Page 272 - Fundamentals of Light Microscopy and Electronic Imaging

DAISY CHAINING A NUMBER OF SIGNAL-HANDLING DEVICES 255

% modulation of system a% b% c%.

The solution for maintaining resolution is to add a magnifying relay lens (a so-called TV
lens) with a magnification range of 2–8 to the imaging system. As seen in the graph
of system MTF, a video microscope containing an 8 relay lens gives close to theoret-
ical resolution. The negative side of using a relay lens is that the field of view is reduced;
image brightness on the camera also decreases in proportion to the square of the magni-
fication difference.

Exercise: Contrast Adjustment and Time-Lapse Recording
with a Video Camera

The objective is to prepare a time-lapse video recording of live cells either in
phase contrast mode or in fluorescence mode after staining with a fluorescent
dye. The exercise will demonstrate the relative ease of video recording and allow
you to experience how time-lapse video imaging can provide valuable informa-
tion regarding cell motility and the dynamics of cellular and intracellular
organelle movements. The exercise will also demonstrate the value and ease of
performing video enhancement of low-contrast specimens, but it will also show
how electronic recording equipment can potentially degrade image quality
(described in quantitative terms as the modulation transfer function, or MTF).

• Prepare the specimen and obtain a live image on the monitor.
1. For this exercise and for use in the laboratory, it is convenient to prepare
a cart containing the video camera, TV monitors, VCR, and other equip-
ment shown in Figure 13-2. The cart can be positioned near the micro-
scope for video recording, but stored in an out-of-the-way place when
not in use. On the video cart, turn on the camera and camera controller
(video tube camera or video CCD camera), the digital image processor
(Hamamatsu Argus-20, Dage DIP, or similar unit), two TV sets for dis-
play of the raw and processed images, the thermal printer, and the time-
lapse S-VHS VCR.
2. Mount a coverslip of living cultured cells with Vaseline as described in
Figure 10-9. Use cell types such as COS-7, U2OS, or another cell type
that has a flat, extended morphology so that organelles can be clearly dis-
tinguished and identified. Alternatively, examine a culture of protozoa
such as Acanthamoeba or Dictyostelium, as these cells move relatively
quickly and give a high contrast image. Examine the specimen with a
100 , 1.3 NA oil immersion objective, and adjust the microscope for
Koehler illumination using phase contrast or fluorescence optics. Exam-
ine the back focal plane with the focusing telescope eyepiece to check that
the oil fills the space between the lens surface and coverslip, that there are
no air bubbles trapped in the oil, and in the case of phase contrast, that
there is precise alignment of the condenser annulus and objective phase
ring. The built-in halogen lamp is suitable for phase contrast, while the
epi-illuminator/mercury lamp is required for fluorescence.