How to Use Auto Zoom/Focus
Auto Zoom and Auto Focus are implemented with stepper motors driving the zoom cell and focus element of the microscope. It is also possible to have a fixed magnification lens with just Auto Focus; determine which configuration you have. The focus function will find the best focus by sweeping the focus through a range of settings and measuring the image sharpness at each step. The best average focus position is then computed from the sharpness data. The zoom setting is a little different. It has two parts: a magnification calibration process and, secondly, a magnification look-up each time zoom is changed.
Homing the Axes
The stepper motors on the Navitar zoom microscope do not have home switches to initialize the stepper motor position. Instead, the following process is used. The stepper motor on a focus-only microscope does not have a home switch, but also does not need any homing process at all. In all cases, the stepper position is written to the configuration file each time it is changed, so it will be remembered correctly unless someone moves the motor manually with their hand, in which case its position is obviously lost. Therefore, the homing process is only necessary when the positions are uncertain or unknown.
The homing process makes use of the fact that there are mechanical hard stops at both ends of the stepper range. As an example, let us assume that the range of the axis is 1500 stepper motor steps, end to end. When correctly homed, it will be possible to drive the stepper from 0 to 1500, and back from 1500 to 0, without hitting either hard stop. When the motor hits the hard stop, it does not do any damage but it does make a very distinct noisy click, different from the normal running sound. Therefore, if we drive to one end and then drive back to the other end without hearing a click at the end, we know we are positioned correctly. More importantly, if we drive end-to-end, we will correctly position the stepper even if it started out incorrect. It will hit the hard stop at one of the ends, but at the conclusion it will be correct. Therefore, driving end-to-end checks and corrects if necessary. The stepper range for each axis is saved in the Driver Setup section for the axis.
When you wish to move the zoom or focus axis under manual control, you enter the increment (change) in motor steps you wish. There are software limits that will not let you go beyond the maximum step position (the range of the axis) or 0 on the low end. If you enter an increment that would carry you beyond the limits, the increment is reduced to a value that will carry you just to the limit.
Initial Setup of Axis Ranges
The range of the zoom and focus axes is determined at the factory and this information is shipped with the instrument in the form of a pre-set configuration file named default.mdb. This will either be on the CDROM or a separate diskette. The new user should copy this file to the application directory, typically as
Each microscope is slightly different, so these values are determined individually.
Should you wish to determine the range of the zoom or focus axis yourself, follow this simple procedure. First, set the maximum range in Driver Setup to the default value. This will be greater than what you will ultimately find and use. Next, choose a value for the range. Enter this, as a positive number in the increment box and Move the axis. The axis will go further towards its high end. It may or may not hit the hard stop. Now move in the negative direction by entering the same increment as a negative number. Move the axis again. If you hit the hard stop in both directions, the trial value is too large, so reduce it. You want to find a value that is too large and then gradually reduce it until the motor can just go end-to-end without hitting the hard stop at either end. If it only hits at one end, re-try because the range setting might be OK with only the starting point wrong. If you prefer not to judge the end point by the sound it makes, you can also manually turn the motor gear in the direction it was going. When the range setting is correct, you can not turn the gear further at either end of travel. If the range setting is any less, you will be able to feel a slight motion at one end of travel. As noted above, this process is not normally required in the field since the range values are furnished with each instrument.
Auto Focus Use
You may always change focus by manually entering a movement increment on the Video tab drop-down list or the Auto Zoom/Focus tab text boxes.
More useful is the Sweep mode. This is available from the Auto Zoom/Focus tab. This process will try out various focus settings and compute the best available focus and move focus to this optimal value. There must be an object in the image that has a sharp edge so image sharpness can be measured at each stepper motor position. You must enter the focus increment and start and stop limits for the Sweep. A good starting point for the increment is 4 steps. This is a compromise between speed and accuracy. For finer work, enter 2 steps. If the focus is approximately correct, the start and stop limits should be about 10% of the range below and above, respectively, the current focus setting. If the range is 500, this would be -50 and +50 from the current value. If the current focus is poor, choose a wider range. Using these example values, the sweep will be through 100 steps and the step increment will be 4, so a total of 100/4 = 25 positions will be measured. It is suggested you have at least ten positions within the range. If your range is too small, the algorithm will recognize it and ask permission to automatically increase the sweep range. Once you have setup these values, click Sweep and the process will begin. It will move to the starting point, wait a few seconds for the sharpness measurement to stabilize, and then begin stepping and measuring sharpness values through the range you chose. As the sweep progresses, the best focus sharpness and position of this best sharpness are displayed on the Auto Zoom/Focus tab. At the end it moves to the best position (which may be between one of the steps you chose).
Auto Focus is useful for people who have difficulty judging focus, but it is absolutely necessary for Auto Zoom calibration, discussed below.
There is an addition capability on the Auto Zoom/Focus tab. This is the Focus/Zoom slope value found in the Driver Setup frame. This allows you to have the focus control track each time zoom is changed. The algorithm multiplies the number of zoom steps by this slope and moves the focus stepper motor if the resulting product is equal or greater than one step. To use this function, find the best focus at the extremes of the zoom range you plan to use, divide the focus steps (focus position) by the zoom steps and enter this value in the text box. Note the value will be a real number, typically small and not an integer.
Auto Zoom Use
Like Auto Focus, you may always change zoom by manually entering a movement increment on the Video tab drop-down list or the Auto Zoom/Focus tab text boxes. What is useful about Auto Zoom is that it can keep track of magnification.
As part of the factory calibration a Zoom Sweep is made which measures the apparent size of a known target at all zoom positions. Since the target size is known, the actual magnification can be calculated and saved. This data is stored in the configuration file. The default name for this file is "default.mdb." You can copy the contents of this file into another name and thus preserve the magnification calibration while changing other settings. The current magnification values for each Auto Zoom step position can be viewed by clicking Show Data in the Zoom section on the Auto Zoom/Focus tab. You can also Print Data.
The key to using automatic magnification calibration is the "AutoZoom sets mag in Calibration" checkbox on the Auto Zoom/Focus tab. When the checkbox is checked, these magnification values will be used as the current magnification. They will be updated with each Zoom move. Thus, when you take a movie, the magnification will already be determined and will be written into the movie. When unchecked, magnification is not updated with each Zoom move.
A further option is that you might so a normal magnification calibration on the Movie's Images | Calibration tab. This leads to the following possibilities:
AutoZoom sets mag checkbox is checked but you make no magnification correction on the Calibration tab: the Zoom Sweep lookup table will be used directly. Every time you change Zoom, the internal magnification calibration is updated.
AutoZoom sets mag checkbox is checked and you do make a magnification correction on the Calibration tab: the internal magnification calibration is linearly scaled by the ratio of your user-set magnification to the lookup table magnification, at this Zoom setting. This forms a "Auto Zoom/Focus Magnification Ratio" that is reported on the Calibration tab. This ratio will be used on all subsequent Zoom settings. This protocol has the following advantage: the Zoom Sweep lookup table characterizes the opto-mechanical response of the microscope's zoom cell. The overall magnification, however, can vary slightly with temperature, contrast, and focus. Thus the ratio will be a slight tweak on the basically-correct lookup table.
AutoZoom sets mag checkbox is not checked: the lookup table is not used, nor is mag ratio, and calibration is done the ordinary, simple way by user entries on the Calibration tab. Internal calibration is not changed when the Zoom motor is moved.
The accuracy of the factory calibration curve is approximately +/-0.25%. This level of inaccuracy will cause a +/-0.5% error in an interfacial tension measurement. It will not cause any error in a contact angle measurement, per se. Drop diameters will be linearly proportional to magnification error and drop volumes proportional to the error cubed. The principal contributors to the magnification error are the imprecision of the stepper motor, ambient temperature changes that affect the microscope itself, and a minor contribution from differing focus values between the original calibration and the time of use.
To summarize, for most work this accuracy is satisfactory, but you can improve it by re-calibrating one zoom position more carefully, say with today's temperature and focus. Then the magnification at other Zoom positions will be automatically scaled if the Auto Zoom/Focus sets mag checkbox is checked.
Auto Zoom Calibration
The Auto Zoom system is shipped with a factory calibration file for your individual microscope. This is normally of sufficient accuracy.
If you wish to re-calibrate the Zoom magnification data, you may do so with a Zoom Sweep. You get to choose what portion of the total Zoom range you wish to calibrate with a particular Sweep. In general, you will want to divide the total range into, say, five or six sub-ranges so the target that you use will mostly fill the image. This lets you use different targets for different portions of the total range. Do not use a small target, one less than 25% of the image width, because you will sacrifice accuracy. Dividing the overall range up also helps keep the focus changes small over the range being measured.
Zoom Sweep automatically runs Focus Sweep at the start and stop values of the Zoom range. It does this first, before it begins measuring each Zoom step in the chosen range. Zoom Sweep will automatically setup Focus Sweep, although it will run faster if you start with a reasonable focus. Zoom Sweep performs a large number of measurements in order to improve accuracy, and for this reason runs slowly. It takes about two hours to cover the entire range, end-to-end.