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