fact that light
passing through the specimen
travels slower than the
undisturbed light beam, i.e. its
phase is shifted

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Phase Contrast Microscopy
• Illumination from Condenser Phase (3)
Ring (“0” Order)  meets phase
ring of objective (1)
• Objective Phase Ring (2) Phase Ring
(2)
• a) attenuates the non-diffracted 0th
Order (red)
• b) shifts it ¼ wave forward
• Affected rays from specimen (blue)
• Expressed by the higher diffraction
orders
• Do not pass through phase ring of
objective >¼ wave retarded
• Non-diffracted and diffracted light Annular Ring
are focused via tube lens into
intermediate image (3)
• Interfere with each other (1)
• ¼+¼= ½ wave shift
• Causes destructive interference i.e.
specimen detail appears dark

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Phase Contrast Microscopy
• S (red) be light passing through
medium surrounding sample
• D (blue) light interacting with
specimen.
• S and D typically interfere to yield
P (green) Phase plate
• What we can usually detect.
• P will be phase shifted compared
to S
• Our eyes cannot detect phase
shifts.
• Phase contrast microscopy
effectively converts this phase
shift into an intensity difference
we can detect

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Differential Interference Contrast
(DIC)
• Changes phase GRADIENTS
across different parts of a
specimen into brightness
differences
• 3-D Image appearance
• Color DIC by adding a wave
plate
• Orientation-specific > orient fine
detail perpendicular to DIC
prism
• Live, unstained speciments
• High Contrast and high
resolution
• Doesn’t suffer from some
artifacts seen in phase contrast
• Uses full NA of objective

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3
7

Differential Interference Contrast (DIC)

• Nomarski-modified
Wollaston prism
• Polarized beam, under 45˚ to
prism, gets split into“ ordinary” Polarizer 2 (Analyzer)
and “extraordinary” beam Wollaston Prism

Wollaston Prism

Polarizer 1
Differential Interference Contrast
(DIC)
• Light Path
1. Unpolarised light → polarised at 45
2. First prism ⟶ separated into two
rays polarised at 90° to each other
3. Condenser ⟶ focuse with a separation of
Polarizer 2 (Analyzer)
around 0.2 μm apart (similar to the
resolution of the microscope) Wollaston Prism (5)
4. Through adjacent areas of the sample 
different optical path lengths where the
areas differ in refractive index or thickness
 change in relative phase
• Many pairs of rays ⟶ an image of the sample
carried by both the 0° and 90° polarised light (4)
• Like bright field images of the sample, slightly
offset from each other (3)
5. Second prism ⟶ rays recombined into o n
e
polarised at 135° ⟶ interference Wollaston Prism (2)
⟶brightening or darkening the image at
that point according to the optical path Polarizer 1 (1)
difference.
• Can adjust so that 0 phase difference cancels
• Wave plate adds color
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Differential Interference Contrast
(DIC)

Polarizer 2 (Analyzer)

Wollaston Prism

Wollaston Prism

Polarizer 1

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Differential Interference Contrast
(DIC)

The 3T3 cell line is an important fibroblast culture, widely utilized in laboratory research,
which was established from disaggregated tissue of an albino Swiss mouse. The fact
that 3T3 cells could apparently grow indefinitely, while being unable to instigate tumor
growth, helped scientists delineate for the first time the differences between cell
mortality and a cell's ability to undergo oncogenic transformation.

http://www.microscopyu.com/moviegallery/livecellimaging/3t3/index.html

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4
1

Phase-Contrast and DIC Comparison

• Phase-Contrast DIC
• Uses wave nature of light • Uses differences in refractive
• One set of light rays are indices
direct and one set are • Uses 2 beams of light
reflected
• Resolution higher
• Makes detailed images of
internal structure of living • Brightly colored image
microorganisms possible • Image appears nearly three-
• Image in greyscale dimensional
Fluorescence Microscopy
• Advantages of fluorescence
• Highly sensitive method
• Simple implementation
• Highly sophisticated fluorescent probes
(fluorophores)
• Fluorophores
• Fluorescent dyes that accumulate in different cellular
compartments or are sensitive to pH, ion gradients
• Fluorescently tagged antibodies to specific cell
features
• Endogenously expressed fluorescent proteins
• Really endogenous
• NADH/FAD: enzymes involved in ATP production
• structural proteins: collagen/elastin
• amino-acids: tryptophan/tyrosine
• After gene modification
• Green fluorescent protein and variants

• Methods
• All fluorescence methods can be done (FLIM, FRAP,
FRET, TIRF, etc)

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Fluorescence Microscopy
Eye
• Light Path
• Light source through Ocular
Lens
condenser passes through an
excitation filter ⟶ narrow
Excitation
excitation band Filter
Emission
• Dichroic mirror directs Light Filter
excitation to the sample
• Reflection and fluorescence Dichroic
from the sample Condenser
Mirror

• Dichroic mirror only passes Lens Objective

fluorescence Lens
• Emission Filter ⟶ emission
band
Specimen

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 Fluorescence Microscopy

Dichroic filter: reflects excitation

and transmits fluorescence

http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html 45
Fluorescence Microscopy
• Common non-laser light
sources
• Arc lamps (Mercury and
Xenon)
• Aligning the light source
• The epi fluorescence
microscope is a reflected light
microscope
• The arc of the lamp imaged at
the back focal plane of the
objective
• Ideally just filling the back
aperature (Koehler
illumination)

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Fluorescence Microscopy
• Dichroic Mirror
• Reflects Excitation Band
• Passes Emission Band
• Sharp cut-off required
• Filter selection
• Broadband filters ⟶ more
excitation, less contrast [more
autofluorescence may be
excited]
• Narrowband filters ⟶
less signal, more contrast
• Note: eye responds to contrast
while detectors respond to
signal

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Fluorescence Microscopy
• Multi-band Imaging
• Image (simultaneously or
sequentially) the same sample at
different excitation emission
wavelengths
• Look at different cell components
• Example
• Cell nucleus stained with blue
Hoechst dye
• Mitochondria stained with
Mitotracker red
• Actin cytoskeleton stained with
phalloidin derivative conjugated
to Alexa 488 (green)

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Limitations of Fluorescence Microscopy

• Photobleaching
• Often limits the number of exposures or the exposure time

Photobleaching is the irreversible photochemical destruction of the fluorescent chromophores

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Limitations of Fluorescence Microscopy

• Autofluorescence
• Autofluorescence can be present in the images
• Image at narrow band or use NIR excitation to minimize this effect (NIR
exogenous fluorophores)

Endogenous Fluorophores
• amino acids
• structural proteins
• enzymes and co-enzymes
• vitamins
• lipids
• porphyrins

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 Limitations of Fluorescence Microscopy

• Resolution is limited in thick specimens

• Detection of out-of-focus fluorescence Excitation
• The excitation beam illuminates uniformly a
wide field of the sample. Emission
• If the sample is thick, fluorescence will be
excited within the focal plane, but also Tissue
within planes above and below the focus.
• Some of this fluorescence will be imaged
onto the detector and will result in a
defocused-looking image

Human medulla Rabbit Muscle Fibers Pollen Grain

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Limitations of Fluorescence Microscopy

• Reject out-of-focus light ⟶ Optical “sectioning” (next class)

• Create 3d images
• Confocal Microscopy, Two Photon Microscopy, SIM

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QUESTIONS???

END