Fluorescence microscopy I Basic concepts of optical microscopy

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Fluorescence microscopy IBasic concepts of
optical microscopy
Martin Hof, Radek Machán
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Further reading

• Introduction to Confocal Microscopy and Image
  Analysis, J. P. Robinson, http//tinyurl.com/2dr5p
  
• Molecular Expressions Microscopy Primer
  http//micro.magnet.fsu.edu/primer/index.html
• Nikon Microscopy Tutorials, http//www.microscopyu
  .com/
• Zeiss Microscopy Tutorials,
• http//zeiss-campus.magnet.fsu.edu/index.html
• Olympus Microscopy Tutorials, http//www.olympusmi
  cro.com/, http//www.olympusfluoview.com/index.htm
  l/
• Stowers Institute Tutorials (especially FCS)
  http//research.stowers-institute.org/microscopy/e
  xternal/Technology/index.htm

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Sources of image contrast
Why do we see the objects?
Because they differ in optical properties from
the background

• Absorption (bright field the basic optical
  microscopy)
• Refractive index (refraction, scattering, phase
  shift)
• Emission (fluorescence)
• Raman scattering
• Others (birefringence, reflection, )

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Bright field microscopy
light form the condenser passes through the
sample, where it is attenuated by absorbing
objects
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Bright field microscopy
light form the condenser passes through the
sample, where it is attenuated by absorbing
objects
Magnification M(objective) x M(eyepiece)
the image formed by the objective in its back
focal plane (the intermediate image plane)
contains all information accessible by the
microscope. Further magnification of the image by
eyepiece or lenses of a camera only change it
size for easier observation or to fit to the chip
of the camera, but do not add any information.
ocular
We will forget about the eyepiece and
magnification. The objective and the resolution
and contrast it can achieve are essential
objective
light
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Köhler illumination conjugated planes
optimal adjustment of the illumination pathway
uses the concept of two sets of conjugated planes
(planes in which the beam is simultaneously
focused) to ensure even illumination of the sample
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Objectives infinity system
tube lens
Inserted optical components (filters, polarizers,
) do not disturb the optical path
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Objectives aberrations and corrections
Chromatic aberration is corrected by combination
of lenses of different refractive index (Achromat
2 different wavelength focused to 1 point,
Apochromat 3 different wavelength focused to 1
point
Flat-Field correction ensures planarity of the
image important for its projection on a chip of
a camera
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Objectives numerical aperture
Dry objective
the width of the acceptance cone of the objective
determines how much light contributes to the
image formation and it is important for the
resolution and contrast of the image
Why refractive index n???
Refraction occurring of the interface of glass
(cover glass of the sample) and air
Immersion liquid reduces the refractive index
mismatch
Immersion objective
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Objectives immersion liquids
immersion oils chosen to match closely the
refractive index of glass nG 1.52
water nW 1.33, worse match, however,
biological samples consist mainly of water and
water immersion is better for imaging thick
biological samples
objectives have corrections for aberrations
introduced by the cover glass of given thickness
and refractive index.
oil vs. water
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Sources of image contrast
Bright field microscopy is based on absorption of
light in the sample.
Most biological objects, however, absorb only
weakly in the visible spectrum. This lead to

• Development of specific staining (nowadays almost
  entirely replaced by fluorescent labeling)

• Development of UV microscopy (Köhler) facing
  technical difficulties due to absorption of UV
  light by glass

• Use of difference in refractive index between the
  object and medium manifested by
• ? refraction (scattering) of light
• ? introduction of phase shift to the
  passing light

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Dark field microscopy

• part-illumination of the specimen
• scattered light collected by objective
• bright object on dark background

Objects with a sharp rise in refraction index
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Phase contrast microscopy
positive phase contrast object of higher optical
path appears darker
Frits Zernike (1888-1966)
uncertainty in image interpretation arises when
objects induce larger phase shift than p/2 or
when absorption appears simultaneously to phase
shift
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Differential interference contrast
brightness profile in the differential image
(eyepiece)
analyser (- 45?)
local phase differences in the overlapping images
revealed by the analyser
doubled image
individual phase profiles in the polarised
components of the doubled image
Wollaston prisms WPO and WPC WPO -
beamsplitter
a prism-induced phase differential between the
two perpendicularly polarised wavefronts
objective
object-induced phase shift
specimen
the lateral profile of the object optical
thickness
condenser
Objects appear as if illuminated from one side
WPC - compensator
iris diaphragm
polariser (45?)
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Phase contrast vs. DIC
Buccal epithelial cell (monolayer)
Phase contrast
Images suffer from a halo of bright light
surrounding some objects caused by a fraction
of diffracted light which has passed the phase
ring
DIC

• Can resolve differences in thickness down to
  about 2 nm

• Small gradients of thickness give little contrast

(with modification http//mikroskopie.de)
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Fluorescence Microscopy
High sensitivity single molecule observation
possible
Possibility of molecule-specific labeling
chemical sensitivity
Fluorescence is sensitive to environment
provides information on polarity, pH,
Example Cytoskeleton (tubulin antibody-Alexa647)
Mitochondria (streptavidin-Alexa488) Nucleus
(Hoechst-DNA intercalator)
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Fluorescence microscope
Epi-Fluorescence setup excitation light passes
through the same objective which collects the
fluorescence
camera
objective sample
sets of filters and dichroics are available for
every common fluorophore
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Fluorescence microscope
Typically the inverted setup objective below
the sample
Sample chamber can be open we can add something
during the measurement
Many cell strands tend to adhere to the bottom of
the chamber
camera
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Photobleaching in fluorescence microscopy
E1
source of artefacts and irreproducibility, low
excitation intensity to avoid photobleaching and
saturation
It can be however used to investigate molecular
diffusion Fluorescence recovery after
photobleaching (FRAP) how fast are
fluorophores, which had been photobleached by a
pulse of high intensity, replaced by new ones
lipid bilayer adsorbed to solid surface mobile
lipids
lipid monolayer adsorbed to immobilized alkyl
chains immobile lipids
D found by fitting the recovery curve with a
model accounting for the size and shape of the
bleached area
I8
I0
IB
Fraction of immobile fluorophores
microscopy.duke.edu/gallery.html
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Microscope resolution Rayleigh criterion
Light from a point source is diffracted by the
objective forming an Airy disc, the size of which
depends on l and NA of the objective
Airy disc
Corresponding intensity profile
Digital contrast enhancement of images may help
resolution of closer points. The improvement may
be, however, overestimated due to smaller
distance between the maxima than between the
centers of Airy discs
Rayleigh criterion points are resolvable if the
maximum of one Airy disc corresponds with the
first minimum of the adjacent Airy pattern
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Microscope resolution Rayleigh criterion
R
Y
a
a
Y
q
a
a
Diffraction sinq 0.61?'/R Rayleigh
criterion Y' a' tanq
Abbe Sine Condition Y n sin? Y' n' sin? ? Y'
n' tan? Ymin 0.61 ? / n sin? considering
that ? ?/ n
Simple geometry yields R/a tan? Y'
0.61?'/tan?
NA
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Microscope resolution Abbes theory
Light passing through a periodic structure in the
sample (a diffraction grating) results in a
characteristic diffraction pattern in the
objective back focal plane. The observable number
of diffraction maxima is determined by NA of the
objective
diffraction pattern mask
Ideal image
image brightness profile
image appearance
Description by Fourier optics Wavefront in the
back focal plane W is a Fourier transform of the
object transmission function O. The image I is
the inverse Fourier transform of W W F (O) I
F-1(W) F-1(F(O))
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Microscope resolution Abbes theory
Description by Fourier optics Wavefront in the
back focal plane W is a Fourier transform of the
object transmission function O. The image I is
the inverse Fourier transform of W W F (O) I
F-1(W) F-1(F(O))
The objective aperture filters out higher order
diffraction maxima from W and, thus, filters out
high spatial frequencies from I
Any aperiodic object O can be theoretically
described as an infinite series of periodic
functions (Fourier series)
Light Microscopy in Biology. A practical
Approach. A.J.Lacey (ed.), IRL Press, Oxford,
1989, p.33.
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Abbes theory and oblique illumination
With oblique illumination higher orders of
diffraction maxima can enter the objective of the
same NA than with axial illumination
? Improved resolution
However, less light enters the objective ? worse
contrast
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Microscope resolution Elastic scattering
The shape of polar scattering diagrams for small
spherical particles depends on the size of the
particle r and l. The smaller r, the more
symmetric is the scattering diagram.
The size of the central scattering lobe
corresponds to the acceptance angle of the
microscope when
r 3 d
r d/3
r d
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Microscope resolution Summary
The lateral resolution of an optical microscope d
The axial resolution (in the direction of
optical axis) dz
Sufficient contrast is necessary for full
utilization of the available resolution
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Acknowledgement
The course was inspired by courses of Prof.
David M. Jameson, Ph.D. Prof. RNDr. Jaromír
Plášek, Csc. Prof. William Reusch
Financial support from the grant FRVŠ 33/119970