MONABIPHOT /PMV / BME

Advanced Optical Methods for

Neurosciences

Benoît C. FORGET
2017-18
MONABIPHOT / PMV / BME

• Nov 9th : B.C. Forget • Dec 7: E. Ronzitti / E. Papagiakoumou

• Refreshers in optics • Structured illumination
• Resolution and the PSF • Holography and optogenetics
• Wavefront Engineering
• Dec 14 : M. Guillon / B.C. Forget
• Nov 16th : M. Guillon / B.C. Forget
• Superresolution and superlocalisation
• Phase imaging
techniques
• Nov. 23rd : W. Suppato
• Non linear microscopy
• Lightsheet microscopy
• Nov 30th : B.C. Forget
• Basic holographic techniques
• Lab visits

2
Summary

• Refreshers in optics
• Geometrical optics
• Rays and images
• Optical Microscope
• Wave optics
• Propagation of waves
• Mathematical description of waves
• More Wave optics
• Resolution and PSF
• Wavefront and wavefront engineering
• Intoduction to phase imaging
• Zernike phase contrast
3
Light propagation and image formation

4
Three theoretical frameworks for optics

Geometrical (ray) optics

• Light rays propagate in a rectilinear path as they travel in a homogeneous medium
• Rays bend (and may split in two) at the interface between two dissimilar media
• Allows the design of efficient (and cool) instruments

5
Three theoretical frameworks for optics

Wave optics
• Scalar waves
• Interferences, diffraction diffusion of light
• High precission interferometric measuring techniques, spectroscopy
• Electromagnetic wave theory (Maxwell)
• polarisation

6
Three theoretical frameworks for optics

Quantum optics
• Emission and absorption of light by molecules and materials
• laser

7
Image and stigmatism

• A and A′ are conjugated points : A′ is the

image of A ;
• Any ray « passing through » point A also
passes through (or seems to pass through)
point A’
• Without stigmatism the image is blurred

8
Clear and blurred images

Is there a bijective relation between "object" and "image" : can we recover

(reconstruct) the "object" from the "image" ?

9
Magnification (lateral or angular)

10
Dioptrics Reflection et Refraction :
at the interface between two media of different index
of refraction a ray of light changes it's direction.

11
Snell-Descartes

• Réflexion : θi  θr

• Réfraction : n1 sinθ1  n2 sinθ2

• Conditions de Gauss : sinθ  θ

12
Snell-Descartes

Réflexion
r  35

n2  1,0 (air)
i  22  i  22

n1  ? Réfraction
n1 sini  n2 sinr

i  22
i  22 n1  n2
sinr
 1,0

sin 35   1,53
sini 
sin 22 

13
« L’effet Vénus » :
Vénus s’admire dans son miroir. Qu’y voit elle ?
Rubens, 1614

Ceux qui la regardent !

Titien, 1555

Velasquez 1647
Principe du retour inverse de la lumière : si un
faisceau lumineux suit une certaine trajectoire
dans un sens de propagation, alors un faisceau
se propageant en sens inverse suivra la même
trajectoire
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Thin lens

1 1 1  1 
conjugation equation      
OA OA OF   OF 

AB OA
lateral magification γt  
AB OA

15
Focal points, planes. Gauss conditions

16
Focal point of a « complex optical system »

17
Afocal system
AB O2F2
γt  
AB O1F1

18
Magnifying glass : angluar magnification

19
Optical microscope, microscope objective

20
Laser scanning confocal microscopy

Butterfly wing epithelium

Excellent online ressource :

https://www.microscopyu.com/techniques/confocal

21
Magnification is not enough

22
What is a wave ?

• In an elastic medium, internal forces tend

to bring it back to its original after a
perturbation
• This perturbation (deformation) moves at a
characteristic speed (celerity) which is
solely determined by the mechanical
properties of the media

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Energy transport without matter transport

It is necessary to separate local (or « particle »)

movement from the wave propagation

25
Mathematical description of waves

A deformation moving while keeping the same shape

Space and time evolution (variables) are 'coupled':

ξ(x,t)  ξ(x  ct) ξ(x  ct) if c  cu x 

26
From a mathematical point of view
• Is there an equation that allows for solution :

Aξ(x  ct)  Bξ(x  ct)

2ξ 2 2ξ
d'Alembert equation ! c 0
t 2
x 2

Dimensionnal analysis: [ξ] 2 [ξ] [L]

 [c]  [c] 
Celerity c is a speed. [T]2 [L]2 [T]

27
From a physical point of view :
Vibrating string

• On s'intéresse au mouvement suivant l'axe x d'un petit segment de longueur Δz

• Il existe une tension (une force) T0 dans la corde. Cette tension se transmet "de
proche en proche", et chaque petit segment Δz la subit

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• On déforme maintenant la corde et on cherche à écrire la déformation ξ(z,t)
• Quelles sont les forces appliquées ? Il s'agit des projetions sur l'axe x des tensions.

Fx (t)  T2 sinθ2  T1 sinθ1

Si les angles sont petits : ξ

sinθ1,2  tanθ1,2 
et si la corde ne se déforme pas : z zz1,z z2
et donc : T2  T1  T0
ξ ξ  2ξ
Fx (t)  T0  T0  T0 Δz 2
29 z zz2 z zz1 z
 Il faut d'appliquer la seconde loi de Newton :

F  ma

ξ  2ξ
ξ  déplacement ;  vitesse ; 2  accélération
t  célérité ! t
La masse linéique (masse par unité de longueur) ρ permet d'exprimer la masse m du segment :
m=ρΔz  2ξ  2ξ  2ξ ρ  2ξ
Fx (t)  ma  T0 Δz 2  ρΔz 2  2  0
z t z T0 t 2

C'est l'équation d'onde ;

c est la célérité de l'onde, c'est bien une grandeur homogène à une vitesse
T0 [MLT 2 ] 1
c  1
 [LT ]
ρ [ML ]
30 et ne dépend que des propriétés mécaniques du milieu
From a physical point of view :
pressure wave in a sound pipe

 2ξ
F  ma  S p(x)  p(x  Δx)  ρSΔx 2
t
p
 Δx
x
p  2ξ p  2ξ
 ρ 2  ρ 2  0
31 x t x t
 Conservation de la masse

V0  SΔx

V  S  Δx  ξ(x  Δx)  ξ(x)

 ξ 
 SΔx 1  
 x 

ρ V0 SΔx
ρV  ρ0 V0   
ρ0 V  ξ 
SΔx 1  
 x 

ρ 1 ξ ξ ρ  ρ0 Δρ
  1   
ρ0 1  ξ x x ρ0 ρ0
x
ou
V ξ ξ V  V0 ΔV
 1   
32 V0 x x V0 V0
 Elasticité : Loi de Hooke

Fk  kΔ

La description qui prend en compte la géométrie 3D relie

linéairement la pression à la variation relative de volume :

ΔV 1 ΔV ΔV
p  cste  p     χp
V0 χ V0 V0

Dans le cas qui nous intéresse, χ est la compressibilité adiabatique.

33
 p  2ξ
• dynamique : ρ 2  0
x t
• élasticité :
ΔV
• continuité :  χp
V0

ξ ΔV

x V0

p  2ξ
ρ 2  0 • C'est l'équation d'onde ;
x t • c est la célérité de l'onde, c'est bien une grandeur
  1 ΔV   2ξ homogène à une vitesse
 ρ 2  0 • et ne dépend que des propriétés mécaniques du
x  χ V0  t
milieu
1   ξ   2ξ  2ξ 1  2ξ
   ρ 2  0  2  0 1 1
χ x  x  t t χρ x 2
c   [LT 1 ]
ρχ [ML3 ]
[ML1T 2 ]
34
What about EM waves ?

ρ  2E
 E   E  μ0 ε 0 2  0
2
ε0 t
1
 B  0 c
μ0 ε 0
B
 E  
t
E Note that vacuum (like materials) has
  B  μ0 j  μ0 ε 0
t electromagnetic properties !

35
trig functions as solutions

Trig functions (sine, cosine) are solution to d'Alembert's equation.

In the form :

with : ω= ck
Vérification ξ( x, t )  A cos(kx  ωt  φ)

   2    
 A cos(ω t  kx  φ)   c  A cos(ω t  kx  φ)  0
t  t  x  x 
 2 

t
  ωA sin(ω t  kx  φ)   c
x
 kA sin(ωt  kx  φ)  0

ω
ω2 A cos(ωt  kx  φ)  c 2k 2 A cos(ωt  kx  φ)  0  c 
k
36
Amplitude, phase, wavelength …

37
Space (λ) and time (T) periodicity are coupled

 x t   2π 2π
cos  2π     φ  k ; ω  2πf   c  fλ
 λ T   λ T

38
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Nombres complexes : représentations
cartésienne et polaire
z  x  iy avec i  1 The EM field E(x,t) is written in complex notation :


A cos(kx  ωt  φ)   E  Ae j ( kx ωt φ) 
  E  Ae jφ
e j ( kx ωt ) 
  E  Ae j ( kx  ωt )


z  a  cosθ  i sinθ)   ae iθ Note: Physical (measurable) quantities can only be

 exp( iθ):rel.dEuler expressed with real numbers.

40
Attention au caractère linéaire

Cette approche fonctionne tant que l'on s'en tient à des opérations linéaires :

{az1  z2 }  az1  z2

{z}  (z )

mais :
{z1  z2 }  z1  z2

41
Propagating wave : the phasor

ξ( x  Δx, t )  Ae j (ωt k ( x  Δx ))
 Ae j (ωt k ( x ))e  jkΔx
 ξ( x, t )e  jkΔx

ωt  kw  φ0
φ0 ω
x   t
k k
φ
 0  ct
k
One particular "point" of phase φ0 travels
along the axis Ox at speed c.
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Interférences

44
A simple example : Young’ slits

45
• D’Alembert’s equation is linear
• Young’ slits
• Fourier series

 πd 
46 I(x)  cos2  x
 λD 
Abbe’s theory of image formation

47
 I(x)

48
Abbe’s diffraction limit

49
Numerical aperture

50
Airy pattern and Rayleigh criterion

2
 2 J (ka sinθ) 
I(θ)  I0  1  1,22λ 0,61λ
 ka sinθ  
2ON NA
2π
k
NA
51
PSF and imaging

NA and image resolution

52
PSF, impulse response … Convolution

C. Pissaro, Enfants au parc (1887)

The imaging system (microscope) is linear invariant system analogous to a LTI :

I  x, y   PSF  x, y  * O  x, y 

53
Deconvolution

Deconvolution is possible to a certain extent ...

54
Résolution axiale et microscopie confocale

55
Laser Scanning Confocal Microscopy

56
TIRF

57
Measuring the real PSF

58
Aberrations

59
Scattering

60
2D and 3D waves : The optical wavefront

ξ( x  Δx, t )  Ae j (ωt k ( x  Δx ))
 Ae j (ωt k ( x ))e  jkΔx
 ξ( x, t )e  jkΔx

ωt  kw  φ0
φ0 ω
x   t
k k
φ
 0  ct
k

One particular "point" of phase φ0 travels

along the axis Ox at speed c.

61
Wave vector
The wave vector indicates the (local) direction
of propagation of the wave.

For the "locally plane" wavefront :

k  r  cst
The wave vector is normal to the wave front

62
Wave vector

k  r  cst  E  Ae j (ωt k r )

63
From wave optics back to ray optics

Diffrent thickness → different phase shift (retardation).

x tanα
Δφ(x)  2π(n  1)
λ
64
65
Wavefront Engineering

66
Tilt
Δg  a  x tanθ x  y tanθy 

67
Lens (defocus)

Δφ  bΔz  x 2  y 2 

68
Tilt and focus : multispots
 N i     
  arg   un e g 
 1 

69
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Improving the PSF with adaptative optics

73
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Seeing water in water

75
Intensity contrast

Brightfield reflectance microscopy is based on intensity constrast

 n1  n2 
2

R
 n1  n2 
2

The cell is not verry "optically different" than the sourounding medium :
Cell : n=1,36 → R ≈ 0,0233
nutriment medium : n=1,335 → R ≈ 0,0206

Imax  Imin
Contrast : C  6%
Imax  Imin

76
Why not measure phase contrast

2π
Δφ  kΔ(nz )  zΔn
λ
z  6 µm;Δn  0,025;λ  0, 5 μm

λ π
Δ(nz )  ;Δφ 
4 2

Détecteur Temps de réponse

Œil ≈0,1 s c  3  108 m/s
Photo film ≈10−4−10−2 s 400nm  λ  700nm
Fast photodiode ≈10−7 - 10−8 s f  6  1014 Hz
CCD ≈10−2 – 10-3 s
77
Amplitude, phase and intensity

Remember the expression of the field (in complex and real notations) :

E  Ae j ( kr ωt )
{E }  A cos(kr  ωt  φ)

Only the intensity (average value over tile of the energy of the wave) can be detected.
1 A2
I E 2
 EE 
*

2 2

Detection of the intensity is phase independant !

78
D'Alembert's equation is linear

The sum of the wave (interference) is also a wave :

E1  Ae j (kr ωt ) E2  Ae j (kr ωt  Δ)

The intensity of this interference is phase dependant :

1
IT  (E1  E2 )(E1  E2 )*  I1  I2  2 I1I2 cos Δ
2

it is a function of the phase difference Δφ between the two intefering waves

79
Phase contrast imaging

Why do we see (weak) contrast here ?

Where is the "second wave" ?

80
Phase mask

81
Microscope à contraste de phase

• "Some part" of the incoming wave interacts

with the sample. This is the "diffracted
wave"
• "Some other part" of the incoming just
"goes right through". This is the "surround
wave".
• The image results from the interference
between these two waves

82
Optimizing phase contrast

IT  IS  ID  2 IS IS cos Δ IT  IS  2 IS ID cos Δ

I2  I1
C  C  1  cos Δ  Δ2
I2  I2
83
Phase shifting of the surround wave
IT  IS  2 IS ID cos(Δ  π / 2)  IS  2 IS ID sinΔ

I2  I1
C  C  sinΔ  Δ
I2  I2
84
Improved contrast

85
Phase contrast microscope

86
Adjusting the phase contrast

87
A first step towards holography

88