Parallel and Flexible Fluorescent Imaging using
Two-photon RESOLFT Super-resolution Microscopy
ARCHNES
with Spatial Light Modulator Control
MASACUSETS INSTITUTE
by
Yi Xue
JUL 3 0 2015
B.E., Zhejiang University (2013)
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Parallel and Flexible Fluorescent Imaging using Two-photon
RESOLFT Super-resolution Microscopy with Spatial Light
Modulator Control
by
Yi Xue
Submitted to the Department of Mechanical Engineering
on May 8, 2015, in partial fulfillment of the
requirements for the degree of
Master of Science in Mechanical Engineering
Abstract
High resolution imaging in three dimension is important for biological research. The
RESOLFT (Reversible Saturable Optical Fluorescence Transitions) fluorescent microscopy is one technique which can achieve lateral super-resolution imaging. Twophoton microscopy naturally generate high resolution in the longitudinal direction
with less background compared to single photon excitation. We combine these two
methods to realize three-dimensional high-resolution imaging. This super-resolution
method also is limited in imaging speed. We use a spatial light modulator (SLM)
as a flexible phase mask of the microscopy. It is used to compensate the system
aberration, as well as increasing the imaging speed. The parallel scanning generates
multiple super-resolution focuses as an array or in arbitrary positions by phase retrieval calculation. This microscopy combined with SLM control could applied to
high throughput 3D imaging or multiple spots tracking in high-resolution.
Thesis Supervisor: Peter T. C. So
Title: Professor
3
4
Acknowledgments
I appreciate the guide and help from my adviser, Prof. Peter So. He gave me advice
in every stage of the research and guide me to the correct direction,, based on his
profound professional experience. Also, I appreciate the help from Dr. Christopher
J. Rowlands, who is a talented researcher in So-Lab. We are in the same sub-group
called 'fluorescence'.
He especially taught me many technique details during the
experiment. I also want to thank all the other members of So-Lab and LBRC (Laser
Biomedical Research Center, MIT), and I am glad to be a member of this fantastic
group. Also, the cooperation lab, Prof. Ed Boyden in Media Lab, helped me about
the biological knowledge. Dr. Kiryl Piatkevich and Mr. Fei Chen provided the early
passage cells and plasmid DNA of GFP. I learn a lot from cooperation with them.
Finally, I appreciate my parents for their support. After these two years study in
MIT, I learned not only the knowledge but also academic attitude. I will continue
my research in So-Lab as PhD caididate. I am looking forward to the new start.
5
6
Contents
1
1.1
1.2
2
RESOLFT fluorescent microscopy . . . . . . . . . . . . . . . . . . . .
17
1.1.1
The mechanism to realize super-resolution
. . . . . . . . . . .
17
1.1.2
Reversibly photoswitchable fluorophore . . . . . . . . . . . . .
19
Multiphoton fluorescent microscopy . . . . . . . . . . . . . . . . . . .
22
25
Optical System Design
2.1
Start from the Laser
. . . . . . . . . . . . . . . . . . . . . . . . . . .
25
2.2
Shutter in the system: Acoustic Optical Modulator (AOM) . . . . . .
29
2.3
2.4
3
17
Introduction
2.2.1
Bragg diffraction and AOM
. . . . . . . . . . . . . . . . . . .
29
2.2.2
Measurement of the switching sequence . . . . . . . . . . . . .
31
Spatial Light Modulator (SLM) . . . . . . . . . . . . . . . . . . . . .
33
2.3.1
Liquid crystal retards phase in pixel resolution . . . . . . . . .
33
2.3.2
Correction of gamma curve by Young's double slits experiment
34
Polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
35
2.4.1
Vectorial Debye theory in high numerical aperture (NA) system
37
2.4.2
Experiments of donut shape focus in different polarization
39
.
.
43
SLM Phase Control
3.1
Adapted Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
43
3.1.1
Optical aberration and Zernike polynomials
. . . . . . . . . .
44
3.1.2
Critical metric in adapted optics for focus quality . . . . . . .
45
3.1.3
LabVIEW control and experiments for the aberration correction
46
7
3.2
4
5
Phase retrieval algorithm . . . . . .... .. .. . . .. . .. .. .. . 51
3.2.1
Gerchberg-Saxton iterative algorithm . . . . . . . . . . . . . .
51
3.2.2
LabVIEW control and experiment for arbitrary position spots
52
Two-photon RESOLFT microscopy imaging
57
4.1
PSF measurement: silver nanoparticle scanning
4.2
Parallel scanning: Gaussian spots array and donut shape spots array
59
4.3
Live cell two-photon imaging
60
. . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . .
Conclusion
57
63
8
List of Figures
1-1
Wavelengths, Timing, and Focusing Scheme in a Point-Scanning RESOLFT
Setup Employing Dronpa-M159T. [1]
1-2
. . . . . . . . . . . . . . . . . .
18
Parallelized scanning (RESOLFT) nanoscopy using orthogonally and
incoherently crossed standing waves [2]. The dark space between the
two standing wave overlapping works as dark center of donut shape
focus.
1-3
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(a) The absorption spectrum of Dronpa (solid line), Dronpa-V157G
(rsFastLime, dotted line) and Dronpa-M159T (dash line).
(b) The
emission spectrum of Dronpa, Dronpa-V157G and Dronpa-M159T [3].
1-4
19
21
(a) The fatigue curve of Dronpa-M159T, under the condition of 1409=2.1
kW/cm 2 , 1491 =1.5kW/cm 2. It can undergo more than 6000 switching cycles before bleaching down to <50% of the initial signal.
(b)
switching-off time comparison among Dronpa, Dronpa-V157G and DronpaM159T. a. b. enlarged figure during one unit cycle of Dronpa-V157G
and Dronpa-M159T respectively. [1,31 . . . . . . . . . . . . . . . . . .
1-5
21
Schematic diagram of the photoswitching of Dronpa. B, deprotonated
form (ON-state); D, an unknown dark state; A 1 , the protonated form
(OFF-state) that originally exists in the sample; A 2 , the protonated
form that is formed by the photoswitching; I, a nonfluorescent intermediate. The photoswitching pathway from B to A 2 is shown by blue
arrows, and that from A 2 to B is shown by red arrows [4J.
9
. . . . . .
22
*2
<gh-photon and two-photon resolution comparison in wide-field mi,rcrscopy (conventional) and confocal microscopy (a) Intensity of the
nage of a thin fluorescent edge (lateral resolution); (b) Intensity of the
&xial response to a thin fluorescent sheet; (c) Intensity of the image of
a thick fluorescent edge; (d) Intensity of the axial response to a thick
; uorescent
2-i
layer. [5] . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
Jhe experiment setup for parallel two-photon RESOLFT microscopy.
Mode-locked Ti:Sapphire laser (Spectra-physics, Tsunami) provides the
source for 'turn-off' light and excitation light. AOM (acoustic optical modulator, ISOMET) synchronized with LED (Thorlabs, M405L2,
center wavelength 405rnm) to control 'turn-on' and 'turn-off' time se-
quence. SLM (Spatial light modulator, HOLOEYE PLUTO) provides
the phase control of the system.
Other main components:
CMOS
(Point Grey, Flea3 USB), OL (objective lens, Zeiss A-Plan, x100, NA
1.25), DM: dichroic mirror, piezo stage (Queensgate, NPS-XY-100A).
2-2
26
The mode-locked laser pulse reflects back and forth between the mirrors
of the resonator. Each time it reaches the output mirror it transmits
a short optical pulse.
The transmitted pulses are separated by the
distance 2d and travel with velocity c. The switch opens only when
the pulse reaches it and only for the duration of the pulse. The periodic
pulse train is therefore unaffected by the presence of the switch. Other
wave patterns, however, suffer losses and are not permitted to oscillate. [61 28
2-3
The common optical path of Ti:Sapphire laser. (Spectra-Physics manual) 29
2-4
Bragg diffraction: an acoustic plane wave acts as a partial reflector of
light (a beamsplitter) when the angle of incidence 0 satisfies the Bragg
condition. [6]
2-5
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . .
The LabVIEW control of three channel digital output: infrared channel, UV channel and camera. . . . . . . . . . . . . . . . . . . .. .. . 31
10
30
......36
2-6
Synchronized time sequence of infrared beam and UV light. Channel
1 is the infrared beam while channel 2 is the UV light. Figure (b) is
the enlarged period of figure (a). The period time is 12ms. Figure (b)
shows more obvious switch-off delay (<500us). . . . . . . . . . . . . .
2-7
32
Molecular orientation of a liquid-crystal cell (a) in the absence of a
steady electric field and (b) when a steady electric field is applied. The
optic axis lies along the direction of the molecules. [6] . . . . . . . . .
2-8
33
The Gamma curve of the SLM under the experiment condition. LUT:
look-up table. This curve represents the relation between gray level
and the voltage on the liquid crystal.
2-9
. . . . . . . . . . . . . . . . . .
34
Experiment measured interference fringes shift in the Young's double
slits experiment, after the gamma-curve correction. The fringes shift
because of the 27r change of phase in one slit. The figure shows each one
fringe linearly shifts one period distance during the 2z- phase change.
35
2-10 The key polarization control optical component in the system. HWP:
half wave plate (thorlab). GT: Glan-Thompson prism. QWP: quarter
wave plate.
. . . . . . . . . . . . . . . . . . . . . . . . . .
2-11 The phase change of s-polarized beam and p-polarized beam in reflection.The reflective media reflective index n>1.
angle.
0
B
is the Brewster's
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
36
2-12 Diffraction of a converging spherical wave at a circular aperture: No-
tation. [7]
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
37
2-13 Normalized intensity of polarized light with a vortex 0-2wr phase plate
at the focal spot in the horizontal (XY) and longitudinal (XZ) planes.
Various polarizations are considered: linear, circular (right-handed and
left-handed), right-handed ellipse (with X and Y as the long axis).
Intensities of 0 and 1 correspond to black and white, respectively. The
axis units are in wavelengths. [8]
. . . . . . . . . . . . . . . . . . . .
39
2-14 The vortex 0 - 27r phase and corresponding donut shape focus. The
center grayscale for the vortex is 58 and it is a left-hand vortex.
11
. . .
40
2-15 (a) The focus of right-hand polarized beam modified by left-hand vortex 0 - 27r phase plate.
(b) The focus of left-hand polarized beam
modified by left-hand vortex 0 - 27r phase plate.
under the same color bar scale.
Two pictures are
. . . . . . . . . . . . . . . . . . . . .
41
2-16 Donut shape focus (a) before polarization modify and (b) after polarization modify. The two images are of the same colorbar scale. The
left figure is the same as figure 2-14 (b).
. . . . . . . . . . . . . . . .
3-1
LabVIEW control panel for system aberration compensation. ......
3-2
The focus spots under different degree of astigmatism (Zernike mode
47
(-2, 2), Table 3.1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3-3
The sharpness, brightness, and the combined metric v.s.
41
48
bias (b)
curves. The combined metric curve will change if change the 'weight'
between sharpness and brightness, depending on which one is major.
3-4
The focus spots under fixed astigmatism (b = -0.9)
compensation
while testing different degree of coma (Zernike mode (3, 1), Table 3.1).
3-5
48
The sharpness, brightness, and the combined metric v.s.
49
bias (b)
curves. In the second round testing, the sharpness and brightness have
similar trend, which means the aberration of the system converge to a
'local minimize' point under these two phases compensating.
3-6
. . . . .
49
(a) The original Gaussian beam focus without aberration compensation to the system. (b) The focus after aberration compensation. (c)
The compensated phase adding to the SLM, which is sum of negative
astigmatism and positive vertical coma. . . . . . . . . . . . . . . . . .
3-7
The donut shape focus under Zernike mode (-1, 1) and bias b = -1
50
: 1
modify, selectively shown every other figures. When b = 0.5, the donut
shape is the most uniform one . . . . . . . . . . . . . . . . . . . . . .
3-8
The donut shape focus under Zernike mode (3, 3) and bias b = -1
: 1
modify, selectively shown every other figures. . . . . . . . . . . . . . .
12
50
51
3-9
(a) The original donut shape focus without aberration compensation
to the system. (b) The focus after aberration compensation. (c) The
compensated phase adding to the SLM, which is sum of positive shift
and a negative trefoil. (a) and (b) are under the same color scale.
3-10 The principle of the iterative Fourier transform algorithm.
51
. .
191 .....
52
3-11 The control panel of flexible position focuses. The aimed focuses are
selected from the 'new picture' Window . . . . . .
. . . . . . . . . . .
53
3-12 . (a) The focuses in random positions, selected from the control panel.
(To easy observation, enlarged the focuses area 10 times, each spot
here should be one pixel size in the calculation.) (b) The phase mask
calculated by G-S algorithm. (c) The camera image of the phase (Rhodamine 123 solution as sample), which is the same as the aimed focuses. 54
3-13 The flexible position of donut spots, for multiple super-resolution particle tracking. The donut shape in the 'input image' is enlarged. Like
the Gaussian focuses, one pixel spot to indicate the coordinate value
of the position is sufficient.
4-1
. . . . . . . . . . . . . . . . . . . . . . .
55
nanoparticle-scanned PSF of the setup. (a) Gaussian focus. (b) Donut
shape focus, with the same intensity scale. (c) Overlay of (a) and (c)
to show the colocalization (pseudo color).
4-2
. . . . . . . . . . . . . . .
58
The PSF comparison between single photon excitation (blue dash line)
and two-photon excitation (red solid line). (a) The Gaussian focus. (b)
The donut shape focus.
4-3
. . . . . . . . . . . . . . . . . . . . . . . . .
59
5x5 spots array, two-photon excitation using Rhodamine 123 sample,
detected by the camera. The period of grating on SLM gradually zoom
in, and the corresponding image 'zoom out'.
4-4
. . . . . . . . . . . . . .
The 5x5 donut spots matrix for parallel super-resolution imaging.
13
.
.
60
60
4-5
HEK 293 cell labelled by Dronpa-M159T on tubulin.
(a) The field
of view (FOV) of the image is 60um x 60um. The scanning step is
500nm. (b) The finc scanning image of the top right region of (a), the
FOV is 30um x 30um. The scauning step is 200nm. . . . . . . . . . .
4-6
61
The position change of live cell. (a) 1st time scanning, FOV is 80um x
80um, and step size is lum. (b) 2nd time scanning for the right bottom
region of (a), noticing the bottom cell orientation changed during this
process. The FOV is 30um x 30um with 200nm step size. ......
4-7
62
HeLa cell labeled by Dronpa-Tubulin. The enlarged region is increased
contrast by ImageJ. The cross profile of the red line region is shown
below. The finest structure we can distinguish is about 400nm. The
image is 50um x 50um, with 200nm scanning step.
14
. . . . . . . . . .
62
List of Tables
1.1
Comparison of common used photoswitchable fluorophore . . . . . . .
19
3.1
Low-order Zernike mode for optical aberration . . . . . . . . . . . . .
44
15
16
Chapter 1
Introduction
High resolution imaging in three-dimension of large area for live specimen is important. For example, we are not only interested in imaging the whole tumor but also
want to observe the metastasis of each tumor cell; we are not only want to take image of single synapse event but also want to observe the synapse circuit in a large
scale [10]. Sometimes the specimen is sparse labeled, high-resolution image only in
the labeled area will be more efficient [11]. Also the requirement for multiple particle tracking simultaneously of high-resolution is important to know the relationship
between different particles [121. Thus 3D high-resolution high-speed fluorescent microscopy with flexibly controlled imaging area is a good choice to facilitate the above
biological research.
1.1
1.1.1
RESOLFT fluorescent microscopy
The mechanism to realize super-resolution
There are many methods to achieve super-resolution imaging. One type of methods
is based on single molecule imaging, such as STORM (STochastic Optical Reconstruction Microscopy)
113] and PALM (PhotoActivated Localization Microscopy) [141;
the other type of methods uses spatial patterned imaging, such as STED (Stimulated emission depletion microscopy) [15], RESOLFT (Reversible Saturable Opti-
17
cal Fluorescence Transitions) [l and SSIM (Saturation Structure Illumination Microscopy) [161. RESOLFT microscopy is one super-resolution technique invented by
Hell's group [1]. The technique use reversibly photoswitchable fluorophores to exploit
long-live dark-state and fluorescent state. Because the comparably long 'on states'
and 'off status' than STED microscopy, the light intensity to achieve sub-diffraction
focal spot is several orders lower. In the imaging process, each scanning point is
'turned on' first, and the periphery of the spot is 'turned off' by a donut shape spot,
then the same position is illuminated by the excitation b:eam and only the residue part
in the center is excited and emits fluorescence (see figure 1-1). This 'on-off-excitation'
cycle is played in each scanning point until the whole field of view is imaged and each
cycle takes about millisecond
[1].
1. kWlcm 2
1
O FF
fluorescence
405nm
3.8 kWIcm
i kwM
Figure 1-1:
2
250nm
Wavelengths, Timing, and Focusing Scheme in a Point-Scanning
RESOLFT Setup Employing Dronpa-M159T. [1]
To make the RESOLFT imaging more efficient, parallel RESOLFT imaging method
is published in 2013 using two incoherently superimposed orthogonal standing light
waves [2] (see figure 1-2). This parallel scanning method could take a 120um by
100um super-resolution image within one second. However, this method did not consider about depth resolution, while it used sCOMS camera. So the depth resolution
is the same as single photon wide-field imaging.
18
Only First Order Selected
Back
*7)Focal Plane
Polarization
direction7
GRID
PBS
LENS
LENS
Focal Plane
Figure 1-2: Parallelized scanning (RESOLFT) nanoscopy using orthogonally and
incoherently crossed standing waves [2]. The dark space between the two standing
wave overlapping works as dark center of donut shape focus.
1.1.2
Reversibly photoswitchable fluorophore
For RESOLFT fluorescent microscopy, reversibly photoswitchable fluorophore is one
of the key part of the technique. Single point scanning RESOLFT much slower than
STED is because the photoswitching time is much longer than fluorescent lifetime.
Reversible photoswitching of individual molecules has been demonstrated for a number of mutants of the green fluorescent protein.
The limited number of switching
and switching speed are always concerned. At the same time, the quantum yield and
absorption coefficient are important optical factors of the fluorophore. Recently, red
color reversible photoswitching fluorophore is also designed [17,18], which extended
the available spectrum range and made multi-color RESOLFT possible. However, the
red reversible photoswitching fluorophore is not as mature as GFP-like switchable fluorophore. The switching speed, absorption coefficient and quantum yield need further
research. For our experiment, because of the laser wavelength we choose (in Chap.
2), we focus on green color reversible fluorophores.
Table 1.1 shows the compari-
son of characteristics of several commonly used reversible fluorophores in RESOLFT
experiments.
Table 1.1: Comparison of common used photoswitchable fluorophore
19
Dronpa
Dronpa-M159T
rsEGFP
rsEGFP2
Absorption maximum (nm)
503
489
493
478
Emission maximum (nm)
522
515
510
503
95000
61732
47000
61300
0.85
0.23
0.36
0.3
Switch-off half-time t,/2off (s) $
263
0.23
~20
5
Off-state relaxation half-time t1/2rela (min)
840
0.5
180
20
7
6000
1100
2100
Fluorophore
Molar absorption coefficient (At - cm-')
Quantum yield
Fatigue times
(<bFL)
t$
f Fatigue time means the number of switching cycles to bleach to 50% of the initial
fluorescence in the on-state.
The switch-off half-time and fatigue switching times depend on experiment conditions, such as the intensity of illumination light. The number is scale to similar
intensity scale according to the references [1-3, 19,20].
According to Table 1.1, Dronpa-M159T and rsEGFP2 have similar performance,
but Dronpa-M159T can be switched three times more than rsEGFP2. Also, DronpaM159T has higher contrast (intensity of dark state/ intensity of bright state) [21, we
choose it as fluorophore in our experiment. Dronpa-M159T is one mutant of Dronpa,
in which Met 5" side chain is replaced by a smaller threonine residue. Because Met"
interferes with the movement of a six-ring structure upon a light-induced cis-trans
isomerization
13], changing it with smaller structure reduces the switch-off half-time.
Figure 1-3 and figure 1-4 show more details of Dronpa M159T.
The mechanism of photoswitching for Dronpa (mutant Dronpa is similar) is shown
in figure 1-5. Photoswitching means Dronpa has two states: 'ON-state' and 'OFFstate'. When the Dronpa is in 'ON-state', it can be excited and emit fluorescence.
Otherwise, it cannot be excited by the same light. From the chemical view, these two
states actually corresponding to two structure. In 'ON-state', Dronpa is deprotonated;
in 'OFF-state', it is protonated. Naturally, pH change also can lead to protonation
(acid) or deprotonation, so the photoswitching of Dronpa can be controlled by pH
also.
The 'turn-on' process is as following: the Dronpa is originally protonated in
A 2 , illuminated by UV light at 390nm.
Electron will be excited, and it has two
20
31,00.8r
0,6-
0*
0.4.
I.
0.2.
0.0-
r
250
wavelength [nm]
540 5;0 5;0 600 6
wavelength [n]
(a)
(b)
350
300
450
400
460
600
50
S0
480
50
520
0640
Figure 1-3: (a) The absorption spectrum of Dronpa (solid line), Dronpa-V157G (rsFastLime, dotted line) and Dronpa-M159T (dash line). (b) The emission spectrum
of Dronpa, Dronpa-V157G and Dronpa-M159T [3].
a
IF U
V
c 0.75.
W,
U
**
b
U
mu
U.
IF
mu
60.50-
c4M0
'a
0
0 2000
0.25.
[Dints
0
2000
4000
C
s600
woUU
Wo
Cydles
t [s]
15W
(b)
(a)
Figure 1-4: (a) The fatigue curve of Dronpa-M159T, under the condition of I405=2.1
kW/cm 2 , 14 91=1.5kW/cm 2 . It can undergo more than 6000 switching cycles before
bleaching down to <50% of the initial signal. (b) switching-off time comparison
among Dronpa, Dronpa-V157G and Dronpa-M159T. a. b. enlarged figure during one
unit cycle of Dronpa-V157G and Dronpa-M159T respectively. [1,3]
choices: one is directly from excited state of protonated form to the excited state of
deprotonated form, called excited state proton transfer (ESPT); the other is falling to
the ground state of deprotonated form (red line in figure 1-5). Because the emission of
fluorescence as excited by 390nm is extremely low (<0.01), it means most electrons go
through the second way. The quantum yield of switch-on is
the 'turn-off' and excitation process are competitive.
21
-
0.37. In 'ON-state',
After Dronpa is excited by
488nm light, 65% molecules perform fast on-off blinking (~
show medium-long off-time (order of tens millisecond).
OqA
ims), and 35% molecules
The fast on-off blinking is
.ttributcd to transitions into triplet states with a triplet lifetime of 1.2ms.
The
medium-long transition may be attributed to an unknown dark state D. The switch-off
time is not related to excitation intensity [4]. In RESOLFT experiment, normally use
longer time illumination to realize switching-off, but using shorter illumination time
to realize excitation [2]. But it is not necessary to be higher intensity to switching-off
then fluorescent excitation [1,21.
ESPT
0.371
65%
'*OSW35
3.2 x10-4\
W
3.6 ns
Aslow
(photoswitched
protonated form)
%
^kB
0.85
(non-fluorescent
intermediate)
B
Reaction Coordinate
Figure 1-5: Schematic diagram of the photoswitching of Dronpa. B, deprotonated
form (ON-state); D, an unknown dark state; A 1 , the protonated form (OFF-state)
that originally exists in the sample; A 2 , the protonated form that is formed by the
photoswitching; I, a nonfluorescent intermediate. The photoswitching pathway from
B to A 2 is shown by blue arrows, and that from A 2 to B is shown by red arrows [4].
1.2
Multiphoton fluorescent microscopy
To improve longitudinal resolution, of course 3D super-resolution is one choice. However, the setup for 3D super-resolution imaging, especially RESOLFT method, is very
22
complicate. On the other hand, multiphoton imaging can provide high-resolution in
Z-axis. Multiphoton microscopy is widely used in biological tissue imaging, including
neuroscience, embryology and oncology [21].
It enables noninvasive study of bio-
logical specimens in three dimensions with diffraction limited resolution, especially
much higher longitudinal resolution compared to single photon wide-field microscopy.
Multiphoton microscopy also reduces specimen photodamage as well as enhances
penetration depth for thick tissue. Recently, multiphoton imaging is used to do in
vivo imaging of subcortical structures within an intact mouse brain [221, as well as
blood flow and neurovascular coupling in the brain [23]. Also, parallel multiphoton
microscopy is realized to increase the imaging speed [24], which used grating to generate a 8 x 8 spots array and collected data by a 8 x 8 PMT array. Recently, spots
array containing larger number of spots is used in multiphoton microscopy, which
can achieve 1kHz throughout rate [25]. Multiphoton microscopy could also combine
with other techniques, which melt the advantages of both methods. For example,
two-photon excitation STED microscopy improved the spatial resolution of standard
two-photon microscopy by a factor of four to six times, which is used to image morphology of dendritic spines and microglial cells of brain slice [26].
One advantage
of two-photon microscopy is that it has naturally 3-D high resolution, especially in
the Z-axis direction, compared to single photon microscopy.
Figure 1-6 shows the
comparison of point spread function (PSF) of single photon excitation and PSF of
two-photon excitation in wide-field microscopy and confocal microscopy, respectively.
The other advantage of two-photon microscopy is that the penetration depth is larger
than that of single photon excitation.
Because two-photon microscopy uses longer
wavelength, according to the Beer-Lambert law, the light is less attenuated by the
specimen. Thus, two-photon microscopy has advantage of imaging thick specimen or
biopsy.
Two-photon iPSF (intensity Point Spread Function) is to the square of singlephoton iPSF. For a 4-f system, the intensity distribution of object can be expressed
as
23
2
(
Io(ri) = Ihi(MAYir1)
*"oitm- otm
cordca
oto
-
ca
0.80-
oovn4onlWQphOtS
\.
0.2
-1
0.6
012
0
4
0
1
5
I0
to1
15
V
U
(a)
(b)
20
2
0
~hc
OA
0.2
0
.1 0
-6
0
10
S
(d)
(C)
Figure 1-6: single-photon and two-photon resolution comparison in wide-field microscopy (conventional) and confocal microscopy (a) Intensity of the image of a thin
fluorescent edge (lateral resolution); (b) Intensity of the axial response to a thin fluorescent sheet; (c) Intensity of the image of a thick fluorescent edge; (d) Intensity of
the axial response to a thick fluorescent layer. [51
where M1 is a matrix including demagnification factors for the objective, and hi
is the 3-D amplitude PSF for the objective lens. Then after the collector lens with
transfer function h 2 and M2 , the image intensity within the detector area D is
hi(r)
=
1hi(Mr) 2 " [h2 (r)|2
D
(
")]
(1.2)
n=1, 2, and 3 correspond to single-photon, two-photon, or three photon excitation,
respectively. From the iPSF equation 1.2, we know that the two-photon iPSF is square
of the single-photon iPSF [27].
24
Chapter 2
Optical System Design
The microscopy is a combination of two-photon microscopy and RESOLFT superresolution microscopy. The experiment setup is shown in figure 2-1. The specimen
is illuminated by the UV 'turn-on' light first. Then, the periphery area of the spot
is 'turned-off' by the donut shape two-photon light. Finally, the residue center area
which is still in 'on-state' is excited by the two-photon Gaussian spot and emit fluorescence. To realize this process, we need to design the optical section of the system.
In this chapter, we will talk about the function of each key optical component, including the Ti:Sapphire laser and LED, the acoustic optical modulator (AOM), the
SLM, and polarization control components. We will show how these components help
us realize the aim of the setup.
2.1
Start from the Laser
The theory of two-photon absorption and emission process is developed by Maria
Goppert-Mayer around 1930 [27]. In her work, the transition probability of a twophoton electronic process is quadratically related to the excitation light intensity.
The process involves the interaction of two photons and an atom via an intermediate
'virtual' state. The first photon induces the transition from the ground state to the
virtual state, and the second photon induces the transition from the virtual state to
the final excited state. Because the possibility of two-photon absorption process is
25
AOM Filter & collimate
DMV
Tub lens
Filter & collimate
OL
Piezo stage
Figure 2-1: The experiment setup for parallel two-photon RESOLFT microscopy.
Mode-locked Ti:Sapphire laser (Spectra-physics, Tsunami) provides the source for
'turn-off' light and excitation light. AOM (acoustic optical modulator, ISOMET)
synchronized with LED (Thorlabs, M405L2, center wavelength 405nm) to control
'turn-on' and 'turn-off' time sequence. SLM (Spatial light modulator, HOLOEYE
PLUTO) provides the phase control of the system. Other main components: CMOS
(Point Grey, Flea3 USB), OL (objective lens, Zeiss A-Plan, x100, NA 1.25), DM:
dichroic mirror, piezo stage (Queensgate, NPS-XY-100A).
26
very low, it requires high intensity laser to achieve a measurable effect. In two-photon
absorption process, the number na of photons absorbed per fluorophore per pulse can
be expressed as
n =
po2a"r p
2
A2
2
(2.1)
.
2hcA
where po is the laser power, 6 is the two-photon absorption cross-section (~1058m4
.
s),
Tr
is the pulse duration (~100fs),
fp
is the repetition rate (~80MHz), A is
the numerical aperture (~1.4) [281. Thus, the photon absorbed in two-photon process
requires high peak power excitation.
For optical domain, the lack of high peak power laser blocks the way.
After
the femtosecond laser is developed in 1980s, Denk, Strickler and Webb demonstrated
two-photon laser scanning fluorescence microscopy in 1990 [28]. They used a collidingpulse, mode-locked dye laser which produced a stream of pulses with a pulse duration
of about 100fs at a repetition rate of about 80MHz. In 1990s, Ti-sapphire laser became
an optimal for two-photon excitation microscopy until now. The Ti-sapphire lasers
are based on Kerr lens mode locking technique to generate femtosecond pulse. Kerr
lens effect is caused by inhomogeneous refractive index change of Ti-Sapphire crystal
under strongly focused beam. Combined with an aperture on the focus of Kerr lens,
only high intensity pulses experience low loss [27].
This is one method to achieve
mode-lock. A laser can oscillate on many longitudinal modes, with frequencies that
are equally separated by the intermodal spacing VF = c/2d where d is the length of
resonator. The modes can be coupled and their phase can be locked together. If M
modes in the locked phase, the pulse length dpise = 2d/M, and the peak intensity is
I = M2| A1 2
=
M 2 1, I is the mean intensity [6]. Thus, the larger M, the higher peak
intensity, and the shorter pulse duration in time. On the other hand, the broader
envelop in spectrum domain.
In a typical Ti-Sapphire laser resonator, when the modes are not locked, the laser
is equal to a continuous wave (CW) laser with low intensity (because no Kerr lens
effect, the loss is high).
Normally the optical system has positive 'chirp' (normal
dispersion), which means the speed of longer wavelength wave is slower. The relation
27
Optical
switch
2d
Reoator
Figure 2-2: The mode-locked laser pulse reflects back and forth between the mirrors
of the resonator. Each time it reaches the output mirror it transmits a short optical
pulse. The transmitted pulses are separated by the distance 2d and travel with
velocity c. The switch opens only when the pulse reaches it and only for the duration
of the pulse. The periodic pulse train is therefore unaffected by the presence of
the switch. Other wave patterns, however, suffer losses and are not permitted to
oscillate.
[6]
between light speed (group velocity) and wavelength is
-nvP=
+
(-)
dAI
.
(2.2)
In normal dispersion region, dn/dA < 0 129]. However, prisms are used to achieve
negative chirp inside the Ti-Sapphire laser resonator, in order to broaden the spectrum
of pulse. For prism, shorter wavelength light has faster speed but also travels longer
distance inside prism, so the chirp can be compensated by a set of prisms. According
to the above mode-lock analysis, when the spectrum of pulse is extended, the pulse
length is shorter in time domain, meaning more mode is phase locked. Figure 2-3
shows the typical optical path inside the resonator of Ti:Sapphire laser. The laser
in our lab is manually mode-locking. That is, we adjust the horizontal mirror and
vertical mirror to achieve the maximum intensity, and the slit is to select wavelength,
as well as a set of prisms to compensate 'chirp'. Once we lose the mode-lock (which
happens often), we need to adjust the laser again. During adjusting, the mirror will
be rotated, so the output beam will have a slightly shift, causing the realignment
of all the following optical path. The laser also needs to be purged by filtered and
dried nitrogen gas, which can eliminate the typical problems associated. with dust and
28
Pmdodl e
HRPr
M
Tug
Beam
'Splitter
otput
Brewster
P2
Owl(n
M3
1er
Blewster
M4 .PZT
(Opfional)
Pumpv-1
2
Tirwptie
MCde9 305
Rod
Duver Eloctronics
-AOM
ResiduvJ
HR
searoumnP
Opfional
Model 3930
Lok-to-Clock Elecitica
Figure 2-3: The common optical path of Ti:Sapphire laser. (Spectra-Physics manual)
contamination, and tuning discontinuities caused by oxygen and water vapor. The
absorption of oxygen and water vapor is right between 900nm to 1000nm most serious,
which is right in our working wavelength range (see fluorophore selection table 1.1).
2.2
Shutter in the system: Acoustic Optical Modulator (AOM)
This section of chapter talks about the time-sequence control of different light path
and camera.
We use an AOM as shutter of the infrared beam, and the LED is
triggered by a TTL signal. The camera is triggered by a falling edge. The change
between Gaussian focus and donut shape focus is controlled by SLM. The specimen
scanning is operated by the piezo stage. These signals are synchronized by NI 6009
card and LabVIEW control. The SLM and piezo stage have commercial VI, so in this
section we focus on the home-made control of the AOM, the LED and the camera.
2.2.1
Bragg diffraction and AOM
For a RESOLFT microscopy, we need to turn-on and turn-off fluorescence in a certain
time sequence. Thus, it is necessary to synchronize different beams. The system is
controlled by LabVIEW program. In hardware, Acoustic Optical Modulator (AOM)
29
is one
the synchronize part, which is the shutter for turni-off and excitation light
from the Ti-Sapphire laser.
The 1.OM is based on the theory of acousto-optic effect. An acoustic wave creates
a perturbation of the refractive index in the form of a wave, thus the medium becomes
a dynamic graded-index medium
[6]. The key component of our AOM is a Bragg cell,
in which the medium reflective index is compressed periodically with a harmonic
sound wave. This structure is equal to a grating with period of sound wavelength A.
If the incident angle satisfies the Bragg condition, the +1 or -1 order of diffraction
light intensity will be maximized. Without the acoustic wave, the light will propagate
along the zero order direction.
Diffracted light
Incident fight
A
~~
Sound
Transmitted light
Figure 2-4: Bragg diffraction: an acoustic plane wave acts as a partial reflector of
light (a beamsplitter) when the angle of incidence 0 satisfies the Bragg condition. [6]
In figure 2-7, the Bragg angle is sin 6 = 2A
The AOM used in our experiment
(ISOMET, VA) has deflection efficiency about 60% in the infrared wavelength. Under designed wavelength, the deflection efficiency can reach 90%. AOM efficiency is
related to the wavelength of incident light and RF power. The modulation frequency
of AOM can reach MHz.
Another choice for fast shutter is electro-optics modulator (EOM), such as Pockel
cells. For some material, the reflective index changes under electric field, so the phase
of incident light will change correspondingly. Thus, the polarization of incident light
change. The operation frequency of Pockel cell is about kHz, and the transmission
30
for the designed wavelength can over 99%.
If the system concern more intensity
than operation frequency, mechanical shutters are more suitable choice.
The fast
mechanical shutters from Uniblitz or Newport can reach 100Hz frequency, the Uniblitz
shutter can reach 400Hz burst frequency of operation.
2.2.2
Measurement of the switching sequence
In the experiment, we use NI USB6009 card to synchronize switching signals. Both
digital and analog output work, but analog output always has about ims delay between different channel (unknown reason). Digital output doesn't have the artificial
delay and also has more channels, so we choose to use digital output. Figure 2-5 shows
the LabVIEW control of the synchronization. The first channel is for the infrared
beam, and the second channel is for the UV light. The third channel is for the camera
trigger. The illumination length can change individually, while the period time of all
the three channels also can adjust together.
Period
[RriAsinle connection wire to the poft~ arthe
kotae oUtpuforTL WfV0*60n WV.
&----A
Ingo-
ntrror
ou
Iftm,
Figure 2-5: The LabVIEW control of three channel digital output: infrared channel,
UV channel and camera.
The maximum trigger frequency of LED is 1kHz, while AOM switching frequency
is much higher.
The limit of system frequency is from the camera.
31
The camera
maximum can achieve 120Hz, and it is controlled by rolling shutter. The minimize
line exposure time is 8us. So for a 1280 x 960 image, the minimum exposure time
is 8ms (corresponding to 120Hz). When we decrease the period of three channel to
about 10ms, we can observe the blinking of focus because the rolling shutter effect.
For the outside triggered mode of camera, the maximum frequency is 60Hz. Also,
the image acquisition time relates to the fluorescent signal intensity, which decide the
minimum exposure time of the camera.
Figure 2-6 shows the switching sequence of infrared beam and UV LED, which
is collected by photodioded and oscilloscope. This is for the basic two-photon microscopy imaging for photoswitchable fluorescent labelled specimen. Channel 1 is the
AOM controlled infrared beam from Ti:Sapphire laser, and channel 2 is the LED
light. From the figure 2-6, both channels have switch-off delay, and the UV channel
has longer delay time. However, both of them are within 500 us, so it won't have
too serious influence. Possibly caused by the frequent turn-on and turn-off, the UV
LED light intensity is not stable within the 'on' period, but the slightly intensity drop
won't influence switching on the fluorophore.
(a)
(b)
Figure 2-6: Synchronized time sequence of infrared beam and UV light. Channel 1 is
the infrared beam while channel 2 is the UV light. Figure (b) is the enlarged period
of figure (a). The period time is 12ms. Figure (b) shows more obvious switch-off
delay (<500us).
32
2.3
2.3.1
Spatial Light Modulator (SLM)
Liquid crystal retards phase in pixel resolution
We use a reflection style phase only spatial light modulator (HOLOEYE Photonics
AG, Germany) in the setup.
The key part of the SLM is a liquid crystal, which
molecule orientation changes under a steady electric field, resulting in the modulation
of light phase.
x
2
E
(a)
(b)
Figure 2-7: Molecular orientation of a liquid-crystal cell (a) in the absence of a steady
electric field and (b) when a steady electric field is applied. The optic axis lies along
the direction of the molecules. [6]
Without external electric field, for a linear polarized light in x or y directions
(parallel and perpendicular to the molecular direction), the wave retardation is P =
27r (ne - n0 ) d/A. When apply the electric field along z direction, the resultant electric
force tends to tilt the molecule along z direction, but the elastic forces at the surfaces
of the glass plates resist this motion. But when the voltage is larger than a certain
threshold, most molecules are tilted except those nearing the glass surface.
relation between tilt angle and voltage is expressed as
aVep
- -V
0
2ta
1 V2> V
x
The
161:
(2.3)
_V)
0
V < V
where V is the critical voltage, and V is a constant. The reflective index is a function
of the tilt angle 0
1
n2(0)
_
cos 2 0
n.
33
sin 2
(2.4)
Thus, the retardation becomes I = 27r (n(O) - n,) d/A. It shows the phase retardation
and the electric field voltage is not linear relation. SLM uses gamma-curve to relate
grayscale with the actual voltage on each pixel.
2.3.2
Correction of gamma curve by Young's double slits experiment
The electric voltage added on the SLM is controlled by gray-scale of image showed
on SLM screen. The relation curve between gray-scale and phase retardation called
'gamma-curve' of the SLM. Gamma-curve relates to wavelength, which can be measured and calculated by Young's double slits experiment. In the experiment, SLM
screen is divided into two regions: the gray-scale of one region is zero, as reference
beam; the gray-scale of the other region is changing from zero to 255, as adding different phase shift to one beam branch of Young's double slits experiment. As the phase
difference between the two branches changing, the interference fringes also shift. After measure the fringes shift, the new gamma-curve corresponding to 27r phase change
for the certain wavelength can be calculated by the software PhaseCam. Reload the
new gamma-curve to the SLM and do the Young's double slits experiment again,
adjusting the gamma curve until achieving linear 27r phase. change.
16---
-
-- '------------
12D
-<0G
A00 A
O
..
40&
5M'0'0-A
8096
12
14
Figure 2-8: The Gamma curve of the SLM under the experiment condition. LUT:
look-up table. This curve represents the relation between gray level and the voltage
on the liquid crystal.
At the same time, SLM is also polarization sensitive, which can only modify light
34
polarized in the same direction of liquid crystal molecule direction. In our setup, the
SLM axis is in the horizontal direction. So we keep the incident light is horizontally
linear polarized. This is the reason we discard the design of perpendicular incident
with polarized beam splitter (PBS) and quarter wavelength plate, because circular
polarized beam cannot be sufficiently modulated, but introduce distortion.
If use
non-polarized beam splitter, 75% of energy will lose during this process, so we choose
incident with an angle without beam splitter.
Figure 2-9: Experiment measured interference fringes shift in the Young's double slits
experiment, after the gamma-curve correction. The fringes shift because of the 27r
change of phase in one slit. The figure shows each one fringe linearly shifts one period
distance during the 27r phase change.
Because the SLM has a layer of glass on the top surface of liquid crystal, this layer
of glass may introduce interference pattern if the incident angle is too large. That
is why we discard the design of 45 degree incident (incident light and reflective light
are perpendicular to each other). After the trade of optical path length and incident
angle, we choose about 10 degree illumination. At the same time, we add two sets of
relay lenses to minimize the phase curvature because of Fresnel diffraction.
2.4
Polarization
The polarization control components are shown in figure ??. The part (a) is to make
sure the incident light to the SLM is the same direction as the axis of liquid crystal
35
molecule in the SLM, which is along the horizontal direction. So, after the part (a)
modulate, the light is purely p-polarized (GT extinction ratio> 100,000:1). According
to figure ??), we know that the phase of p-polarized light after reflection may not
change or change ir degree. Neither case lead to polarization type or direction change.
So the mirrors in the system won't influence the polarization.
HWP
SLM
HWP
GT
QWP
(b)
(a)
Figure 2-10: The key polarization control optical component in the system. HWP:
half wave plate (thorlab). GT: Glan-Thompson prism. QWP: quarter wave plate.
9, 9-O,
0
0
9- Oi
Figure 2-11: The phase change of s-polarized beam and p-polarized beam in reflec0
tion.The reflective media reflective index n>1. B is the Brewster's angle.
However, the SLM provides different phase retardant in different pixel. Thus, the
polarization changed after the SLM. For example, if the SLM works as a vortex 0 -27r
phase plate, the polarization of the reflective light becomes similar to 'azimuthally
polarized beam'
1301
(not exactly the same). The combination of HWP and GT in
section (b) is to get a pure linear polarized beam with maximum intensity. Adding
GT in the system is a trade-off: GT will provide a linear polarized beam rather than
an 'azimuthally polarized beam', which makes a high-purity circular polarized beam
possible after QWP; but part of energy lost. In the former work, some groups use
GT to purify the polarization [311 [321, but some are not [331. Because the circular
36
polarization is crucial for a high-NA system to get uniform donut shape spot (will
discuss more in Chap2-4-1), we choose to use GT to ensure the purity of polarization.
By changing the angle difference between HWP-GT and QWP, we can achieve a
circular polarized beam. However, because we did not put the QWP and HWP right
before the objective lens, there may be some aberration causing polarization distortion
by dichroic mirror. This will be compensated by the spatial light modulator (will
discuss more in Chap. 3).
2.4.1
Vectorial Debye theory in high numerical aperture (NA)
system
Super-resolution fluorescent microscopy usually is a high NA (Numerical Aperture)
system, in which the polarization of light influences the electric field of the focus. The
electric field vector near the focal spot can be calculated from the vectorial Debye
theory [7,8,341.
apartum~
Figure 2-12: Diffraction of a converging spherical wave at a circular aperture: Nota-
tion. [71
Based on the figure 2-12, the diffracted field near the focus is normally evaluated
by Debye integral:
U(P) =
Af
exp(-ikq - R)dQ
(2.5)
where P is the point near focus 0, Q is the solid angle which the aperture subtends
at the focus. q is the unit vector in the direction of OQ, and R is the position vector
37
-f OP. To the paraxial approximation, s -
f
= -q -,R [71, The vectorial Debye
theory considers the electric field at point P as integral of electric field in the surface
Lf a reference sphere EO(P), for linear polarized beam, the revised Debye integral is
(parameter changed to consist two references):
U(P)
U(P) exp(-ikq - R)dQ
=
(2.6)
Write in spherical coordinate,
U(r, 4, z) =
Ui(0, p) exp[-ikr sin 0 cos(p
-
4)- ikz cos 0] sin 0d~d o
(2.7)
Expressing the electric field in spherical coordinate, the incident electric field at
pointP is
Ui(r) = P(r) cos Vap - P(r) sin Va.
(2.8)
a. is the tangent direction unit vector, and ap is the radius direction unit vector.
After the refraction by the high-NA objective, a. does not change its direction, while
ap changes its direction into ao. Thus, P(r) in the above equation becomes P(0), as
equ. 2.9 shows below.
Ja
0
Ui(r) = P(0) cos Va, - P(0) sin a
=
cos 0 cos pi + cos 0 sin Cpj + sin 0k
(2.9)
-sinVi +cosWk
a,
Thus, substituting Ui(r) to equ. 2.7 to get the vectorial Debye integral [341.
According to this theory, only circular polarized beam modified by vortex 0-27r
phase plate can focus as a round donut shape.
For ellipse polarization or linear
polarization, the intensity around the 'dark center' is not uniform or the center has
residue light intensity. Also, the circular polarized beam should be the same spiral
direction with the vortex phase plate.
To achieve the most uniform donut spot as depletion beam, we used a half wave
plate (IIWP) and a quarter wave plate (QWP), as well as a pair of Glan-Thompson
prism to purify the polarization.
The QWP is designed to put right in front of
38
UnwrY
1
.1
ENpse X
1
0
-0
5
.06
,
000 0
0
a0-
0
0
Left44aaded 2-3
ztinsar
00
(D0O'
1
rized g
sd
.a
rriaW
an
00
00
ed
00
050.6
00.0
,O
0
11.
Figure 2-13: Normalized intensity of polarized light with a vortex 0-27r phase plate
at the focal spot in the horizontal (XY) and longitudinal (XZ) planes. Various polarizations are considered: linear, circular (right-handed and left-handed), right-handed
ellipse (with X and Y as the long axis). Intensities of 0 and 1 correspond to black
and white, respectively. The axis units are in wavelengths. [81
objective lens, in order to minimize the polarization and phase distortion caused by
dichroic mirror surface. However, the illumination beam is about 940nm and UV
light, and the fluorescence is about about 515nm. If we also put the QWP right
before the objective lens, it should be a very broad band QWP which is expensive.
So, we still choose to put is only in the infrared path but combined with adaptive
optics to compensate the influence of dichroic mirror. The adaptive optics will be
discussed further in Chap. 3.
2.4.2
Experiments of donut shape focus in different polarization
In the experiment, SLM, a HWP combined with a Glan-Thompson prism and a QWP
mainly control the polarization of the light entering objective lens pupil (see figure
??). Especially the SLM plays an important role. For a fixed SLM phase and HWPGT fast axis direction, as we turning QWP, the polarization after modification could
be left-hand polarized or right-hand polarized, or ellipse polarized or linear polarized
between these two circular polarized statuses.
39
The focus image is detected by a
CMOS camera (figure 2-1). Because of the size of pixels of the camera, the center of
the donut is shown as not zero intensity. To get more accurate PSF of donut shape
focus, we will use sub-diffraction silver bead to scan the PSF and reconstruct the
image, which will be discussed in Chap. 4.
The SLM vortex 0 - 2-r phase and donut focus before modify is shown in figure 214. The gray scale of the phase is modified to get the most uniform circular symmetric
donut spot.
(b)
(a)
Figure 2-14: The vortex 0 - 27r phase and corresponding donut shape focus. The
center grayscale for the vortex is 58 and it is a left-hand vortex.
As we turn the HWP-GT fast axis in different direction, the intensity of the focus
and quality of donut shape are different. As an example, we randomly choose one
direction and show the quality of focuses for left-hand circular polarized beam and
right-hand circular polarized beam, respectively (figure 2-15).
By turning HWP-GT and QWP, we observe. the donut focus quality to select the
best combination of the directions of these two. To measure the exact polarization, we
could also use Polarization Analyzing System (PAX5710, Thorlabs), which shows the
polarization on a Poincare sphere (not available in our lab). Figure 2-16 compared
the donut shape focuses before polarization modify and after polarization modify.
After the polarization modify, the focus is more circular symmetric and intensity is
more uniform distribution. Coincidently, the start polarization direction in this time
test is close to the best combination, so the contrast is not very remarkable. Further
40
(a)
(b)
Figure 2-15: (a) The focus of right-hand polarized beam modified by left-hand vortex
0 - 27r phase plate. (b) The focus of left-hand polarized beam modified by left-hand
vortex 0 - 27r phase plate. Two pictures are under the same color bar scale.
(a)
(b)
Figure 2-16: Donut shape focus (a) before polarization modify and (b) after polarization modify. The two images are of the same colorbar scale. The left figure is the
same as figure 2-14 (b).
phase modification of donut shape focus will be discussed in Chap. 3.
41
42
Chapter 3
SLM Phase Control
The SLM is one of the key components in this system. It plays three roles: first, it
generates the vortex 0 - 27r phase plate in order to create the donut shape focus in the
RESOLFT super-resolution microscopy; second, it generates the compensate phase
aberration mask to optimum the system performance; third, it generates the parallel
spots array or arbitrary spots for parallel and flexible imaging. The first function is
already discussed in the Chap. 2. In this chapter, we will talk about the algorithm
and LabVIEW control for the other two functions of SLM. This chapter relates more
to electric and computer sections of the microscopy.
3.1
Adapted Optics
As we discussed in the Chap. 2, a uniform circular donut shape spot requires high
accuracy in phase, polarization, alignment, and so on. For our setup, one advantage
is that we can use adapted optics to correct the system aberration to achieve better
Gaussian excitation spot and donut shape 'turn-off' spot. Another advantage is that
we can use spherical aberration to slightly reduce the intensity of excitation spot, in
order to avoid 'turn-off' happened before enough excitation (the competition between
excitation and 'turn-off' is discussed in chap. 1).
43
3.1.1
Optical aberration and Zernike polynomials
The aberration of an optical system can be described by Zernike function [291. Because the donut spot and excitation spot are both at about 940nm wavelength, we
don't consider about chromatic aberration correction in this setup.
Also, we only
consider about paraxial system, so the distortion related to high order of departure
of axial image is not considered. The Zernike function describes aberrations from low
order to high order. However, most of the 'obvious' aberration is caused by low order
aberration, while higher order aberration occupies less percentage. Thus we mainly
correct the low order aberrations, as listed in Table 3.1.
Table 3.1: Low-order Zernike mode for optical aberration
Aberration
Mode Zi
Definition
Z2 (-1, 1)
2r cos 0
Figure
i
Tilt
Defocus
Z3 (1, 1)
2r sin 0
Z5 (0, 2)
V/(2r 2 _ 1)
Z 4(-2, 2)
v'-/r2 cos(26)
Z 5 (2, 2)
vf6r 2 sin(20)
Zs(-1, 3)
3/2(3r3 - 2r) cos 0
Z(1, 3)
2/2(3r 3 - 2r) dos 0
I:
Astigmatism
-WI
Coma
iI
44
3.1.2
trefoil
Z7 (-3, 3)
32T/3r' cos 30
trefoil
Zio (3, 3)
2/-3 r3 sin 30
Primany Spherical
Z1(0,4)
v 5 (6r.
r-
6r2 + 1)
0
Critical metric in adapted optics for focus quality
The system aberration is corrected by adaptive optics method [311. To correct the
aberration, M. Booth and his colleagues defined 'intensity' and 'sharpness' as the
critical metrics to judge the quality of focus. The intensity of the focal spot is measured, and then the sharpness and brightness of the spot are calculated. The image
brightness B is the sum of the pixel values of the image, and the sharpness S is defined
as the second moment of the image Fourier transform as
S
2
ijL.,n'
n,m
+ m'2 )/Z1:in,m
n,m
(3.1)
where in,m is the Fourier transform of the image, n' and m' are the coordinate
relative to the center of the image. pn,m is a low-pass filter with the radius w. To
simplify, the combined metric of brightness and sharpness is defined as
M = S + OcB
+
k(S-S)
Sharpness is the main concern unless the sharpness is above the threshold
(3.2)
ST,
which is normally chosen as the 90% of the maximum sharpness. a- is equal to +1 or
-1 decided by theM is the maximum or minimum value. 3 and k are the experiment
value to balance the contribution of sharpness and brightness.
45
-
ml
-.
-=--
Ouins the aberration measurement, add different degree of aberration to the
criginal phase input. For example, when the input phase is vortex 0 - 27r#,,. the
t i.arg phase is 4 = # + bZi, b E {- 1, 1], Z,. is ith Zernike mode representing
aberration. Adding different
# to the SLM
and calculate the metric M of each image,
aod parameter b for the highest M is the compensate factor.
I. 3
LabVIEW control and experiments for the aberration
correction
(Tenerally, different aberration modes, that is, the Zernike modes, are orthogonal to
each other in the approximate paraxial condition. That is why it is convenient to
mneasure them separately but can add together to modify the phase. However, under
high-NA objective lens, the aberration correction causes focus displacement [311. In
the other word, different modes are not orthogonal. So, we cannot measure them
independently and add together. The former work measured the displacement of
each modify mode and calculated the correlation. In contrast, we choose an iterative
Atrategy. We check the influence of each aberration first, and then choose the most
-erious aberration as 'base mode', then add every other mode to it to check the
aew most serious aberration. Iteratively doing this process, we can achieve the least
aberration system.
The first example is the aberration correction for Gaussian beam focus. After
aligning the system, we use, Rhodamine 123 solution as the fluorescent sample, berause of the brightness and uniformity. The illumination light is about 940nm. We
ise the camera (see figure 2-1) as detector. We can observe the change of focus while
changing the aberration degree. We show the influence of astigmatism to the focus
quality (see figure 3-2), which is one of the serious aberration in that test.
From the figure 3-2, we can tell that the intensity of focus is higher when compensating the system by large negative astigmatism. Because we use a 100x objective
lens in the setup, the entrance pupil is smaller than the beam diameter. So only the
top of Gaussian beam can pass through. This way limited the light intensity to the
46
Figure 3-1: LabVIEW control panel for system aberration compensation.
focus, but make it more robust to astigmatism. Even though, according to the phase
compensation experiments, astigmatism is still one of the most serious aberration in
the setup. To quantitative analyze the result, we draw the three curves: brightness,
sharpness, and the combination of these two factors, respectively. (Figure 3-3).
Figure 3-3 shows the sharpness and brightness have nearly opposite trend. The
sharpness curve shows the focus is sharper in positive compensation, but the brightness curve shows the intensity is higher in the negative compensation. If we compare
the figures in figure 3-2, we can tell that the low sharpness of negative phase compensation part may be from the saturation intensity in the center. So we choose the
brightness as major factor and choose b
=
-0.9
as the best compensation for the
astigmatism.
The second step is examine the most serious aberration after the phase correction
of the first step. We put Zernike mode (-2, 2) with b = -0.9
as 'base mode', then
adding every other aberrations in different degree. After our experiment, the coma
47
Figure 3-2: The focus spots under different degree of astigmatism (Zernike mode (-2,
2), Table 3.1).
Adptiv
Adaptive Curve: Combination M
Adaptive Curve: Brightness
Curve: Sharpness
1
--
.i-
-
-
-1
-
-
Di080.8
. ....
....
0.6..0.6-- .
. ...
0.46
..
A..
0.4
0.2 -
001
-05
0.2 - -
..
......
..........
-
0. -...
.
.. ..
-
-
0.
0
Was
0.5
1
-1
-0.5
0
Bias
0.5
1
-1
-0.5
0
Bias
0.5
1
Figure 3-3: The sharpness, brightness, and the combined metric v.s. bias (b) curves.
The combined metric curve will change if change the 'weight' between sharpness and
brightness, depending on which one is major.
(Zernike mode (3, 1), see Table 3.1) is the most serious aberration in this round.
The focus quality is shown in figure 3-4 as the system PSF under the synthesis
compensation of astigmatism and coma.
Because we test the vertical direction coma, it is obvious that the focus is moving
along vertical direction. We can tell the quality of focus is better than the first round
testing generally (figure 3-2). Then we calculate the sharpness and brightness, as well
as combined metric as before. The result is shown in the figure 3-5.
According to the 2nd round quantitative comparison, the aberration is least when
adding b = -0.9
astigmatism and b = 0.4 coma. The combined phase is shown in
figure 3-6 (c). The compare of the focus before and after aberration compensation is
in figure 3-6 (a) and figure 3-6(b). We can tell that the focus is brighter and more
circular symmetric after aberration compensation.
48
This method cannot guarantee
Figure 3-4: The focus spots under fixed astigmatism (b = -0.9) compensation while
testing different degree of coma (Zernike mode (3, 1), Table 3.1).
Adapve Curve: Sharpness
Adapitve
Adaptive Curve: CornbinatanM
Curve: Brightness
1
1
......
.....- -0.8
0.8
0.o...
8-....
00
"
..........
02
0.2
-1
0
0
Bias
0"1
0.6
........
0.2
-0.5 0 0.5 1 -1 -0.5 0 05 1
Bias.
Bias
Figure 3-5: The sharpness, brightness, and the combined metric v.s. bias (b) curves.
In the second round testing, the sharpness and brightness have similar trend, which
means the aberration of the system converge to a 'local minimize' point under these
two phases compensating.
the aberration compensation is the least globally, but can make sure this is the local
least and already corrected most serious aberrations in the system. Under sufficient
time, this phase compensation iterative process could be more accurate.
The next example is the aberration correction for donut shape focus. The method
of trials to determine the 'local best' donut shape focus is the same as Gaussian
focus. However, the quantitative metrics to judge the quality of focus is different.
The brightness and sharpness defined as above cannot accurately describe the characteristics of donut shape focus. The intensity of a uniform donut shape spot is less
than that of a Gaussian focus because of the diffraction loss from SLM. The donut
shape frequency domain is more complicated than the Gaussian focus, so the sharpness calculation is without close relation to uniform donut shape.
49
As we focus on
(b)
(a)
(c)
Figure 3-6: (a) The original Gaussian beam focus without aberration compensation
to the system. (b) The focus after aberration compensation. (c) The compensated
phase adding to the SLM, which is sum of negative astigmatism and positive vertical
coma.
the uniformity of donut shape, the standard deviation around the dark center is an
important metric under consideration. The result is shown below. The experiment
figures as different aberration compensations added are shown in figure 3-7.
The
process is the same as Gaussian beam experiment above. To simplify the result, we
choose to show every other result as bias scanning from -1 to 1. For the
1
" round
test, Zernike mode (1, 1) has obvious distortion, means the displacement matters to
the donut focus.
Figure 3-7: The donut shape focus under Zernike mode (-1, 1) and bias b = -1 : 1
0.5, the donut shape is the
modify, selectively shown every other figures. When b
most uniform one.
Based on the
1"
round result, Zj-1 at b = 0.5, the 2 " round tests all the other
aberration added on this base mode. One representative result is Zernike mode (3,
3), which is the trefoil aberration in Table 3.1. The result is shown in figure 3-8.
When b
=
-0.4,
the donut shape is the most uniform one (skipped in figure 3-8
but shown in figure 3-9).
After these two round modification, the donut shape is
already much more uniform and symmetric than before modification (see figure 3-9).
50
Figure 3-8: The donut shape focus under Zernike mode (3, 3) and bias b = -1 : 1
modify, selectively shown every other figures.
As the same as Gaussian beam, this method will achieve better result if more iterative
testing processed.
(a)
(b)
(c)
Figure 3-9: (a) The original donut shape focus without aberration compensation to
the system. (b) The focus after aberration compensation. (c) The compensated phase
adding to the SLM, which is sum of positive shift and a negative trefoil. (a) and (b)
are under the same color scale.
3.2
3.2.1
Phase retrieval algorithm
Gerchberg-Saxton iterative algorithm
The phase retrieval of arbitrary position multi-focus uses the classic Gerchberg-Saxton
iterative algorithm modified by Fienup [9]. The principle of the iterative Fourier
transform (FT) algorithm is figure 3-10. For the signal changing from kth loop to
k + 1, the iterative equation is
where / is a free parameter, usually chosen close to one. This method has high
efficiency to converge. When we already know the aimed position of multiple focuses
AT, the phase on the back focal plane is calculated iteratively until converge. Add
this retrieved phase to the vortex 0 - 27r phase can generate donut spots in the
51
Ak
Back-propagation IFT
k
Phase space apply
( constraints
(SLM phase input)
Signal space apply
constraints (k)
(Target intensity image)
YES
Ak
function
satisfied?
ak
Propagation FT
NO
Recovered phase
Figure 3-10: The principle of the iterative Fourier transform algorithm. [9]
corresponding position. Thus, it is possible to locally scanning and do super-resolution.
imaging in several sub-regions simultaneous. This flexible imaging method has many
3otential application in biological research.
For example the method can be used
to image sparse aims in the specimen, which is more efficient than scanning the
whole field of view.
For small area scanning, it is possible to do live imaging for
3everal sub-regions, such as do multiple super-resolution tracking for several protein
.ransportation or interaction.
3.2.2
LabVIEW control and experiment for arbitraryposition
spots
According to the G-S algorithm, we programmed the control panel by LabVIEW
(figure 3-11). The aim position is randomly choose from the 'new picture', and the
coordinate value will be collected and process to the phase retrieval calculation. After
-he calculation, the recovered phase is sent to SLM screen, so we can get the aimed
focuses on the camera.
Repeating this process can control the focuses position in
real time. There are several potential applications for this method. For example, the
52
'.Gaussian focuses spot can be used as optical tweezers, which can control the motion
of molecules.
Because we can do several focuses at the same time, it is possible
to do multiple particle tracking, in order to track their transportation as well as
the interaction between each other: Combined with donut shape spots at the same
positions, we can achieve higher resolution. Another application is super-resolution
image for several specific areas. The 'new picture' could be a low-resolutiion image,
and we can select several regions requiring higher resolution, while other sparse area
without scanning again. This can help us get super-resolution images more efficiently.
Figure 3-11: The control panel of flexible position focuses. The aimed focuses are
selected from the 'new picture' window.
In the experiment, we use one pixel point (pulse function) as the aim spot. If
we use several pixels as one spot, we should add the extra limit to make sure the
intensity of all the pixels inside the spot is identical. But actually, the intensity of
different spots are different in the experiment (see figure 3-12 (c)), even the intensity
53
of aimed spots are the same. Adding linit to the difference between focus intensities
should help reduce the effect.
(a)
(b)
(c)
Figure 3-12: . (a) The focuses in random positions, selected from the control panel.
(To easy observation, enlarged the focuses area, 10 times, each spot here should be
one pixel size in the calculation.) (b) The phase mask calculated by G-S algorithm.
(c) The camera image of the phase (Rhodamine 123 solution as sample), which is the
same as the aimed focuses.
If combine the retrieval phase with vortex 0- 27r phase mask, we can achieve donut
shape focuses on the same aimed position. Figure 3-13 shows the process. Compared
to the Gaussian focus, donut shape focus is more difficult because we should consider
about the uniformity of the donut.
Noticed that the intensity of each spot is not
perfectly uniform, this could be improved by more iterative loops, or set the target
rule as the standard deviation of intensity is smaller than a certain threshold. Because
of the aberration compensation in Chap. 3-1 is for the whole field of view, it is hard
to compensate the aberrations for each donut shape focus.
54
Phase recover
Vortex 0-2pi phase plate
Experiment image
(output)
*
*
&
Aim position
(Input)
LabVIEW
SLM
Camera
Figure 3-13: The flexible position of donut spots, for multiple super-resolution particle
tracking. The donut shape in the 'input image' is enlarged. Like the Gaussian focuses,
one pixel spot to indicate the coordinate value of the position is sufficient.
55
56
Chapter 4
Two-photon RESOLFT microscopy
imaging
4.1
PSF measurement: silver nanoparticle scanning
The figures taken by camera is actually the real image sampling by camera pixels.
Because the 'dark center' of donut spot is very small (<50nm), even enlarged by the
objective lens (100x), but still smaller than the pixel size of the camera (about 7um).
To get more accurate PSF of the system, we use nanoparticles to scan the PSF.
Such as silver nanoparticle, the scatter light from the silver nanoparticle is strong
and sensitive to the intensity change. So the measured PSF becomes the convolution
of silver particle with the real PSF. The smaller particle we use, the more accurate
the PSF it. But scatter light intensity from small particle is weaker as well. At the
same time, the scatter light intensity also relates to wavelength. We use 40nm silver
nanoparticle (Sigma-Aldrich) in the experiment. One advantage of the setup is the
Gaussian beam and donut shape beam share the same beam, but with modulation
temporally. So the colocalization of these two beam is accurate.
Figure 4-1 shows the bead-scanned PSF of the Gaussian focus, donut focus and
the overlay of them. The wavelength is about 940nm. The intensity is the square of
the original measured intensity, to show the two-photon PSF distribution (equation
1.2).
57
(a)
(b)
(c)
.gure 4-1: nanoparticle-scanned PSF of the setup. (a) Gaussian focus. (b) Donut
lape focus, with the same intensity scale. (c) Overlay of (a) and (c) to show the
>c4ocalization (pseudo color).
We also measured the PSF of single photon excitation in the wavelength of 532nm.
Figure 4-2 shows the comparison of the PSF between single photon excitation and
two-photon excitation. For the donut beam, two-photon excitation gave darker center
point. But at the same time, the peak-to-peak distance is larger. Considering the
experiment condition, it is very hard to achieve 'zero intensity' in the center of donut
spot, because of the background, or limited size of NA. However, if the residue intensity over 5% of the peak intensity, the super-resolution image signal to noise ratio will
be attenuated [351. From this view, two-photon process can give better quality donut
in the experiment. Another concern we have is whether the donut shape will distort
as imaging depth increases. If the peak-to-peak distance is larger, maybe the donut
spot could tolerate more distortion caused by specimen scatter. This is pending to
prove by experiment.
58
..
-
-
0.8
0.6
0.8
-
1
06
/
0.4-
0.4
0.2
0.2
0
9500
-50
50
500
0
(b)
(a)
Figure 4-2: The PSF comparison between single photon excitation (blue dash line)
and two-photon excitation (red solid line). (a) The Gaussian focus. (b) The donut
shape focus.
4.2
Parallel scanning: Gaussian spots array and donut
shape spots array
One drawback of RESOLFT microscopy is that it is a point scan method, that is, the
imaging speed is proportional to the size of field of view (FOV). However, it is difficult
to achieve large FOV as well as high-resolution simultaneously. Parallel scanning is
one common method to increase the imaging speed for point scan microscopy [?,2,24].
In our experiment, we generated 5x5 spots matrix, also the corresponding donut spots
matrix. By zoom-in and zoom-out the grating on SLM, we can control the separation
and position of the spots arrays flexibly (see figure 4-3).
While the parallel scanning increases scanning speed, it requires more power to
ensure every section of image has good signal to noise ratio. Limited by the maximum
energy, we did not get good signal to noise ratio image of donut spots array in twophoton excitation setup. The flowing donut array images are single photon excited
using
532nm wavelength laser. We also tried even larger spots matrix (like 11x11),
but it is hard to guarantee the quality of every donut shape focus, and the intensity
59
(a)
(bi)
(c)
Figure 4-3: 5x5 spots array, two-photon cxcitation using Rhodamine 123 sample,
detected by the camera. The period of grating on SLM gradually zoom in, and the
corresponding image 'zoom out'.
of each spot is even lower.
Figure 4-4: The 5x5 donut spots matrix for parallel super-resolution imaging.
4.3
Live cell two-photon imaging
Two-photon RESOLFT microscopy is based on two-photpn microscopy.
Without
change the phase mask on SLM, this system can be used as a two-photon microscopy
(single point scan or parallel scan). First we use HEK 293 ce.l as specimen, labelled
60
by Dronpa-M159T (Table 1.1) on tubulin ". The early passage frozen cell and DNA
plasmid are gift from Prof. Edward Boyden's lab. The cell culture follows the typical
mammal cell culture protocol, and the transfection uses TransIT 293 reagent (Mirus)
and follow the protocol of it.
The fluorescent images are taken after 18h-24h of
transfection.
(a)
(b)
Figure 4-5: HEK 293 cell labelled by Dronpa-M159T on tubulin. (a) The field of
view (FOV) of the image is 60um x 60um. The scanning step is 500nm. (b) The
fine scanning image of the top right region of (a), the FOV is 30um x 30um. The
scanning step is 200nm.
Because we use live cell and the imaging process takes minutes, we can observe
the cell position change during continuous two times imaging (figure 4-6).
We imaged HeLa cell line also, which microtubule extends to larger area than HEK
cells. We can distinguish single microtubule labeled by Dronpa under the two-photon
RESOLFT microscopy. The resolution reaches about 400nm (200nm per pixel).
'The color of the image is pseudo color, based on the emission peak wavelength of Dronpa-M159T
is 515nm. The contrast is adjusted.
61
(a)
(b)
Figure 4-6: The position change of live cecll. (a) 1st time scanning, FOV is 80um x
'Oum, and step size is lum. (b) 2nd time scanning for the right bottom region of (a),
oticing the bottom cell orientation changed during this process. The FOV is 30uw
3 9 um with 200nm step size.
150
04
./..
....
Am
0
1000
200
3000
4000
Distante (iXeHlS)
..
......
4/W
5000
0 00
Figure 4-7: HeLa cecll labeled by Dronpa-Tubulin. The enlarged region is increased
contrast by ImageJ. The cross profile of the red line region is shown below. The
finest structure we can distinguish is about 400nm. The image is 50um x 50um, with
200nm scanning step.
62
Chapter 5
Conclusion
We designed a two-photon RESOLFT microscopy with SLM control, which can apply to high speed 3D high-resolution imaging by parallel scanning, as well as highresolution multiple particle simultaneously tracking by flexible position scanning. The
system can achieve less aberration imaging via phase compensation. We showed the
algorithm and experiment results of these unique characteristics in the paper. Iterative phase retrieval algorithm is used to calculate the phase mask on SLM for flexible
position imaging.
aberration.
Phase compensation using adaptive optics is applied to reduce
As for the experiment result, we showed the PSF and time sequence
of the three beams in the RESOLFT microscopy section.
For parallel imaging or
flexible imaging, both Gaussian spots (excitation beam) and donut spots ('turn-off'
beam) experiment images are shown. At the last, the two-photon images of the cell
specimen labeled by reversibly photoswitchable fluorophore are demonstrated.
At
the same time, the design could be further promoted by higher speed scanning, such
as using resonant mirror instead of piezo stage. This will improve the image system
efficiency, making the live imaging possible. The other drawback is the absolute intensity of super-resolution image is low, because the fluorescence was turned off in
the surrounding area and only limited area emits fluorescence.
noise ratio reduced compared to the two-photon images.
Thus, the signal to
sCMOS camera or other
more sensitive detector with less background noise could improve the image quality
further. After these modification, the large field of view, high-speed, high-resolution
63
microscopy could be realized. This microscopy is potentially used for imaging large
area of specimen with 3D high-resolution in detail, such as single synapse event as
well as the synapse circuit in the brain s'icc +n vivo.
64
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