Biomedical Engineering Reference
In-Depth Information
mode, relatively high light levels at the specimen are required to produce images of sufficient
intensity to allow reasonable exposure times. Thus, for multiple day runs with untransformed
cells which are more likely to undergo apoptosis (cell death) from photo-damage, that is,
demonstrate a robust p53 response, phase contrast microscopy is a more reliable choice.
3.2 The Microscope
The comments that follow apply to both upright and inverted microscope stands. The
microscopes of all major manufacturers, be they the older 160 mm tube length systems or
the current infinity corrected systems, offer good quality optics for phase contrast imaging.
Given that live cell imaging should be conducted with monochromatic light (typically green
light), there is no need to buy expensive apochromatic objectives; cost-effective achromats
will work just as well at equivalent numerical apertures.
Focus stability is what sets one microscope apart from another in terms of suitability for
time-lapse imaging. Passive or intrinsic focus stability is sufficient for low magnification/
low numerical aperture imaging. Passive focus stability also works for high magnification/
high numerical aperture applications, but it is imperative to empirically test the focus
stability of the stand under constant temperature conditions. We emphasize constant
temperature conditions because all stands, regardless of design, can show significant
X
,
Y
,
and
Z
movements at the specimen with temperature changes. We find that some of the
30
40 year old stands with gear-driven focus blocks do a fine job. Some of the modern
motorized focus drives are also focus stable. Stands with recirculating ball focus drives are
sometimes not focus stable. We note that all major manufacturers offer for their newest
generation stands optical feedback systems that use an infrared beam to monitor the
position of the coverslip
water interface and drive the motorized focus to maintain a set
focus position with high fidelity. These systems, though costly, work well and can be
important for high magnification applications. We do not recommend the use of
software-based auto focus routines because they can establish an undesired plane of focus
within or outside the cell, and they take time which exposes the cells to more light.
Time-lapse imaging using a microscope with a conventional stage limits observations to a
single field of cells for the duration of the film run. To increase the number of cells observed
during a filming session, one can equip the microscope with a motorized stage and a
motorized focus mechanism, both under computer control. In this way, one can concurrently
film a number of fields, each indexed for
positions. It is important to set up the
acquisition routine so that image sequences from each field are put into separate files.
X
,
Y
, and
Z
3.2.1 Illumination
Attention must also be paid to the illumination parameters. Phase contrast is not light
intensive so tungsten illuminators with voltage stable power supplies work well. For our
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