1. James A Germann, Brian K Canfield, Jason K King, Alexander Terekhov, Lloyd M Davis

2.  Fluorescence Correlation Spectroscopy (FCS) - analysis of time-dependent fluorescence fluctuations  Fluorescence Cross Correlation Spectroscopy – FCS between more than one volume  Correlation functions measure diffusion and velocity through solution  Schwille, P., F.J. MeyerAlmes, and R. Rigler, Biophysical Journal, 1997. 72(4): p. 1878-1886.  Webb, W.W., Applied Optics, 2001. 40(24): p. 3969-3983. Photon at time t

3. y x z 1 2 3 4 x y 1 2 3 4  Four foci in a tetrahedral configuration  Separation of foci provide a standard ruler for flow measurements  Collect four autocorrelation curves and twelve cross- correlation curves (three cross- correlations for each focus)

4.  Advantages Naturally depth selective Low background Large difference between excitation and fluorescence wavelengths  Disadvantages Higher laser power Requires pulsed laser line Single-Photon Two-Photon

5. m5 m3 bs2 laser beam input Ti:Sapphire pulses, 76MHz A B 1 2 m1 m2 50% Beamsplitter bs1 bs3 m4 Four paths: A1 = shortest A2 = A1 + 3.3 ns B1 = A1 + 6.6 ns B2 = A1 + 9.9 ns Microscope objective Four-beam alignment

6. m5 m3 bs2 laser beam input Ti:Sapphire pulses, 76MHz AB m1 m2 50% Beamsplitter bs1 bs3 m4   A1,B2 displaced ↑  Microscope objective 1 2 Four paths: A1 = shortest A2 = A1 + 3.3 ns B1 = A1 + 6.6 ns B2 = A1 + 9.9 ns  Four-beam alignment

7.  m5 m3 bs2 laser beam input Ti:Sapphire pulses, 76MHz A B Microscope objective m1 m2 50% Beamsplitter bs1 bs3 m4   (out of plane)   A1,B2 displaced ↑ A2,B2 displaced →  1 2 Four paths: A1 = shortest A2 = A1 + 3.3 ns B1 = A1 + 6.6 ns B2 = A1 + 9.9 ns  Four-beam alignment

8. m5 m3 bs2 laser beam input Ti:Sapphire pulses, 76MHz A B m1 m2 50% Beamsplitter bs1 bs3 m4   (out of plane)    A1,B2 displaced ↑ A2,B2 displaced → B1,B2 focused   1 2 Four paths: A1 = shortest A2 = A1 + 3.3 ns B1 = A1 + 6.6 ns B2 = A1 + 9.9 ns   Microscope objective Four-beam alignment

9. Photodiode 532 nm diode laser (alignment) Ti-Sapphire beam

10. removable mirror piezo stage pinhole lens mirror Dichroic Mirror SPAD

11. A1 A2 B1B2A1A2B1B2 No crosstalk Crosstalk

12.  Rhodamine B pumped through capillary  One-dimensional flow  Flow velocity dependent on bulk flow rate, cross-sectional area, and distance from capillary walls -T. S. Lundgren J. Basic Eng 86:620-626, 1964

13.  Laminar flow created along the optical axis with crossed channel microfluidics  Microfluidics created by etching a channel on two coverslips and bonded via fusion bonding  Sample fluid driven through the microfluidic at a constant speed. Two crossed microchannels for 4-beam 3-D trapping

14.  Simulated fluorescing particles moving through four foci  Fluorescence photons sorted based on time of counting  Correlation curves created from photon statistics  Three-dimensional Gaussian model applied to correlation functions  Flow velocities globally fit

16.  Three-dimensional Gaussian is not accurate enough.  New model using Gaussian-Lorentzian Simulation Parameters Vx=1 mm/s Vy= 2mm/s Vz=3 mm/s Fit Parameters Vx=.66 mm/s Vy=1.41 mm/s Vz=2.13 mm/s

17.  FCCS performed in three-dimensions simultaneously  Four temporally and spatially distinct foci created using a Mach-Zehnder interferometer configuration  Fluorescence separated with time-gated photon counting  Calibration experiments with square bore capillary  Three-dimensional flow created with cross- channel device  Flow simulated with Monte Carlo simulations