To add an objective:

1. From the main softWoRx window, choose Utilities | Revise Lens Database to open the dvlenses.tab file. Then follow the instructions at the top of this file to add a lens ID to the lens database.

2. Place the new objective in the Objective Turret and set the maximum image size, best camera speed, and axillary magnification slider at 1X.

3. In the Resolve3D window, set Excitation to RD-TR-PE, Emission to FITC, and %T to 0.1%. Then set the eyepiece filter to FITC.

4. Place the silicon target grid on the stage and focus it (9.995 um/square). Align the grid image to the vertical and horizontal axis and maximize the image.

5. Switch the port selector to Camera and click Acquire. Leave Data Collection window 21 open.

6. In the Image window, choose Tools | Measure Distance. Then set the Units to Pixels in the Measure Distance window.

7. Draw a line across the image from a point on the top left square to a point in the same relative position on the top right square.

   Note:  Your measurement will be more accurate if you choose points on the vertices of the squares.

    

8. If the vertical delta is more than four pixels, re-align the slide. Repeat this process at the middle and bottom. Then count and record the number of grid elements (3 in the above image) and record the distance in pixels.

9. Repeat Steps 7 and 8 in the vertical direction.

10. Calculate the pixel size (your calculation should be accurate to four decimal places)for each of the six measurements (top, middle, bottom, left, center, and right) as follows:

11. Average the six pixel sizes to obtain the correct pixel size.

12. From the softWoRx main menu, choose Utilities | Revise Microscope Configuration. Then enter the root password to open the RESOLVE3D.SYS file.  

13. In the Microscope Specifications section:

    • Increase the number of lenses next to MS_Number_Lenses: by 1.

    • Add the name of the objective to MS_Lens_Names: (e.g., 100Xoil, 60Xwater).

    • Add the lens NA value to the MS_Lens_NAs:

    • Add the lens ID to MS_Lens_ID_Numbers:.

14. Enter the pixel size for the new lens.

    For example:

    If the desired lens is 40X/1.35 with ID=10403 (the third lens in the list), then the pixel size is 0.1656.

    MS_Lens_Names: 10X 20X 40X 60X 100X 

    MS_Pixel_Sizes: 0.6680 0.3313 0.1656 0.1103 0.06631 

    MS_Lens_NAs: 0.40 0.75 1.35 1.40 1.40

    MS_Lens_ID_Numbers: 10105 10205 10403 10602 10002