individual molecule localization

School of Photonics
Cortona, March 30 - April 3, 2014
Single molecule localization
and tracking techniques:
from cells to tissue imaging
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
1
Optical resolution vs molecular dimension
GFP
2,5 nm
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
2
Optical resolution vs molecular dimension
microscope point spread function
λ = 500nm
NA = 1.4
GFP
2,5 nm
100 nm
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
3
Resolution
d rad 

2  n  sin( )
2
d ax 
n sin 2 ( )
http://www.compadre.org/OSP/document/ServeFile.cfm?ID=8198&DocID=866
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
4
How to circumvent the diffraction barrier?
Avoiding simultaneous emission
of spatially close fluorophores emitting in the same
spectral range
“Off”
“On”
A
B
SLIDE CREDIT, GIUSEPPE VICIDOMINI, IIT
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
5
SUPER-RESOLUTION TECHNIQUES:
Stimulated Emission
Depletion
JBC Review 2010 by Lothar Schermelleh, Rainer Heintzmann and Heinrich Leonhardt
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
6
SUPER-RESOLUTION TECHNIQUES:
Single molecule
localization techniques
JBC Review 2010 by Lothar Schermelleh, Rainer Heintzmann and Heinrich Leonhardt
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
7
SUPER-RESOLUTION TECHNIQUES:
Structured illumination
techniques
JBC Review 2010 by Lothar Schermelleh, Rainer Heintzmann and Heinrich Leonhardt
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
8
The “on-off” Game
Single molecule
localization techniques
PALM / STORM / GSDIM
JBC Review 2010 by Lothar Schermelleh, Rainer Heintzmann and Heinrich Leonhardt
Francesca Cella Zanacchi
S.W. Hell, et al. Far-Field Optical Nanoscopy, Science 316, 1153 (2007)
Nanophysics - Italian Institute of Technology
www.iit.it
9
RESOLUTION LIMIT IN CONVENTIONAL
MICROSCOPY
Image in conventional
microscopy
System Point Spread
Function

250 nm
PSF ( x, y, z )  Object  I ( x, y, z )
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
10
INDIVIDUAL MOLECULE LOCALIZATION
CONCEPT
Wide-Field
Francesca Cella Zanacchi
Individual molecule
localization
Nanophysics - Italian Institute of Technology
www.iit.it
11
INDIVIDUAL MOLECULE LOCALIZATION
CONCEPT
Wide-Field
Francesca Cella Zanacchi
Individual molecule
localization
Nanophysics - Italian Institute of Technology
www.iit.it
12
INDIVIDUAL MOLECULE LOCALIZATION
CONCEPT
Wide-Field
Francesca Cella Zanacchi
Individual molecule
localization
Nanophysics - Italian Institute of Technology
www.iit.it
13
INDIVIDUAL MOLECULE LOCALIZATION
CONCEPT
Wide-Field
Francesca Cella Zanacchi
Individual molecule
localization
Nanophysics - Italian Institute of Technology
www.iit.it
14
INDIVIDUAL MOLECULE LOCALIZATION
CONCEPT
Wide-Field
Francesca Cella Zanacchi
Individual molecule
localization
Nanophysics - Italian Institute of Technology
www.iit.it
15
INDIVIDUAL MOLECULE LOCALIZATION
CONCEPT
Wide-Field
Francesca Cella Zanacchi
Individual molecule
localization
Nanophysics - Italian Institute of Technology
www.iit.it
16
INDIVIDUAL MOLECULE LOCALIZATION
CONCEPT
Wide-Field
Francesca Cella Zanacchi
Individual molecule
localization
Nanophysics - Italian Institute of Technology
www.iit.it
17
INDIVIDUAL MOLECULE LOCALIZATION
CONCEPT
Wide-Field
Francesca Cella Zanacchi
Individual molecule
localization
Nanophysics - Italian Institute of Technology
www.iit.it
18
Jablonski diagram
The wavelength range at which the dye is excited and emits ﬂuorescence depends on
its electronic structure and the properties of the local environment.
Depending on the total spin of all
electrons Σ the electronic states are
classiﬁed into triplet states Ti where Σ
= 1 and singlet states Si where Σ = 0.
Optical transitions by absorption or
emission of photons are only allowed
between states of equal spin coupling.
A transition between Ti and Sj requires
a ﬂip of the total spin and is thus
forbidden.
Higher order states, (Sn and Tn)
contribute at larger intensities
These processes are termed intersystem crossing (ISC) and for the triplet state a radiative decay
can only occur by a spin-ﬂip, and T1 thus has a prolonged lifetime (few µs).
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
19
SINGLE MOLECULE REGIME
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
20
HOW TO SWITCH OFF THE MOLECULES?
Switching between a
dark and a bright state
(from A to B)
•Photoswitchable
dyes/proteins
Light -induced changes in the
spectral properties of fluorescent
proteins
•Ground state depletion
Switching a regular fluorophore
using its dark states
•Stimulated emission
depletion
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
21
FPALM IMAGE PROCESSING
 Background subtraction
 Threshold to identify single molecules

Gaussian fit of the intensity distribution produced by a single
I  x, z   I 0  e
 ( x  x0 )2  ( y  y0 )2 
 2

r02


molecule
 offset
 Calculation of the localization precision for each molecule
a2
s 
8 s 4b 2
12

 2 2
N
a N
2
 x, y 2
 Apply tolerances and remove duplicates in the final image
 Rendering of the FPALM image: Map of localized gaussian spots
Thompson et al (2002)
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
22
LOCALIZATION PRECISION
Central to the performance of photoactivated localization microscopy (PALM) is the precise
localization of single fluorescent molecules performed by a least-squares fit of an assumed
two-dimensional gaussian point spread function (PSF) to each single molecule image.
The molecule position is given by the mean of the positions of
the individual detected photons and the error in the localization
is provided by the standard statistical error.
x
2
s2

N
The pixelation noise can be taken into account and this adding
in quadrature:
2
 x2 
s2 
a
12
N
The error in the localization adding also the contribution
due to pure background noise is:
a2
s 
4  s 3b 2
12


N
aN 2
2
 x2
Thompson et al (2002)
Francesca Cella Zanacchi
The accuracy is dependent from the
counting statistics of the detected
signal and from the noise introduced
by the detection device and
processing electronic. The two
important categories of noise are the
shot noise of the photons in the spot
and the background noise.
…in the bi-dimensional case:
a2
s 
8 s 4b 2
12

 2 2
N
a N
2
 x, y 2
• s standard deviation of the PSF
• N number of photons
• a effective pixel size
Nanophysics - Italian Institute of Technology
www.iit.it
23
Super resolution imaging of microtubules
12.000 frames
Francesca Cella Zanacchi
Exposure time 20ms/frame
αTubulin-Alexa647
Nanophysics - Italian Institute of Technology
www.iit.it
24
Widefield Imaging
3D STORM imaging of Tubulin in COS7 cell cultures.
α Tubulin immunostained with Alexa 647. Exposure time 10ms/frame.
15.000 frames. Scale bar 1μm.
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
25
3D d-STORM Imaging
Z(nm)
3D STORM imaging of Tubulin in COS7 cell cultures.
α Tubulin immunostained with Alexa 647. Exposure time 10ms/frame.
15.000 frames. Scale bar 1μm.
300
0
-300
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
26
3D Single molecule detection in
mitochondria
Superresolution
image
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
Mitochondria in
fibroblasts
www.iit.it
27
3D-STORM
Huang et al. Nat Methods (2008)
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
28
Stochastic optical reconstruction microscopy STORM
Photoswitchable
dye pairs
Cy3
A405
Slide credit NIKON Instr.
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
29
MOTIVATIONS
Challenges and trade-offs in individual
molecule localization based superresolution microscopy
ADVANTAGES:
Spatial resolution ≈20nm
Information at the
molecular scale
3D
“Complicate
samples”
LIMITATIONS :
 limited temporal resolution
Mainly limited imaging depth
capability
Up to “whole” cell superresolution imaging
...TRENDS:
 improve temporal resolution
 super-resolution imaging of organisms or tissue
How to improve imaging depth capability?
APPROACH FOR 3D SUPER-RESOLUTION OF THICK
BIOLOGICAL SAMPLES
Betzig et al 2006, Hess et al 2006, Rust et al 2006
Francesca Cella Zanacchi
Juette et al., Huang et al., Pavani et al.,
Nanophysics - Italian Institute of Technology
www.iit.it
Shtenhel et al.
30
MOTIVATIONS
Challenges and trade-offs in individual
molecule localization based superresolution microscopy
ADVANTAGES:
Spatial resolution
Information at the
molecular scale
3D
“Complicate
samples”
LIMITATIONS :
 limited temporal resolution
Mainly limited imaging depth
capability
Up to “whole” cell superresolution imaging
...TRENDS:
 improve temporal resolution
 super-resolution imaging of organisms or tissue
capability to perform imaging of large biological
samples and select a compartment for 3D super
resolution imaging
APPROACH FOR 3D SUPER-RESOLUTION OF THICK
BIOLOGICAL SAMPLES
Betzig et al 2006, Hess et al 2006, Rust et al 2006
Francesca Cella Zanacchi
Juette et al.,2008
Huang et al., 2008
Pavani et al.,2009
Nanophysics - Italian Institute of Technology
Shtenhel et al. 2009
www.iit.it
32
TOWARDS in vivo SUPER-RESOLUTION
Individual molecule localization techniques of thick samples
(PALM, STORM, FPALM, GSDIM...)
APPROACHES TO 3D SUPERRESOLUTION OF THICK BIOLOGICAL
SAMPLES
Dealing with
scattering samples
Optical
approaches
New molecules
and dyes
Selective plane
illumination
microscopy
Francesca Cella Zanacchi
From cells… to
whole organisms
or tissue
Two photon
excitation
New
localization
alghorithms
Nanophysics - Italian Institute of Technology
www.iit.it
33
LIGHT SHEET BASED MICROSCOPY
Digital scanned laser
microscopy (DSLM)
Selective plane
illumination
microscopy (SPIM)
ADVANTAGES OF SPIM:
SPIM basics
The basic principle of SPIM
is to illuminate the sample
from the side in a welldefined thin volume around
the focal plane of the
detection optics.
Jan Huisken, et al. Science 305, 1007 (2004)
Francesca Cella Zanacchi
• Optical sectioning capability
• Fast imaging speed
• High signal to noise ratio
• Low photodamage
Keller, et al. Science (2008)
Nanophysics - Italian Institute of Technology
Zsigmondy et al. (1925)
www.iit.it
34
SPIM vs DSLM ???
Selective Plane
illumination microscopy
(SPIM)
Francesca Cella Zanacchi
Digital laser scanned
microscopy
(DSLM)
Nanophysics - Italian Institute of Technology
www.iit.it
35
Selective Plane
illumination microscopy
(SPIM)
Digital laser scanned
microscopy
(DSLM)
• High speed
• High speed
• Low photodamage
• Higher range of
power available
• Easy implementation
• Suitable for beam
shaping and bessel beam
implementation
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
36
3
7
DIGITAL SCANNED LASER BEAM (DSLM)
Philipp J Keller, Annette D Schmidt, Joachim Wittbrodt, and Ernst H K Stelzer. Science, 322 (2008).
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
37
3
8
The wild-type zebrafish embryo was injected with H2B-eGFP
mRNA at the one cell stage
Science 322, 1065 (2008); Philipp J. Keller, et al. Development by Scanned Light Sheet Microscopy
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
38
3
9
LIGHT SHEET BASED MICROSCOPY (LSBM)
Optical sectioning capability
Single plane illumination microscopy (SPIM)
x
y
PSF ( x, y, z ) SPIM  hill ( z, y, x)  hdet ( x, y, z )
2
2
z
NAill = 0.16
ill  488nm
Francesca Cella Zanacchi
NAdet = 0.9
det  515nm
Nanophysics - Italian Institute of Technology
www.iit.it
39
SPIM vs Wide-field Microscopy
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
40
Optical architectures for single molecule
detection in thick samples
• Phisical sectioning (TomoSTORM)
• Optical sectioning (light sheet microscopy)
Francesca Cella Zanacchi
Nanguneri et al. 2012
Nanophysics - Italian Institute of Technology
www.iit.it
41
Optical architectures for single molecule
detection
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
42
SINGLE PLANE ILLUMINATION SET-UP
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
43
SINGLE PLANE ILLUMINATION SET-UP
• visible and IR lasers
• beam shaping unit for
illumination
• water chamber and sample
holder
• detection unit with additional
magnification
• EMCCD camera
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
44
PHOTOACTIVATION IN SPIM REGIME
Activation process primed by
violet laser radiation (405
nm) in single plane
illumination regime:
photoactivation experiments
have been performed on
cells expressing the nuclear
H2B-PAmCherry fusion
protein (A). Readout 561nm.
I act  0.2
kW
cm2
I exc  0.5
kW
cm2
texp  500ms
Sample credit M.Faretta, L. Furia
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
45
IMAGING OF POLYELECTROLYTE
NANOCAPSULES
1 μm
1 μm
5 00nm
5 00nm
1 μm
IML-SPIM 3D super-resolution imaging of nanocapsules.(a,b) Conventional (a) and IML-SPIM (b)
images of polyelectrolyte nanocapsules labeled with photoactivatable caged FITC. The localization
precision (e) and the distribution of photons per single molecule (f) and the axial resolution (g).
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
σ lateral = 35nm
σ axial = 65nm
Huang et al 2008
www.iit.it
46
IMAGING OF THICK SAMPLES
50 µm
human mammary
MCF10A cell spheroids
Debnath et al (2003)
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
47
EFFECTS INDUCED BY SCATTERING
A
50 µm
 Effects induced by
scattering along the
detection path
 Distortion of the
light sheet intensity
distribution within the
illumination path
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
48
SINGLE MOLECULE DETECTION IN DEPTH
Z = 10 μm
SPIM image
Individual molecule detection
10 μm
10 μm
IML-SPIM image of human mammary MCF10A cell
spheroids expressing H2B-PAmCherry.
Francesca Cella Zanacchi
s2
 
N
2
Nanophysics - Italian Institute of Technology
z
www.iit.it
49
SINGLE MOLECULE DETECTION IN DEPTH
Z = 40 μm
SPIM image
Individual molecule detection
10 μm
IML-SPIM image of human mammary MCF10A cell
spheroids expressing H2B-PAmCherry.
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
10 μm
z
www.iit.it
50
SINGLE MOLECULE DETECTION IN DEPTH
Z = 70 μm
SPIM image
Individual molecule detection
10 μm
IML-SPIM image of human mammary MCF10A cell
spheroids expressing H2B-PAmCherry.
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
10 μm
z
www.iit.it
51
SINGLE MOLECULE DETECTION IN DEPTH
Z = 100 μm
SPIM image
Individual molecule detection
10 μm
IML-SPIM image of human mammary MCF10A cell
spheroids expressing H2B-PAmCherry.
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
10 μm
z
www.iit.it
52
SINGLE MOLECULE DETECTION IN DEPTH
Number of photons/molecule (N)
Z = 10 μm
Z = 40 μm
s2
 
N
2
Z = 70 μm
Z = 100 μm
z
Sample credit Mario Faretta
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
53
ILLUMINATION IN SCATTERING SAMPLES
800
A
700
Imaging depth=50m
Illumination
penetration depth 50μm
PHANTOM SAMPLE
MIMICKING SCATTERING
PROPERTIES
Intensity (A.U.)
600
z
500
400
300
200
100
10 μm
y
0
0
20
40
60
80
Distance (m)
800
B
Imaging
depth=100depth
m
Illumination
penetration
100μm
700
Intensity (A.U.)
600
500
400
300
200
100
z
10 μm
0
0
y
20
40
60
80
Distance (m)
800
C
Imaging
depth=200depth
m
Illumination
penetration
200μm
700
Intensity (A.U.)
600
Images of the thickness of
the light sheet for different
optical pathways through
a scattering phantom
sample able to mimic
optical properties of the
sample (Mourant et al.
Applied Optics
37(16)
1998). The illumination
central position has been
centered at 50µm (A),
100µm (B), 200µm (C)
500
400
µs=50mm-1
g~0.98
300
200
100
z
10 μm
y
0
0
20
40
60
80
Distance (m)
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
54
ABERRATION EFFECTS IN
SCATTERING SAMPLES
x
Non scattering
z
y
y
1 μm
x
µs=50mm-1
z
y
y
Point spread function
measurement
performed imaging
sub-resolved
fluorescent
beads
(40nm) in a phantom
sample
mimicking
optical properties of
biological sample of
interest.
1 μm
Objective lens : Leica Plan Apo 60x, 0.9 NA water dipping
Francesca Cella Zanacchi
Imaging depth: 100 μm
Nanophysics - Italian Institute of Technology
www.iit.it
55
IMAGES OF TUMOR CELL SPHEROIDS
IML-SPIM image of human
mammary MCF10A cell
spheroids expressing
H2B-PAmCherry.
The SPIM fluorescence
image obtained by adding
the total signal over the
frames.
localization precision = 28nm
1 μm
I act  0.06
1 μm
kW
kW
texp  40ms
I

5

10
exc
cm 2
cm2
Francesca Cella Zanacchi
Imaging depth: 100 μm
Total acquisition time 3min
Nanophysics - Italian Institute of Technology
www.iit.it
56
LOCALIZATION PRECISION CALCULATION
The molecule position is given by the
mean of the positions of the individual
detected photons and the error in the
localization is provided by the
standard statistical error.
x
2
s2

N
 x, y 2
a2
s 
8 s 4b 2
12


N
a2 N 2
• s standard deviation of the PSF
• N number of photons
• a effective pixel size
• b is the background noise
Mortensen’s model…
 x, y 2
Background noise
 2 a2 
 s  12  
2 2
 16  8 a b 



N
a2 N 
 9
Number of photons
…In the the bi-dimensional case:
2
Localization performances depend on SNR conditions
For practical imaging of large scattering biological samples several
limiting factors, mainly related to scattering and aberration effects,
can contribute to a decreased effective localization precision. To
consider additional errors induced in the localization process, the
precision can be redefined by considering also the standard deviation
ϑ inst of the instabilities of the system (Aquino et al. 2011).
 eff 2 
2   loc 2   inst 2
where the factor 2 takes into account for the excess noise introduced
by electron multiplying process of the EMCCD.
Thompson et al (2002)
Mortensen et al (2010)
Smith et al (2010)
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
57
EFFECTIVE LOCALIZATION ACCURACY
The localization accuracy was
determined from repeated
localization of point–like objects
in the spheroid which provide
SNR conditions typical of IML–
SPIM experiments. The
histogram of localizations was
generated by aligning different
localized clusters by the mean
value. Fitting with a gaussian
function yielded to a standard
deviation of 26 nm in the radial
direction and 60 nm in the axial
one.
The corresponding FWHM
provides an estimation of the
localization accuracy within
cellular spheroids :
FWHM x,y = 63 nm
FWHM z = 141 nm
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
58
SUPER-RESOLUTION OF CELL SPHEROIDS
IML-SPIM imaging of cell spheroids
expressing Connexin43–PAmCherry.
(b) Conventional 2D SPIM images.
(c,d) 3D IML-SPIM image.
Scale bars, 10 μm (a), 5 μm (b) and 1 μm (c,d)
Francesca Cella Zanacchi
Imaging depth: 60 μm
Nanophysics - Italian Institute of Technology
Cella Zanacchi F et al Nature Methods (2011)
www.iit.it
59
3D SUPER-RESOLUTION OF CELL SPHEROIDS
IML-SPIM image of human mammary MCF10A cell spheroids (H2B-PAmCherry)
Cella Zanacchi F., Lavagnino Z., Perrone Donnorso M., Del Bue A., Furia L., Faretta M., Diaspro A. Nature Methods (2011)
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
60
TPE-SPIM BASED APPROACH
 INDIVIDUAL MOLECULE LOCALIZATION
(PALM, GSDIM) IN LIGHT SHEET ILLUMINATION
REGIME
• Improve the resolution to 30nm
• imaging depth up to 150um
TWO-PHOTON EXCITATION IN SINGLE PLANE
ILLUMINATION MICROSCOPY ARCHITECTURE
• 3D imaging of large samples
(>20µm)
Two photon excitation
within the light sheet
illumination scheme to
reduce scattering effects
due to light-sample
interactions.
• Reduced scattering effects
• Higher penetration depth
• Less phototoxicity
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
61
LIGHT SHEET INTENSITY DISTRIBUTION IN CONVENTIONAL
AND TPE-SPIM:
1PE EXCITATION
λ=488nm
Thickness of the fluorescence intensity
distribution produced by the light sheet
illumination in single and two photon excitation .
z
y
50μm
Distortions of the intensity profile
increase in 1PE compared to 2PE,
where the gaussian shape is well
preserved also in scattering
uniform media (50mm-1).
2PE EXCITATION
λ=800nm
Contrast improvement provided by
TPE-SPIM at different imaging
depths in scattering (50mm-1) and
non-scattering media (0mm-1).
z
y
Francesca Cella Zanacchi
50μm
Nanophysics - Italian Institute of Technology
www.iit.it
62
TWO PHOTON SPIM :IMAGING OF MAMMARY CELL
SPHEROIDS
NA = 0.8
P = 450mW
λ = 710nm
Imaging of mammary cell spheroids by 2PE-SPIM
allowed to exploit the improved penetration
depth provided by two photon excitation.
10µm
MCF10A cell nuclei of cells were stained with DAPI (Invitrogen) and imaged by means of 2PE-SPIM. Images at different
depths (A) within the sample have been acquired (Zstep=1µm).
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
63
1P-2P light sheet characterization
z
z
z
y
y
z
y
y
Scale bar = 20µm
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
64
Distortions of the intensity profile increase in 1PE
compared to 2PE
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
65
Contrast improvement:
imaging depth
1PE
2PE
5mm-1
5mm-1-1
5mm
50mm-1 -1
50mm
50mm-1-1
50mm
C
Contrast improvement provided by
TPE-SPIM at different imaging depths in
scattering (50mm-1) and non-scattering
media (0mm-1).
I max  I min
I max  I min
Cella Zanacchi F et al. Plos One (2013)
Francesca Cella Zanacchi
Illumination depth = 350µm
Nanophysics - Italian Institute of Technology
Scale bar = 3µm
www.iit.it
66
Contrast improvement:
scattering coefficient
10mm-1
30mm-1
50mm-1
5mm-1
10mm-1
30mm-1
50mm-1
255
2PE
1PE
5mm-1
0
Scale bar = 5 µm
Imaging depth= 150µm
Illumination depth= 650µm
Scattering coefficient
Contrast improvement provided by
a better confinement of the excitation
volume thanks to TPE-SPIM
Lavagnino et al . Optics Express (2013)
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
67
Photoactivation confinement in
scattering samples
Uv induced photo-activation
Cella Zanacchi F (submitted),
Francesca Cella Zanacchi
Folling et al. 2007,
IR induced photo-activation
Vaziri et al 2008,
York et al. 2011
Nanophysics - Italian Institute of Technology
www.iit.it
68
Super-resolution with two photon
photoactivation in SPIM
1 µm
NB4 cell cultures
pH2AX+FLIP565
(Abberior)
λactivation=760nm
λreadout = 565nm
1 µm
Cella Zanacchi F (submitted)
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
69
Thanks to the ability to localize
single molecule…
Following single
molecule trajectories
time
Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging Andreas Gahlmann & W. E. Moerner Nature Reviews
Microbiology
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
70
Super-resolution imaging and spt Tracking
Super-resolution
image
Following dynamics
Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging Andreas Gahlmann & W. E. Moerner Nature Reviews
Microbiology
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
71
Single molecule tracking
Time lapse imaging of spatially resolved single
molecules, particles or molecular structures.
Obtain sub-pixel resolution by curve fitting to
determine centroids of single molecules
Single molecule tracking of RNA
Polymerase in E.Coli
It results in time trajectories of sub-pixel positions
of single objects that contains information about:
•
diffusion coefficient
• Velocities
• Step sizes
• Track length
• Spatial and temporal confinement
Tracks of RNA Polymerase in
E.Coli
Collaboration: Mike Heilemann, University of Frankfurt
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
72
Pulsed vs cw photoactivation
Continuous photoactivation
Pulsed photoactivation
1ms
photoactivation
500ms
1ms
500ms
1ms
photoactivation
readout
readout
Single molecule tracking of RNA
Polymerase in E.Coli
<10
10-20
Francesca Cella Zanacchi
>20
Nanophysics - Italian Institute of Technology
www.iit.it
73
Probes for Spt approaches
Probe requirements:
• very bright and stable
• Small
• One probe per molecule of interest
• Low non-specific binding
Fluorescent/
photoactivatable
proteins
Image modyfied from B. Christoffer Lagerholm
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
74
Spt approach and diffusion
Courtesy of B. Christoffer Lagerholm
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
75
Illumination architectures for sptPALM
Precisely and accurately localizing single emitters in fluorescence
microscopy Hendrik Deschout, Francesca Cella Zanacchi, Michael
Mlodzianoski,Alberto Diaspro, Joerg Bewersdorf, Samuel T Hess
& Kevin Braeckmans, Nat Methods (2014)
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
76
Spt in the light sheet illumination scheme
SptPALM in thick samples
Widefield
illumination
Light sheet
illumination
Ritter et al Plos One 2010
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
77
Spt in the light sheet illumination scheme
SptPALM in thick samples
Beads dynamically tracked within the nucleus
of a C. tentans salivary gland cell nucleus
Spille et al Optics Express 2012
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
78
Conclusions
SUPER-RESOLUTION IMAGING AND SINGLE
MOLECULE TRACKING IN THICK SAMPLES
Localization based super-resolution coupled with light sheet
illumination to perform 3D super-resolution in thick samples (up to
200μm).
 Two photon photoactivation in light sheet regime
 Single molecule tracking in thick samples
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
79
Acknowledgements
…
…THANKS TO:
E. H. K. Stelzer
Samuel T. Hess (University
of Maine, Orono)
Francesca Cella Zanacchi
Nanophysics - Italian Institute of Technology
www.iit.it
80

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