20 November 2010, R. Ubic
11
Objective Lens Astigmatism Correction
Find the amorphous edge of the sample and increase magnification to
≈
300k - 500kX,
adjusting the
BRIGHTNESS
knob for optimum viewing conditions and using the
ShiftX
and
ShiftY
knobs to center the beam (ensure that the
BRIGHT TILT
button is
illuminated).
Finely focus with the
OBJ FOCUS
knobs.
Press the
OBJ STIG
button (left-hand panel)
Adjust the
DEF/STIG
knobs until minimum contrast is observed in the amorphous
region. It may help to slowly turn
OBJ FOCUS
back and forth through focus to observe
the variation of phase contrast.
Press the
OBJ STIG
button again to shut this adjustment off.
Focusing
If an objective aperture is in place, remove it.
For low magnifications (<
≈
100kX), focusing can be done easily with the image wobbler.
Press the
IMAGE WOBB X
and/or
IMAGE WOBB Y
button (right-hand panel).
Unify the oscillating image with the
OBJ FOCUS
knobs (right-hand panel). If the
image cannot be unified, try correcting for objective astigmatism (above).
Press the
IMAGE WOBB X
and/or
IMAGE WOBB Y
button again to shut it/them
off.
For high magnifications (>
≈
100k X), focusing is best done with either Fresnel fringes or
point images.
Look for the appearance of bright and/or dark Fresnel fringes. If they are very
close together, their appearance results in an increase in contrast.
For widely-spaced fringes, use the
Z
control to bring them together. Only use
OBJ FOCUS
knobs to correct for very small fringe spacings.
For very high magnification (> 300k X), find an amorphous section of the sample
(typically near the edge) and focus the image using the
OBJ FOCUS
knobs. The
point image will have minimum contrast at the point of focus.
Note: The optimum focal condition is not always at exact focus. It is often useful to
slightly underfocus samples, especially biological ones, in order to increase contrast.
