RICS Simulations (PowerPoint) by yurtgc548

VIEWS: 7 PAGES: 17

									RICS Simulations
for two photon excitation
Opening page of GLOBALS for
Images program: Click on the
SimFCS buttom
First page of SimFCS:                                 Under ACTIONS in the
first page of SIMFCS Go to “simulate FCS data”




                                                 Click on Parameter
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
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
                                      Clock     brightness2 of 4000000 as seen
                                   frequency    by channel 2.
                                   determines
                                  the maximum   This help file was created with
                                    Diffusion   Help & Manual (www.ec-
                                     allowed
                                                software.com).
  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.
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”
Save your Simulated Data


                       Select directory to
                       save file




Add comments
Once you have plotted the particles and saved your files, click on the SimFCS
“Analysis” (the background Screen of SimFCS) and select “RICS”
RICS:
 Enter total
 number of
 frames
 collected
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
                                                             Additional Selections for images:

                                                            Must have the frame you want to begin
                                   Image 2:                 on (frame 1 or 2 etc…). You can choose
                                   reserved                 to delete a frame if you don’t want to
                                                            consider it for calculation.
                                   For image of
                                   subtracted
                                   background                  Determines last frame # to be
                                                               analyzed
        Image 1: original file



                                 Reserved for
       Image 3:
       Reserved for
                                 ACF
       Autocorrelation           Of the horizontal
       plot                      and vertical
                                 direction
                                                  Fitting the ACF
                            1           1)This selects the size you want to
                    2                   analyze from frame 3 (SPATIAL
                    3                   CORRELATION FFT)
                                        2) Dwell time at each pixel (1/Hz)
                                        3)Line time Pixel seq = (256x64us)
                        4               4)Pixel size is fixed to 0.05
                                5   6
                                        5)From simulation PSF radial waist
FRAME 3
                                        (go back to slide 4 for value)

                                        6) Perform Fit

                                        7) Results will be plotted on the
                                          next slide

 For Simulations:
 DO NOT Check
 Any of these
For additional simulations:
 Close fit and plot of fitted spatial
  correlation
 Close RICS
 Back to SimFCS first page
 Click on Simulate data
  Simulation: D=5m2/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
Simulation: 20m2/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
Simulation: D=100m2/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
Special conditions:

								
To top