Temporal Focusing-Based Multiphoton Microscopy and Microprocessing

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10-28-2014

Adaptive Photonics Lab, NCKU

- Plasmonic Biosensing & Molecular Imaging - Ultrafast Laser Microscopy & Microprocessing

Temporal Focusing-Based Multiphoton Microscopy and Microprocessing

Shean-Jen Chen (陳顯禎)

Department of Engineering Science

Center for Micro/Nano Sceience and Technology (CMNST) National Cheng Kung University (NCKU), Tainan 701, Taiwan

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Conventional Femtosecond Laser System

- Molecular Imaging: Two-photon excited fluorescence (TPEF) imaging, second harmonic generation (SHG) imaging, FLIM.

- Microprocessing: Microfabrication, nanosurgery, nanomachining.

Mode locked Ti:Sapphire

LabView FPGA

x y

AOM

Exposure Switch

Scanner

Dichroic Mirror

Objective

Bio-sample

Filter PMT

Servo Controller 3D CAD Processing

DXF or BMP Femtosecond Laser Imaging & Processing System

CAD Design

Slicing Program

Transformation Program

3D STL

individual 2D DXF

2D DXF

Photon Counting

Sample XY Stage Objective (Piezo Z Drive) Filter PMT

L3

Galvo x

Mirror 2 Galvo y Dichroic

Mirror

L1 L2 Eyepiece

Scan Lens

Discriminator DIO NI DIO

FPGA

AOM DIO

HWP P Mirror 1

Femtosecond Laser QWP

Rapid on/off switching of the laser and pulse selection

Designing 3D freeform structures: To transform 3D models into 2D processing patterns, and the program convert the 3D model into sequential 2D DXF files.

point-by- point scan

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Biomedical Nonlinear Optical Microscopy

MRC Laboratory of Molecular Biology, UK

2~4 NIR photons

excited state

ground state

shorter λ fluorescence

Dendritic Spine w 2PEF

Tendon Collagens w SHG

Two-photon excitation

One-photon excitation

Y.-C. Hsu et al., J. Hypertension 29 (2011) 2339.

K. Tilbury et al., Biophys. J. 106 (2014) 354.

L.-C. Chung et al., Biomed. Opt. Express 5 (2014) 3427.

Mutli-color 2PEF

Rat Brain w 4PEF + THG

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Cytoskeleton Tubulin w Alex 488 Neuron w Lucifer Yellow

3D Reconstruction Imaging

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S0

S1

S2

12s 10^

T1

nm h 800

Reactive State

- Rose Bengal as Photoinitiator for Two-photon Polymerization & Crosslinking

Multiphoton Fabrication of 3D Microstructures

NCKU Emblem Micro-elephant

10μm

TPEF

2

- Two-photon absorption cross section   F t( )

Excitation wavelength: to the maximum value of relative

two-photon absorption (TPA) of RB at 715 nm.

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Extracellular Matrix (ECM) Biopolymer

The morphological and

cytoskeletal responses of 3T3 fibroblasts were investigated, where the cell morphology and actin cytoskeleton became

increasingly elongated and aligned with the direction of the gradient at increasing concentration.

- Living Cells on Concentric Laminin Gradient & Fibronectin Gradient via Two-photon Crosslinking

Concentric laminin gradient with

800 x 800 microns

Fibronectin gradient with a dynamic range of nearly 40 fold in concentration

X. Chen, et al., Cell. Mol. Bioengn. 5 (2012) 307.

V. Ajeti, et al., Opt. Express 21 (2013) 25346.

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Current Challenges

in Biomedical Nonlinear Optical Microscopy

 Providing Fast Sectioning Images for Real-time Applications -> Temporal Focusing-Based Widefield Microscopy

 Breaking Diffraction Limit for Super-resolution Imaging -> NSIM (Nonlinear Structured Illumination Microscopy),

PALM (Photoactivated Localization Microscopy),

STORM (Stochastic Optical Reconstruction Microscopy), STED (Stimulated Emission Depletion), …

 Imaging Thick Tissues for Deeper Information -> Adaptive Optics System

 Multifunctional (or Selecting) Capability (ex. All Optical Histology)

-> Multiphoton-induced Laser Ablation

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• Motivation & Principle: Temporal Focusing-Based Multiphoton Microscopy and Microprocessing

• High-speed 3D Sectioning Images (over 100 Hz)

• To Head Super-resolution Microscopy

• To Improve Deep Imaging with Adaptive Optics

• Fast Multiphoton Microfabrication (3D Lithography)

• High-throughput Multiphoton-induced Laser Ablation

• Summary

Outlines

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Polygonal Scanner-Based Microscopy Multifocal Microscopy

AOD-Based Microscopy

Temporal Focusing-Based Microscopy

High-Throughput Nonlinear Optical Microscopy

P. T. C. So et al., Biophys. J. 105 (2013) 2641. Adaptive Photonics Lab, NCKU

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P. T. C. So et al., Biophys. J. 105 (2013) 2641.

High-Throughput Nonlinear Optical Microscopy

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Motivation

• Serial scanning microscopy

• Widefield microscopy

• Widefield multiphoton microscopy base on spatiotemporal focusing

Goal: To develop a state-of-the-art femtosecond laser system with fast 3D molecular imaging and microprocessing for bio-

research.

→ Less laser power, lower frame rate

→ Not fast enough

→ No depth-resolved ability

D. Oron et al., Opt. Express 13 (2005) 1468.

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Principle: Spatial and Temporal Focusing

• Spatial focusing: The pulse width remains unchanged, and the lateral beam size is focused.

• Temporal focusing: The pulse width is focused, and the lateral beam size remains unchanged.

Fourier plane Temporal focusing plane

f₁ f₁ f₂ f₂

Collimating lens Objective lens

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• Summary

Outlines

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System Setup

Ultrafast Amplifier

EMCCD

Image Lens Short-Pass Filter

Dichroic Mirror

Water Immersion Objective Lens

Sample

Collimating Lens

Relay Lens Grating

Half-Wave Plate Polarizer

• Amplifier pulse energy is 8000 times higher than the oscillator

• EMCCD offers high SNR and frame rate

• Lateral: 0.4 μm & axial: 3 μm

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Brownian Motion of Micro-Beads

• Frame rate: 100 Hz

• Field of veiw: 50 x 50 μm²

1 μm beads 0.5 μm beads

t = 0 ms t = 10 ms t = 20 ms t = 30 ms

1 μm

L.-C. Chung et al., Opt. Express 20 (2012) 8939. Adaptive Photonics Lab, NCKU

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• Summary

Outlines

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K. Weisshart et al., Adv. Opt. Technol. 2 (2013) 211.

Super-resolution Microscopy

Stochastic Optical Reconstruction Microscopy (STORM)

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Temporal Focusing via Digital Micromirror Device

Ultrafast Amplifier

EMCCD

Image Lens Short-Pass Filter

Dichroic Mirror

Water Immersion Objective Lens

Sample

Collimating Lens

Relay Lens Grating

Half-Wave Plate Polarizer

• 10th order diffraction i.e. 520 lines/μm

• Providing patterned illumination (NSIM)

J.-N. Yih et al., Opt. Lett. 39 (2014) 3134.

DMD with DLP

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0 0.5 1 1.5 2 10⁰

10¹ 10² 10³ 10⁴

k_x (m^-1)

Frequency component intensity (a.u.)

Intrinsic nonlinear two-photon excitation

Nonlinear Structured Illumination Microscopy (NSIM)

10 μm 10 μm ⁰⁰ ¹ ² ³ ⁴ ⁵ ⁶

0.2 0.4 0.6 0.8 1

Lateral distance (m)

Intensity (a.u.)

L.-C. Chung et al., Biomed. Opt. Express 5 (2014) 2526. Adaptive Photonics Lab, NCKU

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FWHM_Original= 397 nm

FWHM_NSIM= 168 nm FWHMExcitation = 3.1 μm

FWHMOriginal = 2.3 μm FWHMNSIM = 1.2 μm

-0.4 -0.2 0 0.2 0.4 0.6

0 0.2 0.4 0.6 0.8 1

Intensity (a.u.)

Original NSIM

-0.4 -0.2 0 0.2 0.4 0.6

0 0.2 0.4 0.6 0.8 1

Intensity (a.u.)

Original NSIM

-4 -2 0 2 4

0 0.2 0.4 0.6 0.8 1

Axial distance (m)

Intensity (a.u.)

-30 -20 -10 0 10 20 30 40

-0.2 0 0.2 0.4 0.6 0.8 1 1.2

Intensity (a.u.)

z-axis (m)

Excitation volum Original

NSIM

Lateral & Axial Spatial Resolutions

Axial resolution Lateral resolution

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Cytoskeleton Image with NSIM

w/o NSIM with NSIM

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Biophys. J. 67 (1994).

Science 319 (2008).

Appl. Phys. Lett. 90 (2007).

Identify the everything

without astigmatism

z-Axis Super Resolution via Astigmatism Configuration

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TFMPEM with Astigmatism Imaging

C.-H. Lien et al., Opt. Express 22 (2014) 27290.

regenerative amplifier

imaging lens

dichroic mirror

sample

collimating lens

relay lenses

DMD

xyz objective

lens

HWP LP

shutter

EMCCD camera

removable cylindrical lens

emission filter

triple-axis motorized stage z-piezo stage

power adjusting

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1000 2000 3000 4000 0

500 1000 1500 2000

Z distance (nm)

Width (nm)

Red: x Blue: y

1000 2000 3000 4000

0 500 1000 1500 2000

Z distance (nm)

W id th ( n m )

Resolution: ~ 60 nm

z step: 20 nm

Inducing Astigmatism to z -axis Localized Optical Section

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100 frames/sec at focal plane

- 500 nm beads in water

200 nm

- Beads in 55wt% glycerol with astigmatism lens

Brownian Motion of Fluorospheres

78 nm

d_rms

with astigmatism lens

123 nm

d_rms 227 nm

d_rms

w/o astigmatism lens

500 nm

BT D 3

d



 

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• Summary

Outlines

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Why Adaptive Optics?

 Image quality is seriously affected by external disturbances such as optical aberrations and environmental turbulence.

 Applications in astronomy, laser weapon, industry machining, microscopy, and free space optical communication.

With Without AO

AO

Binary star image taken by Hale Telescope in Palomar Observatory located in San Diego County, California http://www.astro.caltech.edu/

D. Débarre et al., Opt. Lett. 34 (2009) 2495.

Without AO With AO

Mouse embryo taken by TPEFM

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optical system

What Adaptive Optics System (AOS)?

• Main parts of AOS:

– Wavefront sensors – Wavefront correctors – Multichannel controllers

laser

detector

sample

active optical element

image analysis actuator

C.-Y. Chang et al., Rev. Sci. Instrum. 84 (2013) 095112.

Easily implementable FPGA-based adaptive optics system with state-space multichannel control

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Basic Concept: Temporal Compensation

Grating

Lens Objective

f_L f_L f_O f_O

dispersion

Adaptive Photonics Lab, NCKU compensation

by spatial light modulator (LCOS or DM)

biotissue

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PC with FPGA

EMCCD

blazed grating

L_imag

L₅

L₃

L₄ L_Cx1

DM

PBS L_Cy1

L_Cy2 QWP

HWP P shutter

filter

high-voltage driver

L₂

L₁

~~~~~~

ultrafast amplifier

dichroic mirror

x y

focal plane

Schematic Diagram

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R6G Fluorescent Thin Film

2.8-fold

3.7-fold

2.2-fold

5.3-fold 2.9-fold

With aberration With AOS Calibrated

Int. time: 100 ms Size: 100×100

μm² (512×512) Power: 12.3 mW

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1 μm Fluor. Beads at Different Depths in Agarose Gel

1.7-fold

1.8-fold 2.1-fold

2.0-fold 2.1-fold

z= -38 μm

z= -120 μm

Int. time: 50 ms Size: 25×25 μm² (128×128)

Power: 17.5 mW

Without AOS With AOS

C.-Y. Chang et al., Biomed. Opt. Express 5 (2014) 1768. Adaptive Photonics Lab, NCKU

Without AOS With AOS

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• Summary

Outlines

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3D Multiphoton Microfabrication (Lithography)

 Conventional fabrication technique, such as E-beam lithography, nanoimprinting lithography, etc.

Limited to 2D applications

 Two-photon excited (TPE) microfabrication achieves 3D resolution by spatially focusing light to induce nonlinear excitation within focal volume. Low fabrication speed

TPEF

Goal: To develop a high-speed fabrication technique (mass

production) which can make arbitrary 3D structure. Also, the resolution can achieve sub-micro level.

~50 μm

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Image acquired during

fabrication process Image acquired using serial scanning microscope

Solution: 2 mM RB + 75% TMPTA Objective: 40X oil 1.3

Height: ~40 μm

Fabrication time: 1 s

Multi-objects & Inspection

Image acquired using widefield microscope

30 μm

Y.-C. Li et al., Opt. Express 20 (2012) 19030. Adaptive Photonics Lab, NCKU

30 μm 30 μm 30 μm

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Time: 0.01 sec/layer, Speed enhanced: 300 times

- Gray-Level BSA Microstructures

Multiple BSA structures of different concentrations can be simultaneously achieved by selecting different pulse numbers in the designated regions with an appropriate femtosecond laser power.

Mass-Production via High-Throughput Multiphoton 3D Lithography

C.-Y. Lin et al., Opt. Express 20 (2012) 13669.

Y.-C. Li et al., J. Biomed. Opt. 18 (2013) 075004.

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• Summary

Outlines

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Multiphoton-induced Laser Ablation & Imaging

Goal: To develop high-throughput

multiphoton-induced laser ablation and fast nonlinear optical imaging

simultaneously. (for all optical histology)

 Conventional method for micro-sectioning tomography, such as diamond knife to remove the tissue is very useful.

Limited to sample preparation.

 Point-scanning femtosecond laser achieves automatically and iteratively ablation and imaging at sub-micron level for fresh tissue. Low throughput. Reconstructed Tissue Images

Ablate and Imaging

Ablate and Imaging Stack of Optical Sections

Stain Tissue

Depth, z (μm)

0 -100 -200

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Multiphoton-induced Laser Ablation of Tissues

C.-Y. Lin et al., Biomed. Opt. Express 22 (2014) submitted.

(a)

(b)

100 μm

(c) (d)

SHG images in different depths for chicken tendon after multiphoton-induced ablation machining

SHG images in

different depths @ 10, 20, 40, 60 and 70 μm with the

machining pitches of (a) 30.0 μm & (b) 20.0 μm.

Projective images (c) & (d) from the 3D reconstructed images (a) & (b).

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Bright-field images of reduced GO patterns with different pulse number.

The yellow words indicate the pulse number used for each pattern.

Bright-field images of GO array patterns.

(a) Direct ablation of GO film. (b)

Combination of reduction and ablation of GO film.

20 μm

(a) (b)

20 μm

1000 pulses 2800 pulses 4600 pulses

6400 pulses 8200 pulses 10000 pulses

20 μm

10000 1500 2000 2500 3000

1 2 3 4 5 6 7 8x 10⁴

Raman shift (cm^-1)

Intensity (a.u.)

1000 pulses 2800 pulses 4600 pulses 6400 pulses 8200 pulses 10000 pulses glass signal

GO-based Micropatterns via Multiphoton- induced Reduction and Ablation

Y.-C. Li et al., Opt. Express 22 (2014) 19726. Adaptive Photonics Lab, NCKU

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 High-speed sectioning images (up to 200 Hz) via temporal focusing-based widefield multiphoton microscopy.

 To approach super-resolution microscopy with nonlinear

structured illuminlation microscopy (NSIM) & Astigmatisum imaging.

 To improve deep imaging with adaptive optics (AO).

 High-throughput multiphoton microfabrication: To develop a high-speed fabrication technique which can make

arbitrary 3D bio-microstructures.

 Multiphoton-induced laser ablation: To develop high-

throughput multiphoton-induced laser ablation and fast nonlinear optical imaging simultaneously.

Summary

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• Funding Agencies:

National Science Council (NSC)

University Excellence Program, NCKU

• Contributors:

Yi-Cheng Li, Li-Chung Cheng,

Chia-Yuan Chang, Chi-Hsiang Lien, & Chun-Yu Lin

• Collaborators:

C. Y. Dong (Physics, NTU)

P. Campagnola (Biomedical Eng., UW-Madison) C. Xu (Appl. Phys., Cornell U.)

F.-C. Chien (Photonics, NCU)

Temporal Focusing-Based Multiphoton Microscopy and Microprocessing

Temporal Focusing-Based Multiphoton Microscopy and Microprocessing

Conventional Femtosecond Laser System

Biomedical Nonlinear Optical Microscopy

3D Reconstruction Imaging

Multiphoton Fabrication of 3D Microstructures

Extracellular Matrix (ECM) Biopolymer

Current Challenges

in Biomedical Nonlinear Optical Microscopy

Outlines

High-Throughput Nonlinear Optical Microscopy

High-Throughput Nonlinear Optical Microscopy

Motivation

Principle: Spatial and Temporal Focusing

Outlines

System Setup

Brownian Motion of Micro-Beads

Outlines

Super-resolution Microscopy

Temporal Focusing via Digital Micromirror Device

Nonlinear Structured Illumination Microscopy (NSIM)

Lateral & Axial Spatial Resolutions

Cytoskeleton Image with NSIM

z-Axis Super Resolution via Astigmatism Configuration

TFMPEM with Astigmatism Imaging

1000 2000 3000 4000

0 500 1000 1500 2000

Z distance (nm)

W id th ( n m )

Inducing Astigmatism to z -axis Localized Optical Section

Brownian Motion of Fluorospheres

Outlines

Why Adaptive Optics?

What Adaptive Optics System (AOS)?

Basic Concept: Temporal Compensation

R6G Fluorescent Thin Film

1 μm Fluor. Beads at Different Depths in Agarose Gel

Outlines

3D Multiphoton Microfabrication (Lithography)

Multi-objects & Inspection

Mass-Production via High-Throughput Multiphoton 3D Lithography

Outlines

Multiphoton-induced Laser Ablation & Imaging

Multiphoton-induced Laser Ablation of Tissues

GO-based Micropatterns via Multiphoton- induced Reduction and Ablation

Summary

Acknowledgements