Microscopy of biological samples, fast temperature change.
In microscopy, especially when working with biological material, it is important to ensure a stable temperature within the sample. For example living cells require a constant temperature of 37° C in order to allow for studies under native conditions. Usually, this is done by using heating chambers or open heated microscope stages where the whole sample can be heated to any desired temperature as shown in figure 1.
Figure 1: Live-cell imaging chamber and controlled environ-ment microscope incubator.
Another solution is provided with delta T dishes by the company Biotechs. This system uses ITO coated glass slides, which are heated by an electrical current as shown in figure 2, providing an almost homogeneous temperature over the whole glass surface.
Figure 2: Biotechs Delta T4 live-cell imaging chamber configuration.
Oil immersion microscopy
In high-resolution microscopy, oil immersion objectives are used, where the sample is directly coupled to the objective via a drop of immersion oil to create a higher numerical aperture. However, this connection generates a thermal coupling between the sample and the whole microscope, which drains the heat from the sample and thus causes a thermal gradient with its peak right at the field of view and thus the region of interest. In figure 5) a heated ITO glass slide is shown in combination with an infrared camera. When such a glass slide is brought in contact with an oil immersion objective a strong drop in temperature right at the contact surface is observed (Figure 5e).
In order to compensate for this effect, objective heaters were developed, where a heating source is wrapped around the objective in order to adjust a desired temperature in the objective (Figure 3). As the objective is also thermally coupled to the whole microscope a huge mass has to be heated, which takes a long time.
Figure 3: Objective heater designs for live-cell imaging.
In order to reduce this heating or cooling time, the objective can be thermally decoupled from the microscope using an isolating ring as implemented by Zeiss (Figure 4).
Figure 4: Zeiss objective including an isolation ring, thermally decoupling the upper part and the lower part of the objective.
This reduces the mass, which has to be heated, but the time needed to heat or cool the objective to the desired temperature still remains on the order of several minutes.
Here, we present a new type of temperature controlled oil immersion objective, which can nearly instantly change the temperature within the sample contact surface and thus the field of view.
It is an objective with a conductive nano-coating at the outer surface of the lens heated by an electric current. This objective allows fast heating or cooling processes during observation of the specimen. A temperature change rate of 20° C per second for heating or cooling the sample could be demonstrated.
Using this objective, not only a desired temperature in the sample can be adjusted nearly instantly, also fast temperature changes in the specimen can be induced. If it is necessary to heat the whole sample, this objective can also be used in combination with an already existing heating chamber or heating stage, which was described above. For instance, when working with living cells, the whole cell chamber can be held at 37°C while right at the field of view - which is contemporary the region of interest - a fast temperature change can be induced and it's effect can be observed without affecting the rest of the sample.
Figure 5: Temperature distribution of a heated ITO cover glass with and without contact to an oil immersion objective. An ITO coated cover glass was connected to copper stripes on an isolated aluminum plate containing a hole in the middle (Figure 5 a, b). The surface temperature was recorded using an infrared camera (Figure 5 c). Without any contact with the oil immersion objective the cover glass shows a nearly homogeneous temperature distribution at the center (Figure 5 d). The temperature gradient towards the outer ring occures due to the cold aluminum plate. Bringing the oil immersion objective in contact with the cover glass leads to a strong decrease in temperature right at the contact surface (Figure 5 e).
In order to perform a proof of principle of the idea of a new objective with fast dynamic temperature control we executed the following experiments.
Proof of principle
An aluminum block was built with the same size of a Zeiss objective. This aluminum device represents the objective. The surface of the tip of the aluminum device was coated with an electrically non-conductive varnish. Two copper foils were glued on top of the aluminum cylinder and a round ITO cover glass was glued upside down on the copper stripes. This objective model fulfills perfectly the desired thermal properties as described before.
Figure 6: Aluminum objective model containing a round ITO cover glass with a diameter of 4mm.
In figure 7 the possibility of fast heating and cooling using such a nano coating is shown. An ITO coated glass plate with 4 mm in diameter was placed on a cooled aluminum plate of 9°. An electrical current was applied for 3 seconds and removed after-wards leading to fast increase in temperature and due to the cold aluminum plate to a fast decrease when the current was removed.
Figure 7: Heated ITO glass plate (4mm diameter) underneath a uncoated cover glass (22 x 22 mm2). The whole setup was placed on an aluminum plate, which was cooled down to about 9°C. Heating current was switched on for 3 seconds. Temperature was recorded using a Flir i3 infrared camera.
An objective with a conductive nano-coating at the outer surface of the lens performs fast heating or cooling processes during observation of the specimen.
The temperature change rate is about 20° C per second.
The final temperature (e.g. 37°C in cell experiments) can be adjusted almost instantly. The user has not to wait anymore till the objective and the thermally connected microscope are heated up.
Note that any existing oil immersion objective can be upgraded with such a coating.
Any existing oil immersion objective can be upgraded with such a coating.
Such an objective enables new experiments. For instance the reaction of cells to fast changing temperatures via fluorescent-stained proteins can be directly observed, (heat shock proteins, focal adhesion complexes, etc.) Also diffusion dynamics in lipid bilayers due to local changes in temperature can be investigated currently a topic of great interest.
Patent application filed November 2013.
Ultrafast temperature controlled objective (06.04.2018)