Microscope and Imaging TerminologyYou may not be familiar with the terminology of optics and the types of images taken with these instruments. Some of these terms are defined below.
Magnification: Magnification is the ratio of the size of image that appears on your computer screen to the real object. Because computer screens vary in size, it has become practice to use the term "field of view" when your want to be technically precise. Nevertheless, it is common practice to simply say "magnification."
It is correct to refer to the Calibration quantity "nanometers per pixel" as magnification. In this case, we are referring to the camera pixel as the output reference size rather than the computer screen. This has the advantage of being precise but the disadvantage of being difficult to visualize. Again "field of view" is a more precise term.
Pixel: A digital picture element in the camera's CCD iamge detector. It is a specific region of space on the CCD. It might be something like a 10 micron square. FTA caameras typically have 640 pixels horizontally and 480 pixels vertically.
Zoom: Zoom is the ability to change magnification without having to refocus. Telecentric lens like used in the FTA100 series do not zoom. Most microscopes in the FTA200 and above series are zoom lens.
Field of View: Field of view refers to the size of the image in real space. It normally refers to the horizontal size, although the diagonal is sometimes used. Field of view is also the size of the CCD chip divided by the optical magnification. Notice this does not depend on the size of your computer screen. CCD chips are commonly "1/2 inch" (6.4mm horizontal, 4.8mm vertical) or "1/3 inch" (4.8mm horizontal, 3.6mm vertical) in size. The magnification of zoom microscopes used in FTA systems typically varies from x0.7 to x4.5. The magnification of telecentric lens used in FTA100 series instruments is roughly x0.6. For example, if you have a 1/2 inch camera and a zoom microscope set at x2, the horizontal field of view is 6.4mm divided by 2 = 3.4mm.
Aperture or Iris: An adjustment available on most lens to set the diameter of the hole admitting light to the CCD. It is like the pupil of your eye. The aperture is open when the most light falls on the CCD and closed when the least amount (or none) hits the CCD. Making the aperture smaller (more closed) increases the depth of focus or depth of field, but decreases the total light onto the CCD making the image darker. Always adjust focus with the aperture open. When the aperture has a number scale, open will be something like "2.8." Closed will be something like "32." Telecentric lens have a scale but zoom microscopes do not have a scale on their aperture.
Focus: Focus occurs when the lens accurately projects the image of the object onto the CCD. This occurs when the distances from the lens (i.e., the glass) to the object and from the lens to the CCD are in the correct ratio. An image that is in focus is "sharp." The sharpness can be measured in real time by using the Focus control on the Analysis tab of the Video window. You must adjust the focus to maximize image sharpness. Always adjust focus with the lens aperture open. Then close the aperture partially (say, 1/3 to 1/2) to obtain the best overall image.
Microscope Controls: A telecentric lens has an adjustable aperture (control closest to the camera) and, at its end, a focus barrel which adjusts in and out by turning, like a manual 35mm camera. The numbers on a telecentric focus barrel refer to meters of focal length, not to be confused with magnification. All controls may have small thumbscrews to lock them in place.
A zoom microscope typically (but not always) has an aperture control that will be the closest user adjustment ring to the camera. The magnification control will be in the center of the zoom and the focus ring will be the closest to the sample or farthest from the camera. Only the magnification control will have numbers on it on a zoom microscope.
Magnification Calibration: Magnification calibration consists of telling the software the image size for each pixel in real space. A typical value might be 20,000 nanometers or 20 microns per pixel. (If the physical pixel size in the CCD is 10 microns, and the calibration is 20 microns per pixel, the magnification is exactly 1/2.)
Changing the magnification calibration does not change the size of the image. Instead it changes the value the alogorithms assign to each pixel of distance.
You "calibrate" the optical setup by measuring a known physical object and adjusting the calibration value until the distance across the object in the image is correct. One convenient measure that the FTA software makes is of the dispense tip width or tip diameter. Say this is a #22 needle and its diameter is 0.711mm. Say the current magnification calibration yields a measured value of 0.600mm. This implies that the "nanometers per pixel" value is low because the algorithm perceives the tip to be smaller than it really is. We can correct the value by multiplying the current value by the ratio 711/600. This will make it perceive the tip width is 0.711mm. Using these example numbers, you would enter "0.711" for the Actual Distance and "0.600" for the Measured distance, then click Apply. The calibration would the be updated to the new, higher, value.
Image Orientations: In this work, a "normal" image is one where a pendant drop hangs down from a dispense tip for surface tension or a sessile drop rests on a solid surface for contact angle measurements. The algorithms calcuate correctly for drops in these orientations.
But other orientations are possible. With pendant drops, liquid-liquid measurements are possible using chambers. Then the dispense tip might come up from below and the pendant drop might float up like a bubble. It will float because it is the less dense of the two fluids. Sessile drops may also be upside down in chambers. For example, an air bubble might rest against (float up against) a solid surface immersed in water.