Fast Optical Scanning for Confocal Raman Tweezing Spectroscopy

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Fast Optical Scanning forConfocal Raman
Tweezing Spectroscopy 

• Emanuela Ene

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Abstract
The Confocal Raman Tweezing Spectroscopy (CRTS)
has the ability to provide precise
characterization of a living cell without
physical or chemical contact. In our nanotoxicity
study, CRTS will be employed for monitoring in
real time the chemical and functional changes in
nanoparticle-embedded cells.
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• For a CRTS study a very stable optical trap is
  essential, so that extra cell instability is not
  induced.
• Repeatability and stability of the collected
  Raman spectra during optical trapping may be
  achieved with automatic laser beam steering.
• A two-axis acousto-optic deflector (AOD) and a
  piezo positioner are designed to be included in
  our existing Confocal Raman Tweezing Spectrometer
  (CRTS) in order to achieve fast and precise
  laser trap displacements.

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• A perfect lens OSLO simulation is run for our
  Gaussian beam based CRTS.
• Beam steering OSLO computations, in both
  transversal and axial directions, demonstrate the
  range for scan angles and for linear translation.
  
• For a truncated Gaussian beam, employed in
  optical tweezing, we expect optical aberrations
  even for a perfect lens-like focusing objective.

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Outline
The Confocal Raman Tweezing Spectroscopy
has the ability to provide precise
characterization of a living cell without
physical or chemical contact. The CRTS allows the
analysis of single cells in wet samples, in
contrast with the classical micro Raman
spectroscopy which utilizes dried samples. In a
confocal setting, the collected signal comes just
from a minimum volume around the trapped-excited
object. In our nanotoxicity study, CRTS is
used to monitor the chemical and functional
changes in nanoparticles-embedded cells in real
time .

• Background Our CRTS application
• Experimental setup
• Axial resolution
• Confocal microRaman spectra

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Our Confocal Raman-tweezing system
M Silver mirror P Pinhole LLF - laser line
filter BS beam-splitter BP - broad-band
polarization rotator
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Proposed solution
The problem addressed is monitoring living
cells, via the CRTS technique for nanotoxicity
studies. Both stability of the trap, for around
eight hours of successive spectra collection,
and repeatability are required. Optical
trapping and manipulation can be realized using
mechanical microstages or electric
nanopositioning. The latter method is not only
far more precise, but also assures stability and
repeatability. Nanopositioning systems currently
used for CRTS are galvanic mirrors,
piezo-controllers, and AODs. The automatic
fast laser beam steering will allow moving the
beam focus in 3D to chase the cell that will
be trapped and analyzed. Thus we will eliminate
any mechanical displacement, proven to be a
source of misalignments, instabilities, and
irreversible changes. A two-axis
acousto-optic deflector (AOD) and a
piezo-positioner are designed to be included in
our existing Confocal Raman Tweezing Spectrometer
(CRTS) in order to achieve fast and precise laser
trap displacements.
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• The advantage of choosing to fast steering
  the trap only in the x-y plan simplifies the
  confocal pinhole alignment.
• The pinhole will be initially aligned in
  the conjugate plane of the objective focal plane.
  
• This alignment will be stable while
  scanning the x-y plane in the range of 0-100µm
• (or 0-50mrad) for a pinhole size in the
  range 200-400µm.
• The alignment will be also stable when
  moving the infinity corrected objective on the
  z-optical axis of the setting in the range of
  0-400 µm.
• If the position of the trap on the z-axis
  would be changed by controlling the laser beam
  divergence, as done in classical tweezing setups,
  the conjugate plane of the pinhole can not be
  kept fixed.

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Technical description
The improved CRTS setup is shown in Fig. 1.
Three alternatives for new parts that should be
included are listed in Table 1. The effects of
beam steering with the AODs and of displacing the
objective with the piezo controller are shown in
OSLO simulations.
Potential problems which we may encounter are
due to the thermal sensitivity and to the
electric noise of the driving voltage for the
AODs. We address these two weaknesses by
designing a heat sink for the AODs and by
including the highest precision voltage
controllers on the market.
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Fig.1 the improved CRTS system
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Table 1 Specifications Prices to
electronically control the tweezing position .
Company system Total price () Deflection angle (mrad) Efficiency () Aperture (mm) Delivery time (weeks)
1 Physik Inst. 1D - piezo 6,206 - 100 - 2-4
2 IntraAction 2D -AOD 5,165 42.9 70 10 X 10 Several months
3D price 11,371
3 Isomet 2D -AOD 16,051 50 gt35 9.3 X 9.3 5-8
3D price 22,257
4 Physik Inst. 3D - piezo 16,154 10 for 100µm linear translation 90 2-4
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Preliminary results

• Microscope objectives are complex systems of
  lenses, corrected for geometrical and chromatic
  aberrations such an almost perfect system has
  more surfaces than we may handle in EDU version
  of OSLO in the simulations we enter a PERFECT
  LENS with F2mm and magnification 100X for our
  PLAN APOCHROMAT infinity corrected oil immersion
  objective
• the object to be imaged is the incident laser
  beam
• the laser beam is Gaussian, 632.8nm, is
  collimated, and has an expanded 6.0mm waist size
• the expanded beam fills the 6.0mm-radius of the
  microscope aperture the beam is truncated by
  this aperture to its 1/e2 diameter

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Microscope objectives are complex systems of
lenses, corrected for geometrical and chromatic
aberrations such almost perfect systems have
more surfaces than we may handle in EDU version
of OSLO
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PERFECT LENS

• A perfect lens is that one that forms a sharp
  undistorted image of an extended object on a
  plane surface (from the OSLO Reference manual).
• OSLO uses perfect lenses obeying the exact laws
  of optics. The results when using these perfect
  lenses are different from modeling with paraxial
  lenses.
• If the lens is to obey Abbes sine law, rays must
  emerge from the surface at a different height
  than they enter. A real perfect lens cannot be
  infinitely thin.
• Abbes sine law, valid for aplanatic (coma free)
  lenses
• with
• U, U the angles which the corresponding rays in
  the object and image spaces make with the axis
  of the system
• u, u the slopes of the corresponding rays in
  the object and image spaces

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The 100X on the short conjugate side PERFECT LENS
with F2mm gives for a 632.8nm Gaussian beam a
2µm minimum waist
LENS DATA tweezing INITIAL SRF RADIUS
THICKNESS APERTURE RADIUS GLASS SPE
NOTE OBJ -- 1.0000e03
6.000000 AIR AST
ELEMENT GRP -- 6.000000 AS
AIR 3
PERFECT 1.400000 6.000000 S
OIL M PERFECT 4 --
0.170000 4.804800 S
COVER M 5 -- 0.356946
4.299109 S SAMPLE M IMS
-- --
0.0019921 S
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The beam profile in the image plane for the OSLO
model The cover glass and the solution with
cells change the conditions for a perfect lens
The tweezing spot profile for a Gaussian beam
(Tgt2) Truncation factor TD_beam(1/e2) /
D_apert
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The beam profile in the image plane, the OSLO
model, for truncated Gaussian beams employed in
optical tweezingCalculations based on a paraxial
ray trace may be invalid for a truncated
Gaussian beam
OSLO computes the diffraction image of a point
object (the Point Spread Function) from the
information of the geometric wavefront. For a
truncated Gaussian beam entering our tweezer
the central normalized energy peak is 0.48. The
orresponding trapping force, in the spring-like
trap, is 70 of the full power force.
T1
Truncation factor TD_beam(1/e2) / D_apert
Note the PSF algorithm results depend on the
number of points in the sampling grid
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Trap image (tweezing focus) in the X-Y plane for
a Gaussian beam
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Steering the tweezing focus in the X-Y plane for
a Gaussian beam
AOD objective distance -202mm
Position Axial displacement of the beam center (mrad) Lateral displacement of the focus (micrometers)
1 0.5 0.11
2 1.57 4.88
3 39.6 81
4 49.6 100
Both AODs are driven for equal scan angles on
the X and Y directions
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Summary

• A perfect lens OSLO simulation has shown how a
  perfect lens focuses a Gaussian beam
• Beam steering OSLO computations, in both
  transversal and axial directions, have
  demonstrated the range for scan angles and linear
  translation
• For a truncated Gaussian beam, employed in
  optical tweezing, we expect aberrations even when
  focusing with a perfect lens

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References

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