While maintaining homeostatic temperatures during imaging may seem like a no-brainer, the actual temperature of the cells being imaged is often lower than anticipated. So does a couple of degrees really matter? Yes, many cells types will stall and even reverse the cell cycle at 20–22°C,⁴⁴ see Figure 4. At temperatures between 23 and 35, the timing of mitosis is dilated indicating a substantial stress on the cells. One of the main reasons for lower temperatures than expected is the use of immersion objective lenses without the use of an objective lens heater. The microscope itself acts as a massive heat sink, causing as much as a 10°C temperature differential. Even with dry objectives there can be a significant temperature differential between the edges of the specimen holder and where the cells are being imaged. It is therefore imperative that the temperature where the cells are growing be measured not just the chamber holding them. This can be accomplished with an inexpensive non-contact thermometer equipped w/laser sighting, such as OS-PAL (∼$55.00 from OMEGA Engineering, INC.). When using immersion objectives or where there is not a line of sight to the imaging area (e.g., inverted microscopes), a small thermocouple probe can be directly inserted at the focal point of the microscope. This can be safely done by attaching a small probe to the coverslip (where the cells would normally be attached) and assembling the chamber normally, including filling with media or water. Depending on the chamber type, a small hole may need to be drilled to feed the probe wire through.