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Fluorescence microscopy I Basic concepts of optical microscopy Martin Hof, Radek Macháň CZECH...
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Transcript of Fluorescence microscopy I Basic concepts of optical microscopy Martin Hof, Radek Macháň CZECH...
Fluorescence microscopy IBasic concepts of optical microscopy
Martin Hof, Radek Macháň
CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
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.stowers-institute.org/microscopy/external/Technology/index.htm
Sources of image contrast:
• Absorption (bright field – the “basic” optical microscopy) • Refractive index (refraction, scattering, phase shift)• Emission (fluorescence)• Raman scattering• Others (birefringence, reflection, …)
Why do we see the objects?Because they differ in optical properties from the background:
Bright field microscopy:
light form the condenser passes through the sample, where it is attenuated by absorbing objects
Bright field microscopy:
ocular
light
objective
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.
We will forget about the eyepiece and magnification.
The objective and the resolution and contrast it can achieve are essential
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:
Inserted optical components (filters, polarizers, …) do not disturb the optical path
tube lens
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:
NA = n sin
TR = 41°
Dry objective
Immersion 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
Objectives – immersion liquids:
immersion oils – chosen to match closely the refractive index of glass nG = 1.52
oil vs. water
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.
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:
Objects with a sharprise in refraction index
• part-illumination of the specimen
• scattered light collected by objective
• bright object on dark background
Phase contrast microscopy:
annular diaphragm
image planepositive phase contrast
objectiveback focal plane
&phase plate
specimen (phase object)
condenser
condenser front focal plane
condenser aperture pinhole
positive phase contrast:
object of higher optical
path appears darker
uncertainty in image interpretation arises when objects induce larger phase shift
than /2 or when absorption appears simultaneously to phase shift
Frits Zernike (1888-1966)
the lateral profile of the object optical thickness
object-induced phase shift
a prism-induced phase differential between the two perpendicularly polarised wavefronts
individual phase profiles in the polarised components of the doubled image
local phase differences in the overlapping images revealed by the analyser
brightness profile in the differential image
WPC - compensator
(eyepiece)analyser (- 45)
doubled image
Wollaston prismsWPO and WPC
WPO - beamsplitterobjective
specimen
condenser
iris diaphragm
polariser (+45)
A’ B’A’’ B’’
0
A
0A’’
A’
Differential interference contrast:
Objects appear as
if illuminated from
one side
Phase contrast vs. DIC:Kidney tissue
(tubule with some cells> 100 µm thick section)
Phase contrast
Buccal epithelial cell(monolayer)
DIC
(with modification http://mikroskopie.de)
Images suffer from a halo of bright light surrounding some objects – caused by a fraction of diffracted light which has passed the phase ring
• Can resolve differences in thickness down to about 2 nm
• Small gradients of thickness give little contrast
Fluorescence Microscopy:
Possibility of molecule-specific labeling – chemical sensitivity
Example:
Cytoskeleton (tubulin antibody-Alexa647)Mitochondria (streptavidin-Alexa488)Nucleus (Hoechst-DNA intercalator)
High sensitivity – single molecule observation possible
Fluorescence is sensitive to environment – provides information on polarity, pH, …
Fluorescence microscope:
Epi-Fluorescence setup:
excitation light passes through the same objective
which collects the fluorescence
objective
sample
camera
sets of filters and dichroics are available for every common fluorophore
Fluorescence microscope:
Typically the inverted setup – objective below the sample
camera
Many cell strands tend to adhere to the bottom of the chamber
Sample chamber can be open – we can add something during the measurement
Photobleaching in fluorescence microscopy:
source of artefacts and irreproducibility, low excitation intensity to avoid photobleaching and saturation
microscopy.duke.edu/gallery.html
E1
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
Fraction of immobile fluorophores
Bim II
IIf
0
0
I∞I0
IB
Microscope resolution – Rayleigh criterion:
NAd
61,0
240
200
160
120
80
40
050 100 150 200 250
240
200
160
120
80
40
050 100 150 200 250 300
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
Rayleigh criterion: points are resolvable if the maximum of one Airy disc corresponds with the first minimum of the adjacent Airy pattern
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
R ’
a’
YY’
Diffractionsin = 0.61'/R
Rayleigh criterionY' = a' tan
Simple geometry yields:R/a’ = tan’ Y' = 0.61'/tan’
a
Abbe Sine Condition:Y n sin = Y' n' sin‘ Y' n' tan‘
Ymin = 0.61 / n sin
considering that ‘ = / n’NA
Microscope resolution – Rayleigh criterion:
fedcba
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
Microscope resolution – Abbe’s theory:
Ideal imagediffraction pattern &
mask
image brightness
profile
image appearanc
e
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
Light Microscopy in Biology. A practical Approach. A.J.Lacey (ed.), IRL Press, Oxford, 1989, p.33.
Any aperiodic object O can be theoretically described as an infinite series of periodic functions (Fourier series)
NAd
5,0
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
NAd
61,0
r ≈ 3 d r ≈ d r ≈ d/3
Microscope resolution – Summary:
The lateral resolution of an optical microscope d:
25,0
NA
d
The axial resolution (in the direction of optical axis) dz:
Sufficient contrast is necessary for full utilization of the available resolution
2
4,1NA
ndz
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