Microscopy 101
Figure 3: Ray diagram for an ideal lens. Red represents the parallel incident and transmitted beam, while green and blue represent diffracted beams. The diffraction pattern forms in the back focal plane (BFP) of the objective lens.
background that produces the Ton rings in the FFT. See Figure 1.
iii. If there is varying thickness in the selected area of the sample, such as occurs oſten with nanoparticles on a support film, try to find a portion of the area that is in the center of the thickness to use for determin- ing the sample height. Tis can be gauged somewhat with the focus knob, but remember to set it back to the standard focus when adjusting the height.
iv. It is possible to immediately tell if a sample is way off the standard focus position by changing the condenser lens control (brightness). If the magnification of the image appears to increase or decrease, the sample is not at the correct height. At the correct position and with the stan- dard focus set, there will be no change seen in apparent magnification as the condenser lens is changed.
2) Lower the magnification to observe the desired area of interest, and center the area of interest on the screen. If an objective aperture is inserted, remove it. a. Your sample is now focused, the region of interest is selected, and the diffraction pattern is formed in the back focal plane of the objective lens. If there is parallel illumination, then a spot or ring pattern from the sample is first formed there, as discussed below.
b. Note: Whether doing imaging or diffraction, the sample is now in the calibrated position. Tis should be the posi- tion that both the imaging and diffraction modes were calibrated with using a calibration sample.
3) Center your illumination. a. If using a digital camera, please be careful. Lower your exposure to an appropriate level. (If using DM, three or four clicks of the down arrow will drop the exposure by a factor of 8 or 16 and is usually sufficient.)
b. Condense the beam until it is seen in the camera window as shown in Figure 2A, and center using the beam shiſt controls. If you are not using a digital camera, then cen- ter the condensed beam on the phosphor screen.
c. Spread the beam and restore the camera exposure to what it was (in DM, three or four clicks with the up arrow (see Figure 2B).
4) Introduce the appropriate size of diffraction aperture that selects the area of interest, and center it as shown in Figure 2C.
5) Turn the brightness knob fully clockwise. If you have access to viewing the microscope’s hexadecimal lens values, the condenser lens should read “FFFF.” Regardless, most
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Figure 4: Top: Lines are drawn on the view screen to find the center of the digi- tal image. Center: With a low exposure, the screen is lifted, and the transmitted beam should be close to center. Bottom: Center the transmitted beam to the center as closely as possible. Lower the screen. (In the example here, the expo- sure was a little higher than normally used so that some rings could be seen.)
microscopes beep when you have changed a control to its maximum or minimum. Just listen for the beep. a. At this point the illumination is as parallel as possible, and the diffraction pattern is formed at the back focal plane (BFP) of the objective lens. Ideally, the transmit- ted beam (T) and diffraction spots (g) are points, ignor- ing spherical aberration. Tere is also a plane where the
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