# Microscope Enclosure for Temperature Regulation and Light Isolation
## 1 Introduction  
Fluorescence imaging of biological systems often requires an environment in which the temperature of the sample is regulated. For example, live cell microscopy typically involves imaging at the natural temperature of the biological system [1, 2]. Alternatively, sometimes it is advantageous to image biochemical processes at non-natural temperatures, or to vary the temperature to affect reaction kinetics [3, 4, 5, 6, 7]. Three methods are typically utilized for controlling sample temperature during imaging: (1) whole room regulation, (2) whole image system regulation, and (3) local sample regulation. For imaging at room temperature, the microscope's laboratory room conditions are sometimes sufficient for regulating the sample temperature. To image above or below room temperature, the temperature of the entire room can be increased or decreased using the building's HVAC system or the entire microscope can be placed in a lab-specific hot or cold room. A potential advantage with whole-room regulation is that the entire microscope, table, and image sample are all at approximately the same temperature. In theory, this situation minimizes temperature variations in the microscope which could affect sample temperature or optomechanics stability [8, 9, 10]. However, in practice, depending on the quality of the building's HVAC equipment, laboratory air temperature may vary with spatial position and time. HVAC equipment may also produce air drafts. Together, these conditions can result in unpredictable sample temperature and image instability, including lateral and axial (focal) drift [8, 9, 11]. Without proper compensation, this drift compromises the ability to acquire time-lapse images. To isolate the system from the room conditions, it is advantageous to enclose the microscope in a box. The temperature within the box can then be regulated independently of the room by heating or cooling the air within the enclosure [12]. This mimics whole-room control where the goal is to have the entire system and sample at the same temperature. However, it is typically easier to create a more consistent temperature for the imaging system in a smaller space that is isolated from the building HVAC. In addition, it often is not practical to cool or heat an entire room. Finally, even when not heated or a cooled, a whole-system enclosure provides protection from drafts in a laboratory. Another method to regulate sample temperature is locally, directly at the position of the sample. This can be achieved with devices such as resistive heaters, Peltier coolers, or water heater/cooler jackets attached to the sample holder or the objective [10]. Local temperature control enables quick equilibration and temperature changes, which can be useful for imaging at a series of different temperatures in a relatively short period of time. A disadvantage is the potential for poorly controlled temperature cycling resulting in image instability and inconsistent sample temperature [10]. In practice, and depending on the imaging requirements, a combination of whole room, whole system, and local sample regulation may be used to achieve the best imaging conditions. However, even the best combination of regulation systems may still require some type of active or post-imaging drift compensation [11; 13; 14; 15; 16; 17; 18; 19]. During biological imaging, it is also often necessary to isolate light entering and exiting the imaging system. Eliminating external light is essential for most measurements of luminescence. Even for fluorescence microscopy, an acceptable signal-to-noise requires lowering the background light by isolating the imaging system from external light sources, including room lights and ancillary lab equipment [10; 20]. This situation also minimizes undesirable fluorescence excitation and premature bleaching. Although it may be possible to reduce external sources, such as turning off room lights, this is not always practical, possible, or sufficient.