R. Hui Photonics for bio-imaging and bio- sensing Rongqing Hui Dept. Electrical Engineering &...

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R. Hui Photonics for bio-imaging Photonics for bio-imaging and bio-sensing and bio-sensing Rongqing Hui Rongqing Hui Dept. Electrical Engineering & Computer Science, Dept. Electrical Engineering & Computer Science, The University of Kansas, Lawrence Kansas The University of Kansas, Lawrence Kansas

Transcript of R. Hui Photonics for bio-imaging and bio- sensing Rongqing Hui Dept. Electrical Engineering &...

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)(

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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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Radial and axial two-photon intensity profilesRadial and axial two-photon intensity profiles(excited at 780nm)(excited at 780nm)

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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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G()

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CaM-34-fl G

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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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Silicon substrate

MoldingMolding

PDMS

PDMS

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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

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Clean water

4 times in

crease of yeast concen

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betw

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measu

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10log(N)

10log(V)

Cumulative summation Cumulative summation histogramhistogram

V

dnnHistN )(

V: thresholdn: number of counts