Temperature is one of the main indicators of the state of the biological system. If a person develops an infection, their body temperature rises (usually, but not always), which is a sign of the immune system's response to the threat. In other words, the temperature can be used to determine the approximate state of the body. The problem is that a person is big (literally), but, for example, nematodes are only about 1 mm long. It was extremely difficult to measure the temperature of such a small organism, but scientists from Osaka University (Japan) have developed a method to solve this problem. What means were used to implement the nanothermometer, what practical experiments have shown, and where can this development be used? We will find answers to these questions in the report of scientists. Go.
Research basis
The body temperature of a living organism varies depending on the degree of influence of internal and external factors. We are accustomed to the fact that the ambient temperature directly affects the temperature of cold-blooded people, therefore, its values change with enviable regularity. However, even in warm-blooded animals under normal physiological conditions, temperature fluctuations are observed, which can be associated with homeostatic thermoregulation and energy metabolism.
In other words, the joke is great here: "I'm not messing around, I'm a very busy person at the cellular level." By accurately measuring temperature and its dynamics on a submicron scale, a lot of information can be obtained regarding cellular and molecular activity. The problem is that as the object of measurement decreases, the complexity of its conduct increases (it is difficult to put an ordinary thermometer from a pharmacy into a nematode).
The study authors note that conventional electrical thermometers do not have submicron resolution, and near infrared thermography usually helps determine the surface temperature of biological samples, but not the internal temperature.
Of course, there are already light-emitting nanothermometers (for example, thermosensitive molecular probes) that are able to overcome this limitation. But this technique also has disadvantages. The main one is long-term stability, or rather its absence. Such devices cannot accurately measure temperature changes that take a long time (say a couple of hours). Not to mention the toxic effect on the sample from such a thermometer.
In this work, the scientists describe the concept of a nanodiamond (ND from nanodiamond ) quantum thermometer, which is highly accurate, robust and low in toxicity. The principle of its operation is as follows: the sensor reads the temperature as a frequency shift of the optically detectable magnetic resonance (ODMR fromoptically detected magnetic resonance ) of defective centers of nitrogen vacancies (NV from nitrogen-vacancy ), which mainly arises due to thermal expansion of the lattice. The NV sensor core is deeply embedded in the diamond lattice and is immune to various biological environmental factors. The introduction of this quantum sensor into more complex organisms makes it possible to read their thermal activity at a specific site in real time. But the process of implementing such a technique is fraught with a number of difficulties.
Nematoda (roundworm) of the Caenorhabditis elegans species .
Multicellular model organisms such as Caenorhabditis elegans worms, need a special chamber that can accommodate a millimeter-sized body, and the samples themselves need to be analyzed quickly to maintain their physiological state. Quantum ND thermometers move much faster than in cultured cells, even if the body is dehydrated, requiring a fast particle tracking algorithm. In addition, the positional movement of ND and the complex structure of the body cause significant fluctuations in the detected fluorescence intensity, which is likely to cause temperature measurement artifacts. The solution to these problems at this stage of the study is associated with the adaptation of the device to the individual characteristics of the analyzed sample. The issue of versatility and ease in setting up the future nanothermometer is planned to be considered in future works,in the meantime, attention was paid to the concept itself and the basic principles of work.
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The basis of the nanothermometer is a confocal fluorescence microscope equipped with a microwave irradiation unit (1A).
Image # 1
Nitrogen vacancy ODMR can be measured as a decrease in the intensity of laser-induced fluorescence when spin resonant microwave excitation is applied, since spin excitation activates the non-fluorescent relaxation pathway from the excited state to the ground state ( 1B ).
The sample chamber is a disposable glass bottom dish integrated into the antenna that provides large optical access (12mm diameter) and ease of use ( 1C ) suitable for delicate samples such as stem cells. Time from the capture of the wormCaenorhabditis elegans is only 15 minutes before the actual measurement. This helps keep the worm alive and provides more data on its health.
In addition, the system effectively integrates fast particle tracking and highly accurate real-time temperature estimation from NV center offset ODMR.
In particle tracking, the system measures the ND fluorescence intensity along the xyz axes of the microscope and focuses on the corresponding maximum fluorescence every 4 seconds (shorter tracking interval is possible) during which the temperature is estimated with sampling times from 0.5 to 1.0 seconds. ( 2A ).
Image No. 2
There are several methods of quantum thermometry, but in this work the ODMR four-point measurement method was used. This method assumes that the number of photons detected at all four selected frequencies is scaled linearly according to changes in the detected fluorescence intensity.
However, it was found that each subsequent photon shows a difference in light sensitivity of about ∼0.5%, which actually creates significant artifacts in the estimate of the frequency shift (i.e. ∼300 kHz, which corresponds to several degrees Celsius), especially in the low-photon mode.
These artifacts are most likely due to optical power dependent asymmetry in the ODMR spectrum. To accurately measure the temperature of complex optical dynamic systems (i.e. biological systems), it is necessary to get rid of such artifacts. Therefore, an error correction filter has been added to the four-point measurement method.
To evaluate the operation of the system, coupled with error correction, real-time measurements of the temperature ND were carried out during stepwise thermal events. Sudden changes in temperature could not be used, since sudden changes in temperature cause large defocusing of focal spots and associated fluctuations in the fluorescence intensity.
At 2B shows the time profiles of the total number of photons (I tot) and temperature estimation ND (∆T NV ) when the sample temperature (T S ) changes from 44.3 ° → 30.4 ° → 44.3 ° with a step of ∼2.8 °. The system accurately outputs ∆T NV , corresponding to T S , while the focus position has moved significantly, especially along the z-axis over a distance of more than 30 μm ( 2C ).
With a 3 ° step, a 6 μm positional shift in the z-axis appears within 3-4 minutes, but the tracking speed is high enough to follow the dynamics of 105 nm / s for 96 minutes ( 2C ).
Moreover, ∆T NV clearly demonstrates anticorrelation with I tot... A statistical study of this type of temperature dependence determines the average values for SD: I tot -1 dI tot / dT = -3.9 ± 0.7% / ° and dD / dT = - 65.4 ± 5.5 kHz / ° ( 2D ). The temperature measurement accuracy is ± 0.29 ° and <0.6 ° C, respectively, which gives a sensitivity of 1.8 ° C / √Hz.
After achieving reliable and accurate real-time thermometry as part of the development phase, test local temperature monitoring was carried out on live worms.
Image №3
Pictured 3A shows ND anesthetized worms inside, placed near a microwave antennas. These NDs are well dispersed in water due to the surface functionalization of polyglycerol (PG frompolyglycerol ) and are introduced by microinjection into the gonads (the gonads of the experimental worm).
Graph 3B shows the ODMR spectrum of a single ND (marked with an arrow at 3A ). 3C shows the time profiles of I tot and ∆T NV over a period of 1 hour as the temperature T S changes .
First, T obj was measured at 33.2 ° C, after 6 minutes, a decrease to 25.3 ° C was performed. As a result, Tobj reached 28.6 ° at 35.2 minutes. ∆T NV showed an exact temperature change between two stationary states: 33.2 and 28.6 ° C.
The display of the real dynamics of temperature inside the worms between these two steady states is displayed due to the fact that ∆T NV always lags behind T S and shows a slightly underestimated response due to the finite heat capacity of the microscope objective and the environment. I tot also shows the gradual changes in fluorescence intensity caused by temperature.
Particle tracking was also satisfactory ( 3C ). Within 0-15 minutes, the counted photons show frequent bursts due to positional fluctuations of the ND at approximately 400 nm for several seconds.
The test results clearly indicate the high accuracy of temperature measurement inside the nanoscale biological system in real time. Further, it was decided to conduct additional tests, before which the experimental worms underwent pharmacological treatment with C 10 H 5 F 3 N 4 O (FCCP from carbonyl cyanide-4- (trifluoromethoxy) phenylhydrazone ), causing immobile thermogenesis (roughly speaking, an increase in temperature due to an increase metabolism and without additional muscle activity).
Image №4
Pictured 4A shows ND worms stimulated by FCCP. And graph 4B shows the time profile of ∆T NVND marked with an arrow in the pictures.
At the seventh minute after the start of the measurement, the FCCP solution was used. At the 32nd minute, ∆T NV begins to gradually increase, and at the 48th minute, an even greater additional increase is observed when the level of temperature change increases from 4 to 7 ° C. The fever lasted about 80 minutes.
During stimulation, NDs slowly move a few micrometers over an hour, which confirms the results of separate experiments in which NDs were continuously observed under a microscope.
The control group of worms ( 4C and 4D ), which were not injected with FCCP, showed a uniform ∆T NV response throughout the test without any obvious temperature changes.
To further confirm that FCCP actually induces an increase in body temperature in worms, quantification of ND-labeled worms was performed in both control and experimental groups ( 4E ). The graph clearly indicates an increase in temperature in the worms from the experimental group compared to the control.
Another control experiment, in which no buffer was added and the ∆T NV was monitored statically, shows that the addition of a dopant causes ∆T NV to fluctuate at a certain level, either due to temperature changes or due to ODMR shift artifacts. However, the observation of such a shift is impossible with the addition of FCCP, which additionally confirms the increase in temperature due to FCCP in the experimental group of worms ( 4F).
For a more detailed acquaintance with the nuances of the study, I recommend that you look into the report of scientists and additional materials to it.
Epilogue
In this study, scientists were able to develop a technique that allows you to accurately measure the temperature inside a nanoscale biological system in real time. In exaggerated terms, they managed to measure the body temperature of the worm Caenorhabditis elegans , which is approximately 1 mm long.
It is important to understand that it is much easier to measure anything in a large sample than in a small one. Nevertheless, the use of nanodiamonds injected into the body of the worms made it possible to find out the body temperature of the worm under normal conditions. These nanodiamonds, getting inside the body, begin to move rapidly. A specially developed algorithm and a confocal fluorescence microscope made it possible to track and analyze their movement. The data obtained made it possible to accurately determine the body temperature of the worm and its dynamics, even after the introduction of a special substance that caused an increase in temperature.
This work not only shows that quantum technologies can and should be applied in biology, but also expands the range of possibilities in the aspect of diagnosing various processes at the macro level. Very often, the state of a biological system directly or indirectly depends on the processes occurring inside cells, which were previously extremely difficult to measure in real time. Having received more information regarding the constituent elements of the system, you can better understand the system itself, which, of course, will allow you to more effectively influence its operation.
Thanks for your attention, stay curious and have a great weekend guys! :)
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