Long-Term Recordings of Live Human Cells Using Phase Contrast Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 43

systems without built-in power supplies, we have replaced the simple rheostat-type power
supplies with laboratory grade voltage and current stabilized power supplies. Since most of
the output of tungsten lamps are in the infrared, one should use a good quality “heat cut” or
long wavelength cutoff filter. Digital cameras are to some extent sensitive to infrared
wavelengths and if they do not have a built-in infrared cutoff filter, there is the possibility of
having a poorly focused and poorly corrected infrared image superimposed on the visible
light image. For wavelength selection, we use good quality interference filters, not gelatin
filters that pass a wide range of wavelengths. For our computer-driven microscopes, we
shutter the transmitted light path; this allows us to raise the lamp intensity to allow relatively
short exposures for multiple field acquisition while minimizing the dosage of light given to
the cells. For our simple microscope systems with which we follow only a single field, we
use continuous illumination, cut back the intensity with neutral density filters, and use long
exposures integrated on the camera chip. We normally use green (546 nm) light; for cells
that are extremely photosensitive one could use red light ( B 620 nm) which is substantially
less phototoxic (Galdeen and Sluder, unpublished). Lastly, it is of great importance to
properly align the microscope illumination pathway for K¨hler illumination to ensure even
illumination of the field. Image acquisition parameters set for high gain and contrast stretch
accentuate even subtle variations in illumination intensity across the field of view.
3.2.2 Temperature Control
Live cell imaging of mammalian somatic cells requires precise 37 C temperature control of
the specimen with little temperature fluctuations. If the temperature rises too high, even
transiently, the cells become stressed and viability is compromised. Since stress can be
persistent and additive (see Ref. [1] ), temperature overshoot can sensitize cells to other
environmental or experimental parameters that alone would not be a problem. If the
temperature is even a few degrees too low the timing of events will not be normal and may
not reflect the true kinetics of any given process [2] . Some processes may not be linear with
temperature. For example, shifting HeLa cells from 37 to 33 C doubles their generation
time and further reductions in temperature can differentially affect the kinetics of different
cell cycle stages [2,3] .
There are a number of ways to control the specimen temperature, but here we discuss the
strategy we have found to be effective, safe, and reliable. The web pages listed at the end
of this chapter provide a few sources of information that lay out other control strategies. To
start, the entire microscope is covered with a box leaving the illuminator and camera
outside the enclosure. For our simple systems, we use a corrugated cardboard box cut to
provide doors for access to the stage areas ( Figure 3.1A ).
For our more complicated systems with motorized stages our machine shop built, Plexiglas
enclosures with slider doors high on the front to allow access to the stage and slider doors