RICS Simulations for two photon excitation

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RICS Simulationsfor two photon excitation
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Opening page of GLOBALS for Images program Click
on the SimFCS buttom
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First page of SimFCS Under ACTIONS in the first
page of SIMFCS Go to simulate FCS data
Click on Parameter
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Set up Point Spread Function (PSF)
Pixel size
Is fixed
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
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Click on Particles Select Number of molecules,
brightness and shape (1-4)
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).
Clock frequency determines the
maximum Diffusion allowed
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Exclude Misc., Stick/Disk, and Reaction Click on
Time/Scan
CYCLES Select between 50-100 with 50 being a
good start. When you run complex
simulations, More cycles are useful.
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.
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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
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Save your Simulated Data
Select directory to save file
Add comments
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Once you have plotted the particles and saved
your files, click on the SimFCS Analysis (the
background Screen of SimFCS) and select RICS
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RICS
Enter total number of frames collected
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• 1. For saved data Go to File and open your
  file under open image sequence (bin)
• Set first and last frames to be analyzed (add
  of frames inframes to average for ACF)
• Under tools select Spatial Correlation
• Click on Fit

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Additional Selections for images
Must have the frame you want to begin on (frame 1
or 2 etc). You can choose to delete a frame if
you dont want to consider it for calculation.
Image 2 reserved For image of subtracted backgrou
nd
Determines last frame to be analyzed
Image 1 original file
Reserved for ACF Of the horizontal and vertical
direction
Image 3 Reserved for Autocorrelation plot
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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
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2
3
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FRAME 3
For Simulations DO NOT Check Any of these
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For additional simulations

• Close fit and plot of fitted spatial correlation
• Close RICS
• Back to SimFCS first page
• Click on Simulate data

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Simulation D5?m2/s
Convergence is OK (5) Result of fit Chisquare
0.0016462301 File Size 64 Pixel size
0.05000 Pixel time64 (Frequency15,625Hz) 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 image256
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Simulation 20?m2/s
Convergence is OK (5) Result of fit Chisquare
0.0016739755 File Size 64 Pixel size
0.05000 Pixel time16 (Frequency62,500Hz) 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 image256
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Simulation D100?m2/s(not confined)
Convergence is OK (5) Result of fit Chisquare
0.0010853632 File Size 64 Pixel size
0.05000 Pixel time4 (Frequency250,000Hz) 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 image256
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Special conditions