iCys User Guide

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Administrator Tasks Defining Custom Carriers For example: Carrier Side View Bottom Thickness Base to Well Height Outside edge of carrier { If you do not know the measurements for Base to Well Height or Bottom Thickness, use the method described in “Determining the Microplate Z Parameters” on page C-14. When you have entered these measurements, return to Focus Methods, below. ✧ Retraction Offset—Only used with some Freeform Carriers. For more information, see “Defining a Freeform Carrier” on page C-16. ❍ Focus Methods—Specify the preferred (or required) focus method. ✧ Bottom then Top—For thicker plastic-bottomed plates (greater than 400µ) especially; iCys will focus first on the plate bottom, then translate up by the bottom thickness to the plate top and refocus. ✧ Bottom Only—For glass-bottomed or thinner plastic-bottomed (less than 400µ) plates, iCys focuses directly on the bottom then moves up by the bottom thickness plus other offsets to bring the sample into focus. ✧ Default—To allow your users to select a focus method (as discussed in “Focus and Performance” on page 3-33), select both focus methods and then select the method that you want to use as a default setting. Note: If the Bottom Thickness is less than 400µ, select Bottom Only as the only available focus method. iCys will set the default focus method to match. ❍ Test Autofocus—To test whether you have the values for your carrier set correctly, you can run Test Autofocus. To test under working conditions, CompuCyte suggests that you fill the test wells with water or another buffered solution (such as phosphate-buffered saline [PBS].) Test Autofocus tries to focus the objective correctly on the carrier and produces a result number, as follows: ✧ If the Test Autofocus Result is greater than 800, the parameters are correctly set. ✧ If Autofocus did not work, an error message will display. Check the General tab of the Instrument Setup window (and the Focus parameter in the task bar) to ensure that Autofocus is selected. You may also need to check the values of the X,Y, and Z parameters for errors. ✧ If the Test Autofocus Result is between 0 and 800, try to refine the values for the carrier and retest. 7. When you are happy with the results, click Save to save your changes. Close the Edit Carrier Type window. C-12 iCys Research Imaging Cytometer User Guide Revision B Wells
May 6, 2005 Administrator Tasks Determining the A1 Parameters Finding the A1 Position by Measuring ➣ To determine the position of the top left (A1) well: 1. Measure the distance between the right edge of the microplate (closest to A1) and the center of the A1 well. Enter this value as the X Margin Left. 2. Measure the distance between the top edge of the plate (closest to A1) and the center of the A1 well. Enter this value as the Y Margin Top. 3. Measure the distance from the center of well A1 well to the center of the well A2 well. and Enter this value as the Interval in the Other Wells section. 4. If you cannot accurately measure the distance of the A1 well from the edges, see “Finding the A1 Position using iCys” on page C-13. Finding the A1 Position using iCys ➣ To use iCys to find the A1 position: 1. Load your custom microplate onto the microscope stage, as described in “Loading a Microtiter Plate” on page 2-18. 2. Open the Oscilloscope window by selecting Instrument ⇒ Oscilloscope. Tip: When determining measurements, the oscilloscope is only used to turn the galvanometer on and off. For more information about the oscilloscope, see “Using the Oscilloscope Feature” on page 3-90. 3. From the Oscilloscope window, turn on the oscilloscope by clicking the “Play or Stop” button ( ). 4. Place a piece of thin white paper over the A1 well of the microplate. From the XY tab of the Edit Carrier Type window, click the Move To button in the First well section. 5. Select the Argon Laser *488 from the task bar to open the shutter. Warning: When using paper to find the well measurements, avoid direct eye exposure to the Class 3R radiation. 6. If the beam is not centered in well A1, use the touchpad buttons or the Remote Control unit X and Y knobs to drive the stage such that the beam is centered in the A1 well. 7. Drive the objective lens upwards until you see a line (or pair of lines) on the Autofocus (black and white) monitor. Adjust the focus until you are satisfied with the results. Recheck, and if needed, recenter the beam in A1. 8. When the beam is properly centered and focused, turn the oscilloscope trace off (by clicking on the “Play or Stop” arrow [ ]). Click Set from X,Y in the First Revision B iCys Research Imaging Cytometer User Guide C-13
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Contents Chapter 1 Introduction Man
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Appendix A Installing iCys Research
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