1. RICS Simulations for two photon excitation

2. Opening page of GLOBALS for Images program: Click on the SimFCS buttom

3. First page of SimFCS: Under ACTIONS in the first page of SIMFCS Go to “simulate FCS data” Click on Parameter

4. Set up Point Spread Function (PSF) Pixel size In the PSF page you can select the shape of the point spread function. The 3-D Gaussian has independent radial and axial waist, while for the Gaussian-Lorentzian, the z-extension of the function depends on the radial waist. Also, the 3-D Gaussian is not squared to provide intensity while the Gaussian-Lorentzian is squared. Radial and axial waist have micron units. The radial waist for x and y should be the same (~0.25-0.55) and the axial waist is five times the radial waist. The box size parameters determine the half-size of the box used for the simulation. The conversion factor between physical size and granularity of the box is that each unit of the box corresponds to 50 nm. Generally, this is enough for most optical instruments. A box size of 128 is a good starting point. When you have set up PSF click on particles button Is fixed

5. Click on Particles: Select Number of molecules, brightness and shape (1-4) Clock frequency determines the maximum Diffusion allowed Use the particle editor page to select properties of the particles for simulation. The are a maximum of 50 columns, which represents 50 different types of molecules. The total number of molecules (the sum of all species) cannot exceed 4000. The first column determines how many molecules of a given property are in the box. The example above has 100 particles with a brightness1 (as seen by channel 1) of 4000000 counts/second/molecule, diffusion coefficient of 0.01 um2/s, rotational rate of 10 and brightness2 of 4000000 as seen by channel 2. This help file was created with Help & Manual (www.ec- software.com).

6. Exclude: Misc., Stick/Disk, and Reaction Click on Time/Scan For a RICS simulation change the Clock frequency to correspond to the maximum allowable diffusion rate. For example, a clock frequency of 15,625Hz corresponds to a pixel dwell time of 64  s. In another example a frequency of 62,500Hz corresponds to a pixel dwell time of 16  s. CYCLES: Select between 50-100 with 50 being a good start. When you run complex simulations, More cycles are useful.

7. Now you are ready to begin the simulations. Click on START SIMULATION Click on Diffusion in a volume The simulation will start. The field will be empty. If you want to see the particles select “plot particles”

8. Save your Simulated Data Add comments Select directory to save file

9. Once you have plotted the particles and saved your files, click on the SimFCS “Analysis” (the background Screen of SimFCS) and select “RICS”

10. RICS: Enter total number of frames collected

11. 1. For saved data: Go to “File” and open your file under “open image sequence (bin)” 2.Set first and last frames to be analyzed (add # of frames in“frames to average for ACF”) 3.Under tools: select “Spatial Correlation” 4.Click on Fit 4 Image 3: Reserved for Autocorrelation plot Image 2: reserved For image of subtracted background Must have the frame you want to begin on (frame 1 or 2 etc…). You can choose to delete a frame if you don’t want to consider it for calculation. Reserved for ACF Of the horizontal and vertical direction Image 1: original file Additional Selections for images: Determines last frame # to be analyzed

12. Fitting the ACF 1)This selects the size you want to analyze from frame 3 (SPATIAL CORRELATION FFT) 2) Dwell time at each pixel (1/Hz) 3)Line time Pixel seq = (256x64us) 4)Pixel size is fixed to 0.05 5)From simulation PSF radial waist (go back to slide 4 for value) 6) Perform Fit 7) Results will be plotted on the next slide 1 2 3 4 5 For Simulations: DO NOT Check Any of these FRAME 3 6

13. For additional simulations: Close fit and plot of fitted spatial correlation Close RICS Back to SimFCS first page Click on Simulate data

14. Simulation: D=5  m 2 /s Convergence is OK (5) Result of fit: Chisquare= 0.0016462301 File: Size =64 Pixel size= 0.05000 Pixel time=64 (Frequency=15,625Hz) Pixel seq =16.38 (256x64us) Wo( in um)= 0.50000 G(0) = 5.71675 D = 5.33750 Bkgd = 0.00000 Radius = 40.00000 Amplitude = 0.00000 Blinking t = 0.000000 Analog = 0.000000 X-offset = 0.000000 Y-offset = 0.000000 Using 3-D diffusion formula Using one photon formula Size of original image=256

15. Simulation: 20  m 2 /s Convergence is OK (5) Result of fit: Chisquare= 0.0016739755 File: Size =64 Pixel size= 0.05000 Pixel time=16 (Frequency=62,500Hz) Pixel seq =4 (256x16us) Wo( in um)= 0.50000 G(0) = 5.40853 D = 21.24932 Bkgd = 0.00000 Radius = 40.00000 Amplitude = 0.00000 Blinking t = 0.000000 Analog = 0.000000 X-offset = 0.000000 Y-offset = 0.000000 Using 3-D diffusion formula Using one photon formula Size of original image=256

16. Simulation: D=100  m 2 /s (not confined) Convergence is OK (5) Result of fit: Chisquare= 0.0010853632 File: Size =64 Pixel size= 0.05000 Pixel time=4 (Frequency=250,000Hz) Pixel seq =1.024(256x4us) Wo( in um)= 0.50000 G(0) = 5.89838 D = 97.27914 Bkgd = 0.00000 Radius = 40.00000 Amplitude = 0.00000 Blinking t = 0.000000 Analog = 0.000000 X-offset = 0.000000 Y-offset = 0.000000 Using 3-D diffusion formula Using one photon formula Size of original image=256

17. Special conditions: