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Protocol no. 52
QUANTITATIVE VIDEO MICROSCOPY OF INTRACELLULAR MOTION AND MITOCHONDRIA-SPECIFIC FLUORESCENCE AVEC-DIC

microscopy in combination with mitochondria-specific fluorescence allows a quantitative analysis of cell organelle dynamics and fine structure in cell cultures exposed to test compounds.

CONTACT

Dr. Toni Lindl
Inst. f. Angewandte Zellkultur
Balanstrasse 6, D-8000 Munchen 80 FRG
Tel: Germany - 89 487774 Fax: Germany - 89 487772

RATIONALE

Cytotoxicity testing for potentially toxic compounds in vitro is becoming increasingly important. Although there are many well-validated test systems, most of them are unable to detect the influence of low to moderately toxic compounds and usually measure only one cellular or biochemical parameter. This test system, combining DIC microscopy and image processing by computer, allows the visualisation of more detail inside the living cell than is possible with conventional light microscopy and enables a quantitative analysis of cell organelle dynamics. The progress of a cytotoxic response may thus be monitored.

BASIC PROCEDURE

IMR 90 cells are cultured on cover slips, mounted on slides and incubated for 1-24 hours in the presence or absence of test compounds. Movement of cell organelles is observed by means of video-enhanced contrast microscopy. The cells are maintained at a stable temperature and pH in an incubation chamber. At the same time the lysosomes and mitochondria are specifically stained with fluorescent vital dyes so that their number and morphology may be assessed. The analog video signal is enhanced, digitised and subjected to several steps of image processing. The final images are recorded and later analysed to provide plots of organelle velocity versus incubation time.

CRITICAL ASSESSMENT

Videomicroscopy The full range of parameters that may be measured by videomicroscopy is discussed by Weiss (1989) and listed in Table 1. Videomicroscopy enables cells to be studied while still in the intact living state and before the occurrence of irreversible damage. The development of toxic events may be followed through the continuous observation of one set of cultures, and potentially allows the determination of concentrations of test substances at which impairment of certain cellular parameters is still reversible. This may prove to be a more valid set of end-points than those based on cell death. While videomicroscopy may seem at first sight to be a complicated procedure, it should be viewed not as one difficult technique, but as a bundle of many relatively simple techniques. If a good research microscope is available, the cost of setting up videomicroscopy need not be greater than, for example, that of obtaining a good photometer for the neutral red assay. The full range of image analysis for all parameters listed in Table 1 may, however, require the facilities of a larger research institute. A massive stable microscope, preferably inverted, equipped with epi-illumination fluorescence and transmitted light bright-field, dark-field, and differential interference contrast (DIC) is required. The illumination should be as bright and even as possible and a stabilised mercury arc lamp, preferably with a light-scrambling fibre-optic connection, is recommended. Fluorescence and high magnification DIC work require an exit port projecting 100% of the light to the video camera. Additional magnification may be obtained by the use of high oculars and a video camera objective. The use of at least one heat-absorbing and one heat-reflecting filter is essential, and a narrow-band green filter (546±10nm for mercury arc lamps) and a UV filter are recommended for DIC work. The image is picked up by a high quality video camera and requires processing. First, the analog video signal is subjected to analog contrast enhancement and then it is digitised. Mottle subtraction removes the out-of-focus background pattern. The signal-to-noise ratio may be increased by accumulation or averaging of images, and digital contrast enhancement will select the desired range of gray levels. Video-enhanced contrast (VEC) microscopy The use of the VEC microscopy technique increases contrast and magnification, allowing close observation of cell organelles. The use of image processing permits the resolution of objects of about 150nm and visualisation of even smaller structures such as microtubules and vesicles down to 20nm. In comparison, conventional light microscopy offers a maximum resolution of about 250nm. Motion analysis of movement in cell organelles may be performed by extracting the x and y coordinates of moving objects from a series of video images. The data may be quantitatively evaluated by classical time series analysis, for which the PARTI-MOVI software package (Weiss et al., 1986, 1987) has been developed. Video-intensified microscopy Low light level cameras are used to image the weak signals of fluorescent compounds. Organelle-specific vital dyes will penetrate the living cell without influencing its functions for several hours. Thus, organelle-specific toxic effects may be noticeable before any obvious manifestations of toxicity occur at the level of the whole cell. The use of fluorescent enzyme substrates, chelators or antibodies provide further means for structural and biochemical analysis of cytotoxicity. Comparison with other techniques The test system is more complex than commonly used in vitro tests and allows the evaluation of both biochemical parameters and cellular parameters, including velocity of lysosomal movement, the number, shape and morphology of mitochondria, cytoplasm consistency, appearance of vacuoles, spikes and blebs. The system is more sensitive than conventional methods - lactate dehydrogenase release, trypan blue uptake and form factor analysis - and yields more specific information on the cellular changes caused by compounds as well as detecting damage at lower concentrations and earlier time points. Included in the results section is some comparative data obtained from testing various concentrations of HEMA on fibroblasts cells using conventional cytotoxicity assays. IMR-90 cells are a good choice for the measurement of lysosomal movement by DIC microscopy because they spread out over a wider area thus making observation easier. While they can also be used for comparative cytotoxicity tests using classical methods, they do not grow as well as L-929 cells. For this reason, and since the cytotoxic response of the two cell types is broadly similar, it is recommended to use L-929 cells to assess the cytotoxicity of the test compound by standard in vitro methods. A major advantage is the fact that the system allows observation of intact live cells over a prolonged period, where the cells can also serve as their own controls by being observed prior to treatment. In contrast, many standard systems require the use of heterogenous populations of cells which may also need to be subjected to drastic procedures such as fixing or homogenisation. A potential disadvantage is the apparent complexity of the apparatus, and the fact that some specialised knowledge additional to that used in basic microscopy is needed. There may also be problems in analyzing the results if appropriate software packages are not widely available.

TEST STATUS

In-house

CHEMICALS TESTED

2-OH-ethyl-methylacrylate (HEMA)

REFERENCES

  1. Allen, R.D. (1985) New observation on cell architecture and dynamics by video-enhanced contrast optical microscopy. Ann. Rev. Biophys. Chem. 14, p265-290.
  2. Allen, R.D, Allen, N.S. & Travis, J.L. (1981) Video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: A new method capable of analyzing microtubule-related motility in the reticulopodial network of Allogromia laticollaris. Cell motility, 1, p291-302.
  3. Brugmans, N., Cassiman, J.J., Van der Heydt, L., Oosterlinck, A.J.J., Vlietinck, R. & Vanden Berghe, H. (1982) Quantification of the degree of cell spreading of human fibroblasts by semi-automated analyses of the cell perimeter. Cytometry, 3, p262-268.
  4. Erbrich, U., Naujok, A., Petschel, K. & Zimmermann, H.W. (1982) The fluorescent staining of mitochondria in living HELA- and LM cells with new acridine dyes. Histochem., 74, p1-7.
  5. Herman, B. & Albertini, D.F. (1984) A time-lapse video image intensification analysis of cytoplasmic organelle movement during endosome translocation. J. Cell Biol., 98, p565-576.
  6. Lindl, T. & Bauer, J. (1987) Zell- und Gewebekultur. Gustav Fischer Verlag, Stuttgart. Maile, W., Lindl, T. & Weiss, D.G. (1989) New methods for cytotoxicity testing: Quantitative video microscopy of intracellular motion and mitochondria-specific fluorescence. Journal of Molecular Toxicology, 1, p.427-437.
  7. Weiss, D.G. (1989) Videomicroscopic measurements in living cells: Dynamic determination of multiple end points for in vitro toxicology. Journal of Molecular Toxicology, 1, p.465-489.
  8. Weiss, D.G., Keller, F., Gulden, J. & Maile, W. (1986) Towards a new classification of intracellular particle movements based on quantitative analyses. Cell Motil. Cytoskel., 6, p128-135.
  9. Weiss, D.G., Langford, G.M., Seitz-Tutter, D. & Keller, F. (1988) Dynamic instability and motile events of native microtubules from squid axoplasm. Cell Motil. Cytoskel., 10, p285-295.
  10. Weiss, D.G., Maile, W. & Wick, R.A. (1989) Chapter 8, Video Microscopy. In: Light Microscopy in Biology. A Practical Approach. (ed. Lacey, A.J.) IRL Press, London, pp 221-278.
  11. Zelenin, A.V. (1966) Fluorescence microscopy of lysosomes and related structures in living cells. Nature, 212, p 425-426.

IP-52 © April 1992