Preparation
It is crucial to prepare live cell experiments very carefully, because other than the image acquisition from fixed samples, a lot of additional parameters have to be taken into consideration. It is recommended to test and optimise the conditions, in which to maintain the biological specimens before and during the live cell experiment. A general concern is the effect of phototoxicity caused by the illumination of the sample on the stage and preliminary pilot experiments should be carried out to assess whether the experimental conditions are optimal and whether a live cell imaging experiment is feasible.
Pilot experiments should include tests that report cell viability, bleach effects, cell movement and possible stage drift.
Equipment care
The performance of live cell imaging always requires the use of buffers and salty solutions in close vicinity of complex and very expensive miscroscopes and electronic equipment. This might prove disastrous if liquid spills onto parts of the equipment are not being dealt with immediately. The objective lenses and the lens caroussels of inverted microscopes are particularly vulnerable and possible spills should be removed instantly and the affected areas rinsed with an excess of deionised water to remove all residual salts. Once a salty solution has dried in on an objective lens, this often renders it useless and requires reapir that can easily cost several thousands of pounds. Thus, if you intend to carry out live cell experiments please indicate this to the facility manager and you will get a separate induction. It is essential that you make yourself familiar with the use of the live cell experimental equipment and potential problems that might occur and how to deal with them.
Equipment available
All communal microscopes in the CALM facility are prepared for live cell imaging. However, due to technical differences and experimental requirements, the choice of the system should be considered carefully and if necessary discussed with the facility manager. The following gives a brief description of the equipment available. In general, both types of system, confocal scanners and widefield miscoropes, can be used for live cell imaging.
The Olympus widefield system is suitable for low magnification phase contrast time lapse imaging (for more technical details see Olympus system).
The Leica SP5 is fitted with a Solent incubation chamber that encloses the whole stage and controls humidity, temperature and an atmosphere that contains 5% carbon dioxide. However, the system needs a few hours of equilibration time and the fitted motorised stage needs to be excahnged with a mechanical one that is suitable to accommodate the heated chamber. Therefore, unless you need to use the 405 nm laser line for excitation, it is recommended to use the Zeiss LSM510 META.
The Zeiss LSM510 META is equipped with an incubation chamber that encloses a heated stage insert (POC Chamber System). The system controls the temperature, air humidity and a range of carbon dioxide concentrations and is very easy to use. Whereas, the CALM facility provides the heated stage insert, users are asked to bring their own adapters that are necessary to mount the circular coverslips onto the heated stage insert. These adapters are available in different diameters and shapes and thus can be chosen according to the experimental requirements. A detailed description of the different adaptors and configurations you can find on the Pecon website. This website also includes detailed information about each component, user guidelines and catalogue numbers. The system can be used as 'open' and 'closed' cultivation systems, including the option of microperfusion.
Specimen cultivation
In preparation for a live cell experiment, the biological samples should be pre-cultivated under optimised conditions and if the expression of exogenous proteins such as fluorescent fusion proteins is required, cells or tissues should be transfected or transduced accordingly. As for general cell and tissue culture all components must be sterilised prior to use.
Adhering cells should be seeded at a suitable density prior to the experiment (usually 24 - 72 hours) on coverslips suitable for microscopy. Poorly adhering cells or non-adhering cells that grow in suspension can be 'fixed' onto the coverslip by coating the glass with e.g. ECM protein mixes, collagen, D-Lysine or compounds such as CellTak. However, care must be taken that these coats do not produce an autofluorescent signal (test on control coverslips).
Tissue slices such as organotypic cultures can be imaged but care must be taken that the specimen does not move during the image acquisition. If the slice is grown on a mesh this must be turned upside down on an inverted microscope.
It is important that during transfer of the cells from the culture facility to the microscope, the samples are not exposed to excessive movement, light and temperature changes etc.