Download Microscopy Basics

CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
Fluorescence microscopy I
Basic concepts of optical microscopy
Martin Hof, Radek Macháň
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.olympusmicro.com/,
http://www.olympusfluoview.com/index.html/
• Stowers Institute Tutorials (especially FCS)
http://research.stowersinstitute.org/microscopy/external/Technology/index.htm
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, …)
Bright field microscopy:
light form the condenser passes through the sample, where it is attenuated by
absorbing objects
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
Köhler illumination – conjugated planes:
A. Köhler
(1866-1948)
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
Objectives – infinity system:
tube
lens
Inserted optical components (filters, polarizers, …) do not disturb
the optical path
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
Objectives – numerical aperture:
Dry objective
TR = 41°
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
NA = n sin
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
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
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
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
Phase contrast microscopy:
positive phase contrast:
image plane
positive phase contrast
object of higher optical
path appears darker
objective
back focal plane
&
phase plate
specimen (phase object)
condenser
Frits Zernike
(1888-1966)
condenser
front focal plane
pinhole

condenser aperture

annular diaphragm
uncertainty in image interpretation arises when objects induce larger phase
shift than p/2 or when absorption appears simultaneously to phase shift
Differential interference contrast:
brightness profile in the
differential image
(eyepiece)
analyser (- 45)
doubled
image
A’’ B’’
A’ B’
0
A’’
Wollaston prisms
WPO and WPC
A’
0
WPO - beamsplitter
local phase differences in the
overlapping images revealed
by the analyser
individual phase profiles in
the polarised components of
the doubled image
a prism-induced phase
differential between the two
perpendicularly polarised
wavefronts
objective
object-induced phase shift
specimen
condenser
the lateral profile of the
object optical thickness
A
WPC - compensator
Objects appear as
iris diaphragm
if illuminated from
polariser (+45)
one side
Phase contrast vs. DIC:
Buccal epithelial cell
(monolayer)
Kidney tissue
(tubule with some cells
> 100 µm thick section)
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)
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)
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
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
E1
Photobleaching in fluorescence microscopy:
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
I0
I∞
IB
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
Fraction of immobile
fluorophores
fim 
microscopy.duke.edu/gallery.html
I0  I
I0  IB
Microscope resolution – Rayleigh criterion:
Light from a point source is diffracted by the objective forming an Airy disc,
the size of which depends on  and NA of the objective
Airy disc
Corresponding intensity profile
240
0,61 
d
NA
200
160
120
80
40
0
50
100
150
200
250
Rayleigh criterion: points are resolvable if the
maximum of one Airy disc corresponds with
the first minimum of the adjacent Airy pattern
240
200
160
120
80
40
0
50
100
150
200
250
300
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
Microscope resolution – Rayleigh criterion:
Y

R
a
Diffraction
sinq = 0.61'/R
Rayleigh criterion
Y' = a' tanq
Simple geometry yields:
R/a’ = tan’
Y' = 0.61'/tan’
’
Y’
q
a’
Abbe Sine Condition:
Y n sin = Y' n' sin‘  Y' n' tan‘
Ymin = 0.61  / n sin
considering that ‘ = / n’
NA
Microscope resolution – Abbe’s 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
Ideal image
a
b
c
d
e
f
diffraction
pattern &
mask
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))
Microscope resolution – Abbe’s 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
0,5 
d
NA
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.
Abbe’s 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
Microscope resolution – Elastic scattering:
The shape of polar scattering diagrams for small spherical particles
depends on the size of the particle r and . 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
d
r≈3d
0,61 
NA
r≈d
r ≈ d/3
Microscope resolution – Summary:
The lateral resolution of an optical microscope d:
0,5  
d

NA
2
The axial resolution (in the direction of optical axis) dz:
1,4  n
dz 
NA2
Sufficient contrast is necessary for full utilization of the available
resolution
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