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Laser scanning confocal microscope

Photonics for bio-imaging and bio-sensing Rongqing Hui Dept. Electrical Engineering & Computer Science, The University of Kansas, Lawrence Kansas. Fluorescently labeled tissue. 3-demensional translation stage. Detector pin-hole. Photo detector. Laser source. Lens. Lens. Beam splitter.

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Laser scanning confocal microscope

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  1. Photonics for bio-imaging and bio-sensingRongqing HuiDept. Electrical Engineering & Computer Science,The University of Kansas, Lawrence Kansas

  2. Fluorescently labeled tissue 3-demensional translation stage Detector pin-hole Photo detector Laser source Lens Lens Beam splitter Lens Focal plane Out of focus Laser scanning confocal microscope Advantages: out-of-focus background can be removed by the small aperture in front of the detectorAllows 3-D imagingDisadvantages:Photon bleach because the use of visible light (400 – 600 nm)sensitive to background light

  3. Excited state Excited state ~800nm Fluorescence Fluorescence ~400nm ~800nm Ground state Ground state One photon excitation Two photon excitation Concept of Two-photon excitation

  4. Optical power spreading Better focus Two-photon microscopy Use near infrared wavelength -less photon bleach and less scattering when penetrating through tissueDetection at wavelength far away from excitation – no background noise due to Raman and direct fluorescenceTwo-photon excitation is proportional to the square of the power density – smaller focus point, minimum off-focus excitation and no need of a pin-hole in front of the detector

  5. Why two-photon microscopy is not popular so far ? • Requires very high peak optical power because of the low 2-photon excitation efficiency • Ti:Sapphire lasers have to be used to provide kW level peak optical power: big in size, very expensive, needs tweaking from time to time • Difficult to deliver femtosecond optical pulse from laser to microscope

  6. Saturable absorption mirror Faraday rotator Doped MM fiber Faraday rotator Partial reflection Pump High power femtosecond fiber laser Popular wavelengths: 1550nm: Erbium doped fiber 1064nm: Ytterbium doped fiber 780nm: Frequency-doubling the output of 1550nm fiber laser using periodically polled LiNbO3 (PPLN)

  7. Enabling technologies • Improvement in rear-earth doped optical fibers • Excite only the fundamental mode of a doped multi-mode fiber: breakthrough power limitations • Use Faraday rotator: eliminate polarization sensitivity • Saturable absorption mirror: pulse shaping

  8. Femtosecond fiber laser at 780nm(fixed wavelength)

  9. Highly nonlinear Photonic crystal fiber • Periodic air holes in the core • Very high nonlinearity • Zero-dispersion wavelength shifted to 700nm • Support Raman shifted soliton in NIR

  10. 6m photonic crystal fiber Optical spectrum analyzer Femtosecond fiber laser Mechanical translation stage Wavelength shift using photonic crystal fiber

  11. Power spectral density (linear) Wavelength (nm) Pulse wavelength shift due to power change(from 1mW to 4mW average power) Fundamental soliton condition:

  12. Pulse wavelength shift due to power change(Computer simulation)

  13. Microscope Source PCF-1 Filter 780 nm fiber laser AOM Pulse compressor Sample PCF-2 1050 nm fiber laser AOM Detector Digital control Register Image @ l1 Memory & signal processing Image @ l2 Control and signal processing Image @ ln Fig.3. Block diagram of the proposed wavelength switchable two-photon equipment Multi-color two-photon fluorescent microscopy using TDM

  14. Absorption and emission spectrum of Alexa fluorescent dyes

  15. At depth 2 At depth 1 Measured Two-photon fluosphere images

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

  17. Radial and axial two-photon intensity profiles(excited at 920nm)

  18. Observation volume area Focal volume area Fluorescent correlation spectroscopy (FCS)(measured at 780nm)

  19. Bio-assay based on flow cytometryCount and analyze individual particles in a fluid channel

  20. UV Photo-mask Spin-coat SU-8 photo-resistor Silicon substrate Silicon substrate Flow-cytometer on chip:Create channels

  21. UV Photo-mask Spin-coat second layer SU-8 Silicon substrate Flow-cytometer on chip: Create grooves Silicon substrate

  22. PDMS Molding PDMS Silicon substrate

  23. Input fiber Detection fiber Buffer Buffer Detection fiber Sample Picture of a flow chip

  24. Buffer Buffer Sample Demonstration of sheath flow Sheath creation

  25. Computer D/A converter Laser PMT Fluid pump (water) Fluid pump (water) Waste Fluid pump (sample) Measurement

  26. PMT output (V) Time (s) Measurementpassing diluted yeast solution through the cytometer

  27. Clean water 4 times increase of yeast concentration between each measure

  28. 10log(N) 10log(V) Cumulative summation histogram V: threshold n: number of counts

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