Chapter 9: Procedures and Technical References
Adding an Objective
Selecting Lens from Database
• Find the appropriate lens in the Utilities, Revise Lens Database and record the
lens ID number
Pixel Size Calibration
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Max image size, best camera speed, magnification slider at 1X.
Focus on the silicon target grid (9.9 µm/square).
Align the grid image to vertical/horizontal. Maximize the image.
Open Tools - Measure Distance.
Set the Units to Pixels.
Draw a line across the screen from a point on the top left square to the same
point on the top right square. If a delta of more than four (4) in the vertical is
measured re-align the slide.
Repeat this process at the middle and bottom.
Count and record the number of grid elements and record distance in Pixels.
Repeat the above three steps in the vertical direction.
Calculate Size
Pixel Size (um) = ___________________9.9um/box__________________
Σ (Measured pixels)/(Number of grids per measurement)
Add Lens Information to Resolve3D
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Open softWoRx - Utilities - Revise Microscope Configuration. (Root password
required.)
Caution: When modifying and system files DO NOT use tabs or other unusual
characters. Spaces should be used to separate items for clarity.
• Find the microscope specifications
• Pick a spot in the list that is convenient. Make sure all relevant information is
added to the same relative location.
• Increase the number of lenses (MS_Number_Lenses:) by 1.
• Add the desired name of the objective to names (MS_Lens_Names:) Ex.
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100Xoil, 60Xwater
Add the lens NA (this is not used but may not work well with out the place holder
Add the lens ID
Save and Exit. Quit and restart Resolve3D.
Example of a typical Resovle3D.sys file:
# Microscope Specifications:
# ``````````````````````````
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Chapter 9: Procedures and Technical References
MS_Mag_Factor: 1.0
(additional magnifiers, e.g. IMT2's have a 1.5
optivar)
MS_Number_Lenses: 6
MS_Lens_Names: 100X 60X 60w 40X 20X 4X
MS_Pixel_Sizes: 0.0671805 0.11340 0.1091912 0.16795125 .3360 1.550
MS_Lens_NAs: 1.35 1.40 1.35 1.35 0.75 0.16 (obsolete, see Lens ID
numbering system)
MS_Lens_ID_Numbers: 10003 10602 10603 10403 10205 10106 (refer to Lens
database)
#------------------------------------------------------------------------------
Collecting a Point Spread Function
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Set the excitation and emission filters on Rh., the eyepiece to T-Red (or Rh.) and
the ND to 0.000.
Set the exposure time to 0.5 sec.
Place the Bead Slide in Position and focus on a bead. There are two different
sizes of beads. The larger is to assist in focusing and the smaller will be used for
the psf.
Center the small bead in the eyepiece and acquire an image. Center a bead with
Resolve3D. If there are more than one beads in the imagine change the image
size to 128 x 128 or less to include only one bead in the image.
Move through Z sections to find the greatest intensity. Increase the exposure time
to read approximately 2000. (If this intensity cannot be reached with an exposure
time close to 0.5 try finding another bead.)
Select the proper oil using the following experiment.
Take a Z section at 0.30 for 40 images. Set the excitation and emission filter
wheel to Rh. Ensure the correct exposure time is shown.
Click Save and Exit, Run, Do It.
Name the experiment with the object magnification and oil NA.
Repeat until the best oil is determined.
Once the proper oil has been established, run the final PSF below.
Design an experiment to take a Z section at 0.10 for 128 images. Set the
excitation and emission filter wheel to Rh. Ensure the correct exposure time is
shown.
Click Save and Exit.
Click Edit. Modify the experiment macro by scrolling down to the Z Sectioning
Loop. Replace the line where it says “CCD<space>Exposure time” with
“AVG<space>4”
Click Save and Exit, Run, Do It.
The name you give the file is very important. It needs to be in this format:
.Lens Maginification_NA_psf
Ex. 60X_140_psf
Click OK
When the experiment is done running move the file and the log file to
/usr/local/softWoRx/data
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Chapter 9: Procedures and Technical References
Creating an OTF
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Select Conversion from the Main Menu and Convert PSF to OTF. Select the
correct file for Input and Do It.
The new OTF file will have the extension .r3d_otf. Copy the file from
/usr/local/softWoRx/data to /usr/local/OTF and rename it in the following format:
Lens Magnification_NA.oft
Ex. 60X_140.otf
The system comes with OTF’s for the existing lenses. It is best to not overwrite
the existing OTF’s. If a lens has been replaced, rename the old .otf before
overwriting it. If the lens is in addition to those already present the file needs to
be added to the configuration file (/usr/local/softWoRx (or
dv2.10)/config/sytem.dvrc. See below)
Modify softWoRx to use New Objective and OTF
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Add the otf file to the system.dvrc file for automatic entry into the deconvolve
function (when the lens is properly selected during the experiment the correct
OTF file will automatically come up.)
Open a unix shell and type "su" and the root password.
Type nedit /usr/local/softWoRx/config/system.dvrc
Find the section labeled "Lens-to-OTF matching"
Follow the instructions give for the OTF file as shown below.
Example of a typical lens section of the system.dvrc file:
# Lens-to-OTF matching. These are of the form LENS_<lensIDNumber>_OTF and
# are defined to be the file name in the OTF directory of the OTF that
# is to be used for this lens ID.
LENS_12_OTF
60X_140_sample.otf
LENS_10602_OTF 60X_140.otf
LENS_10003_OTF 100X_135.otf
LENS_10403_OTF 40X_135_sample.otf
LENS_10603_OTF 60Xw_120.otf
LENS_10205_OTF 20X_0.75c.otf
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