Temperature control for microscopy imaging : objective heater collar
The challenge with live-cell imaging is to maintain the cells in a physiological-like environment. Cell temperature is one of the many parameters to control during image acquisition. There are several temperature controllers available for users to choose from, and depending on the length and type of experiments some systems will be better suited than others. Objective heater/cooler collars are used as a complement of already set-up temperature controller devices.
Introduction
Objective heaters can be adopted as complementary systems in order to mitigate the heat sink produced by the objective lens when working with immersion lenses.
How does an objective heater work?
When working at high resolution, the objective lens is in contact with the glass coverslip of the sample trough the immersion oil. The oil establishes a thermal bridge that leads to a significant heat transfer from the warmer side to the colder one. The immersion objective acts thus like a heat sink, creating local temperature mismatches in the sample. The magnitude of those temperature discrepancies depends on several factors (room temperature, system adopted to thermalize the sample, difference between sample and room temperature, etc…) and can be easily underestimated. Most of temperature controllers are not designed to specifically correct/mitigate the heat sink produced by immersion objectives. Thus coupling objective collars to the standard thermalization systems is a requisite to prevent large temperature discrepancies and gradient setting. Based on the adopted technology it is possible to divide objective collars into two main classes: electronic or fluid based collars.
Electronic objective collars wrap the objecive and are able to heat it in order to avoid the heat sink phenomenon. Often they include a thermal sensor that takes into account the heating effect produced by the light passing across the lens of the objective. This feature is particularly useful during long acquisition experiments.
Collars based on circulating fluids allow more accurate and stable control of the objective temperature and can easily go below room temperature. Fluidic collars are composed of a metallic hollow ring fixed on the objective in the proximity of the lens. A thermalized fluid is perfused inside the ring by an additional device that provides the thermalized water flow.
Features
Large temperature range
The majority of objective collars ensure a temperature control from RT to 50°C. When the experiment requires to thermalize below room temperature, fluidic-based collars are adopted.
Enhanced temperature homogeneity of the sample
Objective collars improve temperature uniformity across the sample when working with immersion objectives. They mitigate the thermal gradient
Drawbacks
Not self-sufficient
Objective collars do not thermalize the sample, they must be coupled to another temperature control system (thermalized stages or top stages).
Difficult coupling to other systems to perform temperature shifts
Performing temperature shifts without generating gradients is much complicated given the fact that objective collars must be coupled with other temperature control devices. If the two systems are not coordinating the differential thermalization speed this will ultimately produce a gradient accross the sample.
Must be operated by experienced personnel
Strong knowhow is required to understand artifacts generated by temperature controlled objective collars and to make the complete system work properly, especially when temperature shifts are needed.
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