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R. Hui
Photonics for bio-imaging and bio-Photonics for bio-imaging and bio-sensingsensing
Rongqing HuiRongqing HuiDept. Electrical Engineering & Computer Science,Dept. Electrical Engineering & Computer Science,
The University of Kansas, Lawrence KansasThe University of Kansas, Lawrence Kansas
R. Hui
Laser scanning Laser scanning confocal microscopeconfocal microscope
Photo detector
Detector pin-hole
Focal plane
Laser source
Out of focus
Lens
Lens
Lens
Beam splitter
3-demensional translation stage
Fluorescently labeled tissue
Advantages: Advantages: out-of-focus background can be out-of-focus background can be removed by the small aperture in front removed by the small aperture in front of the detectorof the detectorAllows 3-D imagingAllows 3-D imaging
Disadvantages:Disadvantages:Photon bleach because the use of Photon bleach because the use of visible light (400 – 600 nm)visible light (400 – 600 nm)sensitive to background lightsensitive to background light
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Concept of Two-photon excitationConcept of Two-photon excitation
~400nmFluorescence
Ground state
Excited state
One photon excitation
~800nmFluorescence
Ground state
Excited state
~800nm
Two photon excitation
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Two-photon microscopyTwo-photon microscopy
Optical power spreading
Better focus
Use near infrared wavelength -less Use near infrared wavelength -less photon bleach and less scattering when photon bleach and less scattering when penetrating through tissuepenetrating through tissue
Detection at wavelength far away from Detection at wavelength far away from excitation – no background noise due excitation – no background noise due to Raman and direct fluorescenceto Raman and direct fluorescence
Two-photon excitation is proportional Two-photon excitation is proportional to the square of the power density – to the square of the power density – smaller focus point, minimum off-smaller focus point, minimum off-focus excitation and no need of a pin-focus excitation and no need of a pin-hole in front of the detectorhole in front of the detector
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Why two-photon microscopy is not Why two-photon microscopy is not popular so far ?popular so far ?
Requires very high peak optical power because of Requires very high peak optical power because of the low 2-photon excitation efficiency the low 2-photon excitation efficiency
Ti:SapphireTi:Sapphire lasers have to be used to provide kW lasers have to be used to provide kW level peak optical power: big in size, very level peak optical power: big in size, very expensive, needs tweaking from time to timeexpensive, needs tweaking from time to time
Difficult to deliver femtosecond optical pulse from Difficult to deliver femtosecond optical pulse from laser to microscopelaser to microscope
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High power femtosecond fiber laserHigh power femtosecond fiber laser
Pump
Saturable absorption mirror
Faraday rotator
Faraday rotator
Partial reflection
Doped MM fiber
Popular wavelengths: 1550nm: Erbium doped fiber1064nm: Ytterbium doped fiber780nm: Frequency-doubling the output of 1550nm fiber laser
using periodically polled LiNbO3 (PPLN)
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Enabling technologiesEnabling technologies
Improvement in rear-earth doped optical fibers Improvement in rear-earth doped optical fibers
Excite only the fundamental mode of a doped multi-Excite only the fundamental mode of a doped multi-mode fiber: breakthrough power limitationsmode fiber: breakthrough power limitations
Use Faraday rotator: eliminate polarization Use Faraday rotator: eliminate polarization sensitivitysensitivity
Saturable absorption mirror: pulse shapingSaturable absorption mirror: pulse shaping
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Femtosecond fiber laser at 780nmFemtosecond fiber laser at 780nm(fixed wavelength)(fixed wavelength)
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Highly nonlinear Photonic crystal fiberHighly nonlinear Photonic crystal fiber
Periodic air holes in the corePeriodic air holes in the core
Very high nonlinearityVery high nonlinearity
Zero-dispersion wavelength Zero-dispersion wavelength shifted to 700nmshifted to 700nm
Support Raman shifted Support Raman shifted soliton in NIRsoliton in NIR
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Wavelength shift using photonic crystal fiberWavelength shift using photonic crystal fiber
Femtosecond fiber laser
Optical spectrum analyzer
Mechanical translation stage
6m photonic crystal fiber
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Pulse wavelength shift due to power changePulse wavelength shift due to power change((from 1mW to 4mW average powerfrom 1mW to 4mW average power))
Power spectral density (linear)
Wavelength (nm)
Fundamental soliton condition:
1)(
22
20
D
PTc
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Pulse wavelength shift due to power changePulse wavelength shift due to power change((Computer simulationComputer simulation))
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Multi-color two-photon fluorescent Multi-color two-photon fluorescent microscopy using TDMmicroscopy using TDM
780 nm fiber laser
PCF-1
AOM
Sample
Detector
Register
Image @ 1
1050 nm fiber laser
AOM
PCF-2
Digital control
MicroscopeSource
Memory & signal processing
Image @ 2
Image @ n
Filter
Control and signal processing
Pulse compressor
Fig.3. Block diagram of the proposed wavelength switchable two-photon equipment
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Absorption and emission spectrum of Alexa Absorption and emission spectrum of Alexa fluorescent dyesfluorescent dyes
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Measured Two-photon fluosphere imagesMeasured Two-photon fluosphere images
At depth 1 At depth 2
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200400
600800
10001200
1400 0200
400600
8001000
12001400
0
50
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0500
10001500
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50
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Radial and axial two-photon intensity profilesRadial and axial two-photon intensity profiles(excited at 780nm)(excited at 780nm)
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200400
600800
10001200
1400
0
10
20
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0
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600800
10001200
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Radial and axial two-photon intensity profilesRadial and axial two-photon intensity profiles(excited at 920nm)(excited at 920nm)
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0.1 1 10
0.00
0.02
0.04
0.06
0.08
0.10
0.00
0.02
0.04
0.06
G()
(ms)
CaM-34-fl G
()
fluorescein50% Glycerol
Focal volume area
Observation volume area
Fluorescent correlation spectroscopy (FCS)Fluorescent correlation spectroscopy (FCS)((measured at 780nmmeasured at 780nm))
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Bio-assay based on flow cytometryBio-assay based on flow cytometry Count and analyze individual particles in a fluid channelCount and analyze individual particles in a fluid channel
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Silicon substrate
Spin-coat SU-8 photo-resistor
Photo-mask UV
Silicon substrate
Flow-cytometer on chip:Flow-cytometer on chip:Create channelsCreate channels
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Silicon substrate
Spin-coat second layer SU-8
Photo-mask UV
Silicon substrate
Flow-cytometer on chip: Flow-cytometer on chip: Create groovesCreate grooves
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Picture of a flow chipPicture of a flow chip
Input fiberDetection fiber
Detection fiber
Buffer
Buffer
Sample
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Demonstration of sheath flowDemonstration of sheath flow
Buffer
Buffer
Sample
Sheath creation
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MeasurementMeasurement
Laser
Fluid pump(water)
Fluid pump(sample)
Fluid pump(water)
PMT
D/A converter Computer
Waste
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MeasurementMeasurementpassing diluted yeast solution through the cytometerpassing diluted yeast solution through the cytometer
Time (s)
PM
T o
utp
ut
(V)
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