PowerPoint Presentation Optimization of Illumination Pulse Duration Increases Flexibility and Perfor

1
Extending the Flexibility of Multiphoton
Microscopy for Multidisciplinary Research
Optimization of Femtosecond Pulse Duration and
New Approaches to Precision Beam Control
Karl Garsha Imaging Technology Group, Beckman
Institute for Advanced Science and Technology,
University of Illinois at Urbana-Champaign
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
2
Purpose

• Overview construction of a device for
  pre-compensation of positive group velocity
  dispersion of femtosecond TiSapphire laser
  pulses
• Introduce ongoing research into the effects of
  pulse width compensation with regard to diverse
  imaging situations
• Discuss strategies for permitting directed
  application of MP excitation energy with
  sub-micron resolution in 3D

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
3
Acknowledgements

• The Center for Microanalysis of Materials at UIUC
  and Dario A. Farias
• Prof. Keith Singeltary and Allison Ellington,
  Department of Nutritional Food Sciences, UIUC
• Art Camire (Spectra Physics)
• Michele Garsha and Dr. J. Campanelli

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
4
Laser Scanning Multiphoton Microscopy
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
5
Laser Scanning Multiphoton Microscopy
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
6
Advantages of Multiphoton Excitation

• Longer wavelength is used
• less specimen damage
• better penetration into light scattering subjects
• less distortion of optical path length in
  refractive index mismatched systems
• Excitation is confined to the focal volume
• less (3-D) photo bleaching
• less radiation damage and photo toxicity to
  living specimens
• ability to ablate (micro dissection) or
  polymerize materials with precision in three
  dimensions
• No pinhole
• PMTs can collect more light
• optical path length distortion has less profound
  of an impact on signal to noise ratio

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
7
Advantages of Multiphoton Excitation
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
8
Advantages of Multiphoton Excitation
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
9
Role of MP Capability in the Context of a Laser
Scanning Microscopy Facility

• Extends/enhances the existing capabilities of a
  confocal system
• Routine MP (MP excitation must be effective
  enough to provide a decent signal/noise ratio)
• Adds additional capabilities
• High-Performance MP (Efficiency of MP
  excitation must be optimized for acceptable
  results)

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
10
Considerations for Optimizing MP Imaging
Performance

• Wavelength
• Bandwidth/Pulse-Width
• Average Power Levels
• Pulse-Shape
• Bardeen et al. (1999). Effect of Pulse Shape on
  the Efficiency of Multiphoton Processes
  Implications for Biological Microscopy. J.
  Biomed. Opt., 4(3), 362.
• Brixner et al. (2001). Photoselective adaptive
  femtosecond quantum control in the liquid phase.
  Nature 414, 57.
• Chirp
• Bardeen et al. (1998). Quantum Control of
  Population Transfer in Green Fluorescent Protein
  by Using Chirped Femtosecond Pulses. J. Am.
  Chem. Soc. 120, 13023-13027.
• Brakenhoff et al. (1999). The Measurement of the
  chirp-dependent fluorescence response a window
  to excited-state population dynamics. Proc. SPIE,
  3605, 40.
• Buist et al. (1999). Probing microscopic
  chemical environments with high-intensity chirped
  pulses. Optics Letters, 24, 244.

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
11
First Generation Methods of Increasing the
Probability of MP Absorption/Excitation

• Increase the average laser power (tweak
  alignment, adjust the EOM, get a bigger laser)
• Pulse Dispersion Pre-Compensation

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
12
Pulse Dispersion Pre-Compensation

• Advantages
• Increased MP effect for all power levels
• Ability to manipulate amount of chirp
• Helps to characterize the imaging system
• Disadvantages
• Principles are foreign to most imaging facility
  administrators
• Commercial solutions are expensive and possibly
  ineffective
• Some engineering effort is involved
• Space
• Beam divergence

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
13
Increasing average Power

• Advantages
• Easy
• Disadvantages
• Pre-supposes increased power is available
• Small amounts of linearly absorbing matter may
  make disproportionately large contributions to
  specimen heating
• More collateral damage is associated with optical
  breakdown (ablation)
• Kim et al. (2001). Influence of pulse duration
  on ultrashort laser pulse ablation of biological
  tissues. J. Biomed. Opt. 6(3), 332
• Oraevsky et al. (1996). Plasma Mediated
  Ablation of Biological Tissues with
  Nanosecond-to- Femtosecond Laser Pulses Relative
  Role of Linear and Nonlinear Absorption. IEEE J.
  Quant. Electron. 2(4), 801.

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
14
Construction of a Pre-Compensation Unit

• Polarization of laser source (horizontal vs.
  vertical)
• Placement
• Prism material (refractive index)

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
15
Design Considerations

• Brewsters angle
• Minimum deviation
• Translate prisms through apex
• Mechanism to adjust inter-prism spacing
• Retro-reflector coating

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
16
Chirp Champ I (Chirp Chump)
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
17
Optimization of Pulse-Width using MP excitation

• Fluorescence standard
• Adjust distance
• Displace prism
• Watch for increased fluorescence/decreased
  fluorescence
• Repeat until optimized

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
18
Results Pulse Width

• _at_ laser 42 femtoseconds min w/ frequency
  distribution centered about 785nm wavelength
• Post scan head (pre-objective) with no
  compensation 535 femtoseconds
• Post scan head w/62cm inter-prism distance 82
  femtoseconds

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
19
Results Pulse Width

• Estimated broadening due to objective 3200 (fs)2
• Estimated pulse-width post-objective w/ 62cm
  inter-prism distance 139 fs
• Estimated pulse-width post-objective w/ 90.5
  inter-prism distance 1Tin 42 femtoseconds
• Guild et al., (1997). Measurement of group delay
  dispersion of high numerical aperture objective
  lenses using two-photon excited fluorescence.
  Applied Optics. 397-401.
• Spectra-Physics Tsunami
  documentation A-6 to B-9

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
20
Results Fluorescence Intensity
PMT Gain 650 Offset 0
PMT Gain 650 Offset 0
Beckman Institute for Advanced Science and
Technology
5mw 63x NA1.32 3x Beam Expand 90.5cm
interprism Mean Amplitude 60.96
5mw 63x NA1.32 3x Beam Expand No Prisms Mean
Amplitude 29.47
Imaging Technology Group www.itg.uiuc.edu
21
Results Fluorescence Intensity
PMT Gain 650 Offset 0
PMT Gain 650 Offset 0
Beckman Institute for Advanced Science and
Technology
5mw 63x NA1.32 3x Beam Expand 90.5cm
interprism Mean Amplitude 60.96
30mw 63x NA1.32 3x Beam Expand No Prisms Mean
Amplitude 50.53
Imaging Technology Group www.itg.uiuc.edu
22
Ongoing/Future Studies

• Absorption/heating and ablation thresholds in
  live cell imaging applications
• Engineering practical 3-D ablation/polymerizatio
  n solutions
• Practical pulse width measurement

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
23
Absorption/Heating and AblationThresholds in Live
Cell Imaging Applications Live Cell Dose
Dependant Mortality
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
24
Absorption/Heating and AblationThresholds in Live
Cell Imaging Applications Ablation vs. Excitation
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
25
Summary Live Cell Damage Thresholds

• Optical Breakdown, Specimen Heating, and
  Phototoxicity contribute to cell damage
• The relative contributions of these factors to
  the dose response vary with pulse width,
  specimen, and average power.

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
26
Ongoing/Future StudiesEngineering practical 3-D
ablation/polymerization solutions

• Microfabrication (Micro-Electromechanical
  Systems)
• Sun et al. (2002). Appl. Phys. Lett. Jan 14, 321.
• Optically active 3-D structures
• Fluorescent doped polymer resins
• Sun et al. (2001). Appl. Phys. Lett. 79,1411.
• 3-D Defects in Colloidal Crystal Structures as
  Photonic Bandgap
• Braun Group (W. Lee, S. Pruzinski)

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
27
Beam Control ROI Scan
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
28
Beam Control ROI Scan

• Beam scan raster remains active beam is
  attenuated by EOM outside of regions of interest
• Dwell time is dependant on scan speed (not much
  control is available)
• Pixel overlap is dependant on objective and scan
  resolution (not much control)
• Control of minimal ROI size is difficult
• ROI selection is limited to 2-D

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
29
Beam Control Alternative Strategies

• Beam Steering point by point polymerization of
  the substrate
• Stage Scanning beam remains stationary while
  high precision stage moves with respect to beam
• Combination Beam Steering in concert with stage
  scanning

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
30
Beam Control Point Based Steering

• Beam is attenuated by shutter minimum dwell time
  is determined by the shutter speed
• Range of the z-axis is determined by the galvo
  stage range (160um)
• XY range is limited to field of view (zoom
  dependant)

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
31
Beam Control Stage Scanning

• Dwell time is controlled by stepper motor rpm
• Dwell time varies with and - acceleration of
  the stage at the beginning and end of a vector
• Stage resolution and repeatability are limited
  (one line approach)

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
32
Beam Control Combination Approach

• Tiled consecutive fields of view may require
  some overlap or intersection scheme to
  accommodate stage accuracy and precision limits
• one line approach to center of next field of
  view to permit continuous structures branches
  are polymerized through point by point beam
  steering

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
33
Create/Ablate Enabling Beam Control
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
34
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
35
Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu
36
Summary

• Overviewed construction of a device for
  pre-compensation of positive group velocity
  dispersion of femtosecond TiSapphire laser
  pulses
• Introduced ongoing research into the effects of
  pulse width compensation with regard to diverse
  imaging situations
• Discussed strategies for permitting directed
  application of MP excitation energy with
  sub-micron resolution in 3D

Beckman Institute for Advanced Science and
Technology
Imaging Technology Group www.itg.uiuc.edu