**6. Illumination and visualisation**

The circumferential position of the gas exchange membrane facilitates another feature of the culture chamber. Because there is nothing on either planar side of the vessel, it is possible to provide uniform illumination from one side and observe the culture from the other side. Fey *et al.* demonstrated that photographs of spheroids in culture could be used to determine the amount of soluble protein (or DNA or number of cells) by measuring their shadow area and using a look-up table [39, 42]. Thus, if the culture chamber is uniformly illuminated from one side and a camera is placed on the other side (**Figure 7**), it is possible to measure the sizes of the clusters without removing them from the incubator.

Even though most modern incubators have a double door, with the inner door made of glass, it is difficult to see the cell clusters clearly often because of poor illumination. The use of an integrated back-light and a camera to inspect the cultures brings yet another advantage: it is not necessary to open the incubator to see the cultures. If these shadow area measurements are repeated over time it becomes possible to follow the growth curve of the clusters. The only manipulation required is to change the media (typically this would be every 2–3 days). If the media is changed for example on a 2, 2 and 3 day (weekly) cycle, then the incubator will need to be opened only 10 times during a 21 day culture. Since a practiced person can change the media within 1 minute each time, this means that the cells need to be out of the incubator for less than 10 minutes in the 21 day period.

Thus, the construction using illumination and a camera for each cell culture vessel minimises the number of times that the incubator needs to be opened. This in turn further reduces the risk of infection, in addition to the effect gained by running the incubator 'dry'.

An extra source of illumination has been included on the same side as the camera so that it is possible to see the clusters by direct inspection and not just their silhouettes.

All of the images are displayed on (and can be captured from) a tablet that also serves to regulate the temperature, CO₂ and rpm of the culture vessels (**Figure 8**).

**Figure 7.** *A clinostat incubator, containing 6 culture vessels (one back illuminated).*

#### **Figure 8.**

*The tablet display showing regulation of the rpm and set value (left panel), culture vessel (main panel), clinostat incubator number (A-1, main panel top left), actual rpm (top right), actual temperature and CO₂ (bottom left). The front access port plug can be seen at the bottom right of the culture vessel.*
