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

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

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

R. Hui Two-photon microscopy Optical power spreading Better focus Use near infrared wavelength -less photon bleach and less scattering when penetrating through tissue Detection at wavelength far away from excitation – no background noise due to Raman and direct fluorescence Two-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

R. Hui Why two-photon microscopy is not popular so far ? n Requires very high peak optical power because of the low 2-photon excitation efficiency n 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 n Difficult to deliver femtosecond optical pulse from laser to microscope

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

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

R. Hui Femtosecond fiber laser at 780nm (fixed wavelength)

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

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

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

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

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

R. Hui Absorption and emission spectrum of Alexa fluorescent dyes

R. Hui Measured Two-photon fluosphere images At depth 1 At depth 2

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

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

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

R. Hui Bio-assay based on flow cytometry Count and analyze individual particles in a fluid channel

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

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

R. Hui Silicon substrate Molding PDMS

R. Hui Picture of a flow chip Input fiber Detection fiber Buffer Sample

R. Hui Demonstration of sheath flow Buffer Sample Sheath creation

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

R. Hui Measurement passing diluted yeast solution through the cytometer Time (s) PMT output (V)

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

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