Heat sink played by immersion oil objectives

The thermal path generated by immersion objectives lenses affects the sample’s temperature

The function of immersion oil is to avoid changes in the diffraction index during the light path from the sample to the objective (Fig. 1). Changes in the diffraction index can be detrimental since light path can be significantly deviated.

Figure 1: Scheme of immersion (A) and dry (B) setting in an inverted microscope. Immersion oil is used to obtain high quality images ta high magnifications. The main drawback is that immersion oil will establish a thermal bridge (heat sink) between the objective and the sample. Black broken arrows = light path; red broken arrows = light diffracted; red arrows = bi-directional heat flow.

Figure 1: Scheme of immersion (A) and dry (B) setting in an inverted microscope. Immersion oil is used to obtain high quality images ta high magnifications. The main drawback is that immersion oil will establish a thermal bridge (heat sink) between the objective and the sample. Black broken arrows = light path; red broken arrows = light diffracted; red arrows = bi-directional heat flow.

On the other side immersion oil, although is not a good thermal conductor, it establishes a thermal bridge (heat sink) between the objective and the sample support (Fig. 1A).

Although the heat sink produced by oil immersion and the following changes in sample temperature is a well-known problem, very few systematic investigations do exist in the literature. Most of the thermalization solutions focused on keeping the sample at the right temperature but often poorly consider the effects of heat sink induced by the thermal bridge formed by immersion oil. On the other side several companies detected the need to take into consideration the heat sink factor and developed solutions to mitigate the heat sink generated by immersion oil. So far it appears that immersion oil-induced heat sink is much more sensed at the technical level rather than the scientific level.

The importance of an accurate system that control sample temperature and mitigate heat sink produced by immersion oil

Also when mitigation strategies or specific devices are used during microscopy imaging, it is rather common to have temperature gradients >3 degrees within the sample (Tab. 1, elaborated from (1))

Table 1: Advantages and drawbacks of thermalizing systems. Different factors, comprised the possibility to mitigate the heat sink induced by immersion oil, are here summarized. * = perfusing the sample with thermalized media is not applicable in case of not adherent cells/specimens. Some devices have fixed geometries and thus less flexibility in term of experimental design. Table extrapolated from (1).

Table 1: Advantages and drawbacks of thermalizing systems. Different factors, comprised the possibility to mitigate the heat sink induced by immersion oil, are here summarized. * = perfusing the sample with thermalized media is not applicable in case of not adherent cells/specimens. Some devices have fixed geometries and thus less flexibility in term of experimental design. Table extrapolated from (1).

Also in the case of fixed samples it is well know that the efficiency of several fluorophores is significantly improved leading to strong signal. Moreover, since the introduction of two-photons microscopy based on long wavelengths (1200 to 1700 nm) lasers, heating induced by water absorption need to be considered (2). In this view an accurate and very efficient temperature control that take into account the thermal bridge produced by immersion oil is likely to be useful to prevent cells/fluorophores problems associated with an excess of heating.

Another significant advantage of rapidly change and controlling with precision the sample temperature is the possibility to perform physiological temperature dependent experiments. In this aspect, considering the sensitivity of specific temperature-dependent physiological process it is mandatory to properly taking into account the disturbance induced by heat sink of immersion oil.

Especially in this latter case but also in general, a system that take into account the effects of objective temperature and the associated disturbances generated by immersion oil heat sink is able to exclude environmental thermal disturbances and produce more reliable and repeatable data.

The possibility to exactly control sample temperature also considering the heat transfer induced by immersion oil have several advantages (Fig. 2)

Figure 2: Main advantages arising from the possibility to rapidly changes temperatures with high accuracy (avoiding heat sink effect caused by immersion oil).

Figure 2: Main advantages arising from the possibility to rapidly changes temperatures with high accuracy (avoiding heat sink effect caused by immersion oil).

Conclusions: the need to monitore and correct the heat-sink artefact

The heat sink established when working with immersion oil is a critical, well-known but often underestimate factor. While several companies have developed systems to control temperature of samples only few of them take into consideration the disturbances induced by the immersion oil heat sink. Among those devices very few systems are able to rapidly switch sample temperature also taking in consideration this factor.

A fast and reliable system able to control sample temperature and the heat sink produced by immersion oil provides several advantages in terms of repeatability and quality of data.

The CherryTemp heater/cooler device is a unique temperature controller for microscopy embedding a 4-points electrode deposition which continuously takes into account the heat sink played by the objective in contact with the sample.

Thanks to PT-100A 0.1°c precise temperature sensors and continuous monitoring of both the room temperature variations and the objective temperature itself, our algorythm compensates the impacts on the sample’s temperature leading to an unprecedented stability and precision of temepratuer control on a microscope.

CherryTemp heater/cooler for live-cell imaging

Ultrafast shifts: dynamic 10 seconds temeprature changes


Ultra-precise: the most precise temperature calibration


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Bibliography/Sources

(1) Velve Casquillas G, Fu C, Le Berre M, Cramer J, Meance S, Plecis A, Baigl D, Greffet JJ, Chen Y, Piel M, Tran PT. Fast microfluidic temperature control for high resolution live cell imaging. Lab Chip. 2011 Feb 7;11(3):484-9. doi: 10.1039/c0lc00222d. Epub 2010 Nov 19.

(2) Singh A, McMullen JD, Doris EA, Zipfel WR. Comparison of objective lenses for multiphoton microscopy in turbid samples. Biomed Opt Express. 2015 Jul 30;6(8):3113-27. doi: 10.1364/BOE.6.003113. eCollection 2015 Aug 1.

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