Imaging the division process in living tissue culture cells

Abstract

We detail some of the pitfalls encountered when following live cultured somatic cells by light microscopy during mitosis. Principle difficulties in this methodology arise from the necessity to compromise between maintaining the health of the cell while achieving the appropriate temporal and spatial resolutions required for the study. Although the quality of the data collected from fixed cells is restricted only by the quality of the imaging system and the optical properties of the specimen, the major limiting factor when viewing live cells is radiation damage induced during illumination. We discuss practical considerations for minimizing this damage, and for maintaining the general health of the cell, while it is being followed by multi-mode or multi-dimensional light microscopy.

Fig. 1

Excessive illumination during imaging of live cells arrests the division cycle. In this example a mid-prophase PtK₁ cell, in which chromosome condensation was already well advanced, decondenses its chromosomes and arrests in late G₂ in response to multi-mode imaging. Top part of each frame presents DIC and bottom shows GFP-γ-tubulin epi-fluorescence. This visible change in nuclear morphology provides a convenient visible assay for over-illumination. From [18].

Fig. 2

Vibrations generated by shutters and filter wheels can be eliminated by mounting such devices external to the microscope chassis. In this example, the shutter/epi-light source assembly and filter wheel (bottom), as well as shutter/trans-illumination source assembly (top), are all mounted onto a vibration-isolation table using stainless steel rods. The whole assembly is disconnected from the microscope body, which in turn rests on the same vibration-isolation table.

Fig. 3

We maintain cells for long-term microscopic observations in modified Rose chambers. A side view of the fully assembled chamber is seen in the middle, just above the 3″ (8 cm) mark on the ruler. The chamber is constructed from two 25 mm² coverslips (top left and right corners), a silicon spacer (middle, above assembled chamber), a metal planchet milled to accept a condenser lens (left side, middle), and another planchet milled for objectives (right side, middle and bottom). The whole assembly is held together by four screws (bottom left). The chamber can be filled and drained using two 25G needles and a syringe. It can also be constructed using different objective planchets depending on the viewing conditions. For high-resolution oil-immersion work the top planchet, which is more extensively milled and thinner, is used (see also [48]).

Fig. 4

Enclosing the microscope and some of its peripherals in a plexiglass box allows for precise control of the specimen temperature during live-cell imaging. The temperature inside of this box is maintained by a heat blower (bottom right hand corner of image), positioned well away from the specimen stage, which cycles on and off in response to a thermistor positioned near the specimen. Note that the oculars protrude from the box (but are sealed by cotton), and that focusing can be done externally. See text for details.

Fig. 5

(A) Peltier-based heater to keep the viewing chamber at a desired temperature (also see [44]). This heater, in which our modified Rose chamber is mounted, can be firmly attached to the microscope stage by clamps (B).

Fig. 6

An overview of a thermal-stable microscope that we use in our research. The microscope proper is mounted on a vibration-isolation table and is equipped with filter wheels, multiple shutters, and a Rose-chamber stage heater. All of these devices are covered by a cardboard box (covered with aluminum foil), to shield the entire assembly from airflow and light. Electronic controllers for all of these devices are placed outside of the microscope to prevent their overheating. The whole system is driven by a workstation (on the left) that runs image-acquisition software (in this case Isee, Isee Imaging, Raleigh, NC, USA).

Fig. 7

The dependence of camera sensitivity on the readout speed. In this example, the images of a live PtK₁ cell, expressing α-tubulin/GFP, were recorded on an Orca II camera operated in “fast” (10 MH; top row), and “precise” (1.25 MH; bottom row) modes. As evident from comparison of (A and A′), the background noise (no light to the camera) is dramatically lower when the slow readout speed is used. This difference does not affect camera performance when the exposure time can be adjusted to use the entire dynamic range (cf. B and B′). However, under identical conditions, but in a light-limiting situation, slow (precise) readout provides better image quality (cf. C and C′ ~10 times less light than in A and A′). The difference becomes even more dramatic under extremely low light conditions (cf. D and D′, ~5 times less light than in B and B′) when slow readout provides an acceptable image while the fast-readout mode does not.

Fig. 8

A comparison between wide-field fluorescence (A and B) and spinning-disk confocal (A′ and B′) images of interphase (A and A′) and mitotic (B and B′) cells expressing α-tubulin/GFP. As is clear from the comparison, confocal imaging provides a much clearer picture of how microtubules are distributed in interphase cells (cf. A and A′). However, the improvement in image quality is not as dramatic when mitotic spindle is imaged (cf. B and B′). Note that the confocal images presented here were recorded at approximately 75% higher excitation light intensity, than were the wide-field fluorescence ones.