JPH11258159A - Method for measuring temperature of single cell in cell specimen or tissue biopsy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To realize a method for detecting the temp. and/or metabolic state of a single cell contained in a cell/tissue specimen. SOLUTION: In a method for detecting the temp. of a single cell, (a) cells are arranged to a metabolism probe containing a solid substrate including a temp.-sensitive phosphor sealed in a polymer (the phosphor emits a detectable fluorescent signal when it is excited by proper ultraviolet rays, visible light or infrared rays and, when living cells are arranged to the metabolism probe, the fluorescent signal is affected by the metabolic speed of cells) and (b) the phosphor is excited by proper ultraviolet rays, visible light or infrared rays and (c) the fluorescent signal is detected (the intensity of the detected fluorescent signal is the index of cell temp.).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates generally to a novel method for measuring the temperature of a single cell or group of cells in a tissue sample. Also included is the use of this method as a diagnostic.

[0002]

BACKGROUND OF THE INVENTION Living cells require a stable rate of metabolism. Therefore, in both cells and organisms containing cells, such regulation is under strict control. Approximately 50% of total energy in carbohydrates is AT
P and the rest is released as heat [Alberts
Et al., Molecular Biology of the Cell, (1989)]. ATP
Are, in turn, used during growth for biosynthesis and otherwise used for work, the energy of which is released as heat. This release of heat is used to warm the living body in the form of temperature. The inability to maintain stable metabolic levels at the biological level is a fever-pathology called elevated temperature of the organism. Changes in metabolic levels are seen in a variety of conditions, including bacterial or viral infections, in cancer, and during general weakness seen in sepsis or cachexia. It is often understood that the temperature of an organism is an indicator of the organism's health.
Even a slight shift in temperature can indicate a lesion. However, individual cell temperatures cannot be analyzed due to current technology limitations. Thus, it is not yet known how much the cell temperature changes over time within a cell or between two different cells. However, based on studies of large populations of cells, there is good evidence that certain symptoms can be detected by measuring temperature at the single cell level.

[0003] Cellular metabolism can be considered a balance sheet for all of the enzymatic reactions that occur in cells. In this context, the next exothermic activity is seen as black body radiation, the heat we analyze when we measure the temperature of the cells. Previous studies of biological metabolic activity have used levels of whole organisms, or at least calorimetry applied to individual organs. Microcalorimetry can be applied to as few as 10 cells. However, the limitations of current techniques for elucidating thermogenesis from single cells have significant limitations [Kemp,
Thermal and Energetic Studies of Cellular Biologic
al Systems, Edited by AM James (Bristol: Wright), 147-1
66 (1987)]. The most sensitive technique applied to measure metabolism in tumor cells is the Cartesian diver, which can resolve hundreds of cells [Lutton and Kopac, Cancer
Res., 31: 1564-1569 (1971)]. However, this technique is still too coarse to elucidate a heterogeneous population of cells, which requires dissociation of the cells. Much of normal cell physiology is a dynamic process that requires cell interactions in a three-dimensional matrix. Many cell activities are altered by contact between cells. All of this is lost when the cells are dissociated. Recently, metabolites in living cells, such as
Techniques have been developed for measuring ATP, glucose and lactate [Hossman et al., Acta Neuropathologica, 6
9: 139-147 (1986); Okada et al., Journal of Neurosurger.
y, 77: 917-926 (1992)]. These techniques are based on photographic film [Hossman et al., 1986, supra; Okada et al., 1992, supra] or a photon counting camera [Tamulevicius.
And Streffer, British Journal of Cancer, 72: 1102-11
12 (1995)] and demonstrated substantial heterogeneity in metabolism in tumors when analyzed at millimeter spatial resolution.

Unfortunately, some important issues cannot be addressed at present. This is because the metabolism of individual cells cannot be measured. For example, we cannot currently study a heterogeneous population of cells and elucidate the activity of individual cells rather than the average of a mixture. For example, tumors are made up of cancer cells, normal cells, and infiltrating immune cells, each of which metabolizes at very different rates.
In this case, the measurement of the average metabolism of the tumor may not reflect the actual metabolism of individual cells. In most cells, aerobic respiration is responsible for almost all of the production of ATP, and anaerobic glycolysis is the rest. It was observed more than 50 years ago that anaerobic respiration was significantly increased in ascites tumor cells versus non-tumor cells [Warburg et al., The Metabolism of Tumors,
Edited by O. Warburg, Constable & Company LTD., London,
129-170 (1930a); Warburg et al., The Metabolism of T
umors, O. Warburg Editing, Constable & Company LTD., L
ondon, pp. 254-265 (1930b)]. This has led to the hypothesis that aerated breathing is impaired in tumor cells [Warburg, S.
cience, 123: 309-314 (1956)]. As much was learned about metabolism in the intervening years, aerial respiration was found to be normal in tumor cells, but anaerobic respiration was increased. The mechanism responsible for this increase in anaerobic respiration is currently unknown. One major limiting factor in learning the mechanism is the inability to analyze the relative metabolic levels of individual cells. As noted above, one particular problem is that tumors consist of a mixture of normal, malignant, and immune cells. Current techniques only allow the measurement of the average metabolism of this mixed population of cells. The second problem is considerable heterogeneity within the tumor cells. For example, in tumors, oxygenation is often rate-limiting for tumor growth [Kallinowski et al., J. Cel. Physiol., 138: 183-
191 (1989)]. Thus, in rapidly growing tumors, growth is limited by angiogenesis, the growth of new blood vessels for the delivery of oxygen and nutrients.

[0005] Chemotherapy is a powerful tool used to fight tumors. However, tumor cells frequently develop resistance to chemotherapy. This resistance was observed as reduced sensitivity to a broad spectrum of chemotherapeutic drugs, and such cells were classified as multidrug resistant [Simon
Proc. Natl. Acad. Sci. USA, 91: 3497-3504 (1994)
]. While these cells were initially considered “supercells” that could withstand any therapeutic challenge, there is now increasing evidence that multidrug-resistant cells behave more like normal cells and avoid chemotherapy. It is evident in some aspects that these cells have undergone "reverse" transformation in their nature [Biedler et al., Cancer
& Metastasis Reviews, 13: 191-207 (1994)]. Once the chemotherapeutic agent is removed, these cells recover their aggressive malignancy. It is not known what happens to the metabolism of multidrug resistant cells during this period. The indirect result is
This indicates that the level of anaerobic respiration in these cells has returned to the level seen in untransformed cells [Miccadei et al.
Oncology Research, 8: 27-35 (1996)]. Therefore, there is a need to measure the metabolism of individual cells that are multidrug resistant to diagnose when tumors are shifting from their quiescent multidrug resistant phase to a more malignant phase. Furthermore,
There is a need to provide a method of measuring the temperature of individual cells to measure metabolic changes that occur in normal or tumor cells. In addition, there is a need for a method of measuring the temperature of cells from a fresh biopsy of live tissue to quickly diagnose the tissue for the presence of tumor cells.

Although the emission of almost all phosphors is affected by temperature, the use of phosphors that are particularly sensitive to
dner et al. [Appl.Phys.Lett. 40: 782-784 (1982); Appl.Ph
ys. Lett. 42: 782-784, (1983)] as the first technique for calibrating temperature. Kolodner uses Eu (TTA) ₃ encapsulated in phosphor, polymer and PMMA
Temperature resolutions of 0.07 ^o K and a spatial resolution of 10 μM were obtained.
Thus, this temperature sensitive phosphor could be used to quantify temperature. Various phosphors, including Eu (TTA) ₃ , are solubilized in various solvents and polymers and are described in John P. Sulliv
an (see Table 1 adapted from Pursue University, [Campbell et al., 1994, supra]) and was used to "paint" the wings of airplanes. The change in fluorescence
For example, it can be used to monitor airplane wing temperature changes in a wind tunnel. To date, temperature sensitive phosphors have been used for integrated circuit diagnostics [Kolodner et al., Appl. Phys.
Lett., 42: 117-119 (1983)], detected the boundary layer transition of a two-dimensional wing and visualized the interaction between the leading eddy and the surface of the delta wing [Campbell et al., Sixth Intern. Symp. . on Appli
cations of Laser Techniques to Fluid Mechanics, Li
sbon, Portugal (1992); Campbell et al., Temperature Mea
surement Using Fluorescent Molecules, Abstract (19
92); Campbell et al., Temperature SensitiveFluorescent.
Paint Systems, 18: 94-2483 (1994); Hamner et al., A Scan
ning LaserSystem for Temperature and Pressure Sens
itive Paint, 32: 94-0728 (1994)]. Citation of any reference herein shall not be construed as an admission that such reference is available as "prior art" in the present application.

[0007]

SUMMARY OF THE INVENTION In its broadest embodiment, the present invention provides a method for measuring cell temperature by using a temperature sensitive phosphor. The present invention uses metabolic probes that include a solid substrate that includes a temperature sensitive fluorophore encapsulated in a polymer. When the phosphor is excited with the appropriate ultraviolet, visible, or infrared light, it emits a detectable fluorescent signal. When a living cell is placed on a metabolic probe, the intensity of the fluorescent signal is affected by the metabolic rate of the cell.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The metabolic probe is a fluorophore capable of emitting a stable fluorescent signal while encapsulated in a polymer adjacent to an aqueous solution for at least one hour after excitation with ultraviolet light at 37 ° C., pH 7.5. It is preferred to include. In a particular embodiment of the invention, cells placed on metabolic probes produce a 2.5% change in the intensity of the fluorescent signal per 1 ° C. increase in temperature. In one embodiment, the temperature sensitive fluorophore included by the metabolic probe is Eu (TTA) ₃
It is. In another embodiment, the polymer of the metabolic probe is poly (methyl methacrylate). In a preferred embodiment, the polymer is poly (methyl methacrylate) and the temperature sensitive phosphor is Eu (TTA) ₃ .
The solid substrate of the present invention may be made from any inert material, but is preferably glass or plastic. In a further preferred embodiment of this type, the solid substrate is a glass coverslip. The present invention provides a method for detecting the temperature of a cell. One such embodiment involves placing the cells on a metabolic probe that includes a solid substrate that includes a temperature sensitive fluorophore encapsulated in a polymer. When the phosphor is excited with the appropriate ultraviolet, visible, or infrared light, it emits a detectable fluorescent signal. When a living cell is placed on a metabolic probe, the intensity of the fluorescent signal is affected by the temperature of the cell. In one particular embodiment of the method, the polymer is a non-aromatic synthetic polymer. In one particular embodiment of this method, after excitation with ultraviolet light, the phosphor is capable of emitting a stable fluorescent signal at 37 ° C., pH 7.5 for at least one hour.

The present invention also includes a method for detecting metabolic activity of a cell. One such embodiment is to place a cell on a metabolic probe of the invention, detect the temperature of the cell by exciting the probe's fluorophore with the appropriate ultraviolet, visible, or infrared light and detecting the fluorescent signal. The intensity of the detected fluorescent signal is an indication of the temperature of the cell. The measured temperature is then correlated with the metabolic activity of the cell. In a preferred embodiment of this type, the cells are tumor cells. The present invention also provides for placing a cell on a metabolic probe of the present invention (as described above) and exciting the metabolic probe's fluorophore with appropriate ultraviolet, visible, or infrared light,
A method for detecting the presence of tumor cells in living tissue, comprising detecting the temperature of the cells by detecting a fluorescent signal. The intensity of the detected fluorescent signal is correlated to the temperature of the cell. It is known that the metabolism of tumor cells is significantly higher than that of non-transformed cells. The present invention allows for the detection of tumor cells in a mass of normal (eg, non-transformed) cells by measuring the temperature of the cells. In a preferred embodiment of this type, the live tissue is obtained from a tumor biopsy. In a related embodiment, the invention provides that the cell is placed on a metabolic probe of the invention (as described above) and that the fluorophore of the metabolic probe is appropriately ultraviolet light,
Abnormal metabolism of cells surrounded by healthy tissue (in a normal cell mass) characterized by detecting the temperature of the cells by excitation with visible or infrared light and detecting the fluorescent signal Provide a method of detecting presence. Differences in cell temperature relative to control cells indicate that the cells are undergoing abnormal metabolism. In one such embodiment, abnormal metabolism of the cell is indicative of a cell having ultraviolet damage. In another embodiment, abnormal metabolism of the cell is indicative of a cell undergoing cachexia. In a third embodiment, abnormal metabolism of the cell is indicative of a cell undergoing apoptosis.

In one particular embodiment of the present invention,
The metabolic probe is placed on the microscope, the fluorophore is excited,
The luminescence is detected using a microscope. In one such embodiment, the microscope is a laser scanning confocal microscope equipped with an ultraviolet laser for excitation. In another such embodiment, the microscope is a fluorescence microscope equipped with a UV excitation filter and a Hg / xenon lamp with a visible emission filter at 615 nm ± 10 nm. The present invention also includes a method for producing the metabolic probe of the present invention.
One such embodiment involves contacting the polymer with a temperature-sensitive phosphor in a miscible solvent to produce a solution of the phosphor and the polymer. The solution is then placed on a solid substrate, and the polymer is then annealed to the solid substrate. Preferably, the solid substrate used in this method is glass or plastic. In a further preferred embodiment of the method, the solid substrate is a glass coverslip. In a preferred embodiment of this method, the glass coverslip is immersed in a strong acid (eg, 70% nitric acid) before placing the polymer-phosphor on a glass slide. In another embodiment of the method, the solid substrate is rotated during annealing of the polymer to the solid substrate.

[0011] In yet another embodiment of the method, the polymer used is poly (methyl methacrylate). In yet another embodiment of the method, the temperature sensitive phosphor is Eu (TTA) ₃ . In yet another embodiment,
The solvent is nitroethane. In a specific embodiment of this type, the metabolic probe is obtained by a method comprising contacting a solution of 0.1 mM poly (methyl methacrylate) in 96% nitroethane with 200 mM Eu (TTA) ₃ in 96% nitroethane. Can be The phosphor / polymer solution is then placed on an acid-washed glass slide, which has already been soaked in 70% nitric acid, washed with distilled water, rinsed with ethanol and then dried in a vacuum oven . The glass slide is then mounted on the rotor and rotated. The glass slide is then baked in a vacuum oven at 100 ° C. for 1 hour to anneal the polymer. These and other aspects of the invention will be better understood with reference to the following drawings and detailed description.

The present invention provides a method for monitoring the temperature and / or metabolic changes of a single cell or group of cells using the metabolic probes described herein. The cells may be dissociated from other cells or parts of the cell sample. If the cells are tumor cells, such monitoring will assist in understanding the cell biology of the tumor cells. The metabolic probes of the present invention and related methods can also be used to determine whether a tumor cell is present in a fresh biopsy of living tissue during the time frame that results in a diagnosis during a tumor biopsy. In this method, biopsy analysis may be performed immediately after tissue removal, and the health physician immediately determines whether additional tissue needs to be removed, or whether more surgery is needed. Can be determined. Such a procedure has many advantages over current techniques that require an initial biopsy, and then typically a 7-10 day evaluation of the tissue, often followed by additional surgery. Having. Thus, the present invention allows a medical physician to perform surgery and immediately respond to tissue analysis, thereby preventing further tissue degradation during the analysis time span.

[0013] The present invention also includes a method for detecting cell temperature using the metabolic probe of the present invention. The metabolic probes of the present invention include a solid substrate comprising a temperature sensitive fluorophore encapsulated in a polymer. When the phosphor is excited with the appropriate ultraviolet, visible or infrared light, it emits a detectable fluorescent signal. This fluorescent signal is affected by the metabolic rate of the cell placed on the metabolic probe. Thus, by exciting the fluorophore with the appropriate light and then detecting the intensity of the fluorescent signal, the temperature of the cell can be measured. Therefore, as appearing herein, the following terms should have the definitions set forth below. As used herein, a "metabolism probe" includes a solid substrate comprising a temperature sensitive fluorophore encapsulated in a polymer, wherein the fluorophore is excited at the appropriate wavelength of light (eg, ultraviolet, visible or infrared). At times, the fluorophore emits a detectable fluorescent signal, and when a living cell (alone or as part of a cell sample) is placed on a metabolic probe, the fluorescent signal emits heat (eg, of the cell) (Measured as temperature and / or metabolic state). As used herein, a "solid substrate" may be made of any solid material, preferably plastic, metal, or glass, more preferably a glass lens. As used herein, "about pH 7.5" refers to pH 7.0-pH
Including 8.0. In one application of the invention, the temperature of cells obtained from a cell sample from a patient suspected of suffering from cachexia is measured. Cachexia is a condition that depletes the body and is often fatal. The cachexia induced by tumor necrosis factor is associated with increased production of lactic acid and is the result of activating the futile substrate cycle between fructose 6-phosphate and fructose 1,6-bisphosphate. Conceivable. It burns cellular ATP [Zentella et al., Cytokine, 5: 436-447 (1993)]. Thus, the measurement of elevated temperature on cells obtained from a patient suspected of having cachexia is consistent with a diagnosis of cachexia.

In another aspect of the invention, the mechanism of fever induced by macrophage inflammatory proteins can be explored. Fever is a factor released by macrophages: macrophage inflammatory protein [Myers et al., Neuroc
Chemical Research, 18: 667-673 (1993)]. Although this factor is known to regulate plant intake by animals, it is still unknown where or how they have their effect on cells to increase metabolism and increase temperature. Thus, determining which particular cells (or cell types) release excess heat and produce a fever can be achieved by detecting the temperature of individual cells (or cell types). In a particular aspect of the invention, the temperature of the cultured cells is measured. In one such embodiment, a population of breast cancer cells is used. In another such embodiment, drug resistant breast cancer cells are used. In yet another embodiment, non-transformed breast cells are used. In a preferred embodiment, two of these cell types are used. In a further preferred embodiment, all three cell types are used. In a preferred embodiment, the breast cancer cells are from a human source. In one particular embodiment,
Brown adipose tissue cells from the tumor cell line HIB 1B are used. In another embodiment of this aspect of the invention, a section of breast tissue is used as a cell source. Sections from the breast include adipocytes, which are filled with large fat globules with very low metabolic levels, and duct cells that are actively metabolizing and secreting. The basal metabolic rate of tumor cells is significantly higher than that of "normal" cells. Assays using skin tumor surface temperature measurements show a temperature difference (after irradiation) of up to 3.3 ° C.

[0015] In current medical practice, excised tissue obtained during a biopsy is prepared for a tissue assay that requires 7-10 days. Thus, the patient is no longer in the operating room when the results are measured. Therefore, it is often necessary that subsequent surgery be scheduled. A method for detecting the temperature of cells in a section using the method of the present invention involves the localization of "hot spots" of metabolic activity (cells that emit more than normal amounts of heat) as a fingerprint of the tumor cells. Allows cells of a tissue section to be immediately analyzed using a conventional fluorescence microscope to determine whether the cells are being stained. Such measurements allow the surgeon to immediately determine whether additional tissue should be removed. Yet another aspect of the invention involves measuring the temperature / metabolism of individual cells to monitor the shift of quiescent multidrug resistant cells to a more malignant state. In this case, an increase in temperature would be consistent with such a shift. Such monitoring is particularly beneficial during chemotherapy and more particularly after termination of such treatment. In a particular embodiment, a cell or tissue biopsy is placed on a metabolic probe and then equipped with a laser scanning confocal microscope equipped with an ultraviolet laser for excitation, which allows simultaneous emission measurements from temperature sensitive fluorophores. Is detected by In another such embodiment, detection is performed on a fluorescence microscope equipped with a Hg / xenon lamp with an ultraviolet excitation filter and a visible emission filter.

As mentioned above, one preferred phosphor is Eu.
(TTA) ₃ , which has an excitation maximum at 360 nm and an emission maximum at 614 nm. More preferably, Eu (TTA) ₃ is combined with polymer poly (methyl methacrylate), (PMMA) and annealed to a solid substrate to form a metabolic probe. However, other suitable combinations are contemplated by the present invention. Such potential combinations are illustrated in Table 1. These and other combinations can be tested to determine their suitability for use as components of metabolic probes. In particular, the key criteria for selecting a phosphor compound are that the phosphor has the ability to (i) exhibit a maximum change in fluorescence with temperature change at 37 ° C, and (ii) be encapsulated in a polymer and released from aqueous solutions. And (iii) non-toxic to living cells. Thus, such tests show the sensitivity of the phosphor to temperature changes at 37 ° C., the stability of the phosphor in aqueous environments, the stability of the phosphor during encapsulation in polymers, and the resistance of the phosphor to photobleaching. Such resistance may be required for long-term observation and subsequent calibration). The greater the sensitivity of the phosphor to changes in temperature at 37 ° C., the greater its stability under the above conditions, including resistance to photobleaching, making the phosphor more suitable as a component of a metabolic probe. Similarly, the polymer is selected to have an optimal effect on the temperature sensitive phosphor. Finally, the phosphor / polymer combinations need to be compatible with living cells, ie, they should not adversely affect cell viability. The following examples describe these and / or other factors. The invention will be better understood with reference to the following non-limiting examples, which are given by way of illustration of the invention. The following examples are provided to more fully illustrate the preferred embodiments of the present invention. However, it should not be considered as limiting the broad scope of the invention.

[0017]

EXAMPLE: Fluorophores and polymers suitable for metabolic probes
Determining introduced to select a fluorescent material and a polymer suitable for metabolic probes of the present invention, it was introduced several criteria. For example, 37 ° C
Is an important criterion, and a large sensitivity is desirable. Similarly, the phosphor is selected for its stability in an aqueous environment. Because surviving cells are most stable in an aqueous environment. In addition, the phosphor is selected for its stability during encapsulation in the polymer. For example, in a cell sample, changes in fluorescence are the result of temperature release from cells,
Or should be separated from the phosphor by about 50 nm thickness of the polymer to ensure that it is not the result of secretion of iron, which can alter the properties of the phosphor. Furthermore,
A polymer is selected that optimizes the properties of our temperature sensitive phosphor. Such polymers should be selected for their considerable resistance to photobleaching (the properties required for long-term observation and subsequent calibration). Furthermore,
Polymers and fluorophores should be compatible with living cells to ensure that the polymers and fluorophores do not adversely affect cell viability.

Materials and Methods Coating Materials for Coverslips with Temperature-Sensitive Phosphors Dye: Europium thenoyl trifluoroacetonate abbreviated as Eu (TTA) ₃ or EuTTA (Advanced Materials, New Hill, NC) Polymer: abbreviated as PMMA Poly (methyl methacrylate) (Aldrich, Milwaukee, WI 18,226-5) Solvent: 96% nitroethane (Aldrich, Milwaukee, WI 13,020-6) standard polymer (PMMA) concentration in dye / polymer mixed solvent (nitroethane) is 0.
1 mM (100 mg PMMA in 5 ml nitroethane). Eu (T
Various concentrations of TA) ₃ were used. The best fluorescence signal is 200 mM Eu (TTA) _{3 in} solvent (EuTTA 100m in 5 ml nitroethane).
g). Stir with a magnetic stirrer until the dye / polymer mixture is completely dissolved, then
The solution was filtered through a 0.45 μm pore filter (German Sciences, Acrodisc 13CR PTFE) to leave a completely clear solution. To minimize hydration problems, the dyes and polymers were stored under a nitrogen atmosphere, and the solvent was stored with molecular sieves. The layer thickness can be adjusted by changing the polymer concentration (higher, thicker) and spinning speed (slower, thicker).

Preparation of Coverslips All coverslips were acid washed in nitric acid. The coverslips are immersed in 70% nitric acid for 5 minutes and dH ₂ O
Rinsed, rinsed with 95% ethanol and then dried in a vacuum oven at 100 ° C. An acid wash was required for the polymer-phosphor to remain firmly and stably fixed to the glass. Spin coating process. A coverslip slide was attached to the rotor and covered with a polymer / phosphor / solvent sample. The coverslip was spun at a typical 600 rpm speed for 1 minute. The coverslip was then baked in a vacuum oven at 100 ° C. for 1 hour to anneal the polymer. Characterization of coatings. The cover slip coating was characterized by several methods. Uniformity was analyzed on a phase contrast microscope using a 20x or 40x objective. In addition, homogeneity was analyzed with a fluorescence microscope using a 40x objective (Nikon Diaphoto) or a laser scanning confocal microscope using a 60x objective (Ultima, Meridian Instruments, Okemos, MI). 36 absorption of cover slips
Analysis was performed on a diode-array spectrophotometer (Milton Roy Spectronic, Array 3000) at 5 nm. The temperature sensitivity of the various mixtures of phosphor, polymer and solvent was first tested in a fluorescence spectrofluorimeter (Amicon Bowman 2, SLM Amicon) in a temperature controlled cuvette. The coverslip was cut and fitted in a water-filled cuvette at a 45 degree angle. This arrangement was used for the following measurements. Optimal excitation and emission spectra by recording excitation and emission scans; photobleaching rate and stability of the layer in water (tested by recording the average fluorescence signal over time); Temperature coefficient (change in intensity per degree) measured by recording the change in fluorescence intensity during temperature change

[0020]Cell proliferation on coverslips Human breast cancer line MCF-7 cells were grown on the coating. Coating
Growth rate on top is less than on polystyrene surface of culture dish
Although late, the viability was higher than 80%. Cell culture MCF-7 / ADR cells are chemotherapy-resistant cells derived from human breast cancer
System. Moisturizing incubator (forma
37 ° C and 5% pCO in Scientific, OH)_TwoIn
Enol red, bovine insulin 10 μg / mL and L-glu
Tubes in modified Eagle's medium containing Tamine and 10% FBS
Manage. In addition, MCF-7 / ADR cells were
Manage continuously in mycin. Xenon Run Cells
, Uniblitz Shutter (Vincent and
Associates, Rochester NY) and excitation and emission
Olympus fluorescence microscope with side filter wheel
Observe with. Control light source shuttering by computer
I will. Excitation at 450nm and 490nm with filter holder
Manufactured to hold filters. Add data
Pine 4972 cooling and loading equipment (Hamamatsu Photonics)
Collected and listed in the National Instrumentas Lab View
Digitize with software. Constant perfusion of cells with medium
To visualize in a temperature controlled room. Λex at 355 nm
And 614 at λem. The ratio was taken in a continuous image
In that case, no cells could be detected. But
Increase the proton permeability of mitochondria,
A proton-ionophore that increases the hydrolysis of ATP
After the incorporation of some FCCP, increased
There is fluorescence emission. The maximum rise in temperature is about 100 mK,
This is almost consistently observed in the cell growth cone (Fig.
2B and 2D).Fluorescence measurement Fluorescence measurement using UV excitation filter and emission filter
Using a fluorescence microscope equipped with an Hg / xenon lamp
Was. Eu (TTA)_ThreeExcites at 360 nm and emits at 614 nm.

Results Selection of Fluorescent Compound: It was necessary to exclude any water in the solvent and polymer before coating the coverslip. Unfortunately, water adversely affected the stability of the phosphor / polymer mixture, and many of the reagents commonly used to dehydrate the solvent adversely affected the phosphor. One important point was to use phosphors that could be treated with dehydrating reagents without adverse effects. One type of potential phosphor (Eu-FOD) was found to undergo a significant change in properties when we baked it onto a glass coverslip. Eu (TTA) ₃ did not have this problem and was therefore chosen as a good phosphor. Polymer Selection. The studied phosphors change their fluorescence in response to temperature. However, their fluorescence may be sensitive to particular protein, carbohydrate, pH or ionic changes. Thus, the phosphor is encapsulated in the polymer used to coat the coverslip. Polystyrene was our first choice for the support polymer. It is stable to many different treatments, it is non-toxic, and it can be used as a substrate for growing cells. Unfortunately, the response of EuTTA to temperature was weak in polystyrene.
It is clear that the benzene ring in polystyrene results in a loss of activity as if it had adversely interacted with the group in EuTTA. On the other hand, PMMA is a non-aromatic phosphor. This polymer was known to be sensitive to the UV wavelengths used to excite EU-TTA. In fact, ultraviolet light is often used to etch PMMA. However, after changing the concentration of PMMA: EuTTA: solvent: dehydrating agent, a mixture of EuTTA: PMMA that was very stable on the coverslip was determined. This mixture can be immersed in an aqueous environment without adversely affecting mechanical stability. The fluorescent signal is stable for at least one hour during UV excitation. Water does not adversely affect the fluorescent signal. And its fluorescence is
2.5% change. Furthermore, the noise is very low (about 100:
1), enable 0.01 ° C change resolution. A plot of the change in fluorescence with temperature is shown in FIG. In Tables 1 and 2, the Eu (TTA) ₃ / PMMA mixtures were tested for their maximum Log slope / ° C.
With respect to other phosphor / polymer pair samples.

[0022]

[Table 1] Phosphor Polymer Maximum Temperature Log Slope / ° C Anthracene Cellulose Acetate Coumarin PMMA -0.004 60 CuOEP GP-197 -0.0113 -100 Erythrosin B Polycarbonate Europium Model Airplane Dope -0.036 0 Thenoyltrifluoroacetonate (EuTTA) Europium PMMA- 0.049 0 Tenoyl trifluoroacetonate (EuTTA) Europium marathon -0.0095 Tenoyl trifluoroacetonate (EuTTA) HC-295 GP-197 La ₂ O ₂ S: Eu (1%) Solid -0.0031 -150 Perylene model airplane dope -0.0134 25 Perylenedicarboximide (PDC) PMMA -0.007 75 Perylenedicarboximide (PDC) SOA -0.06 75 PtOEP GP-197 -0.0033 10 Pyrene Cellulose acetate Pyronine B PMMA -0.046 60 Pyronine Y Model airplane dope -0.055 50 Pyronine Y PMMA -0.072 75 Pyronine Y polycarbonate -0.0168 50 Quinizarin polystyrene -0.084 90

[0023]

[Table 2] Phosphor Polymer Maximum Temperature Log Slope / ° C Rhodamine B Cellulose Acetate -0.0167 -125 Rhodamine B PVC -0.014 -5 Rhodamine B PVP +0.057 -30 Rhodamine B Polyurethane -0.0175 80 Rhodamine B Model Airplane Dope -0.018 Rose Bengal Ethyl cellulose -0.09 80 Rubrene PMMA -0.034 5 Rubrene SOA Ruthenium compound DJ-171 GP-197 Ruthenium compound DJ-201 GP-197 -0.042 0 Ruthenium compound DJ-275 GP-197 -0.018 0 Ruthenium compound VG-220-1-2 GP-197 -0.0573 10 Ruthenium compound VG-225-2 GP-197 -0.0212 -10 Ruthenium compound VH-127 GP-197 -0.0071 -150 Ruthenium compound SC-324 GP-197 -0.0233 0 Ruthenium compound SC-393 GP- 197 -0.0343 0 Ruthenium (bpy) / zeolite PVA -0.0269 40 Ruthenium (trpy) Ethyl cellulose -0.0131 -140 Ruthenium (trpy) GP-197 -0.0134 -140 Ruthenium (trpy) Model airplane dope Ruthenium (trpy) PMMA ruthenium (trpy) / zeolite PVA -0.0096 -100 sulforhodamine B model airplane doped -0.0375 105 TTMHD solid -0.0293 25

The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. It is further to be understood that all base or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides, are approximate and are provided for description. Various publications are cited herein, the disclosures of which are incorporated by reference in their entirety.

[Brief description of the drawings]

FIG. 1 shows the change in fluorescence intensity with temperature for Eu (TTA) ₃ / PPMA. Excitation wavelength is 365 nm and emission wavelength is 6
It was 21 nm.

FIGS. 2A and 2C are the cells described in the examples observed by absorbance spectroscopy. FIGS. 2B and 2D show the fluorescence difference spectra obtained from the cells of FIGS. 2A and 2C, respectively, after addition of FCCP. Cells were excited at 355 nm and emission was monitored at 614 nm.

Claims (10)

[Claims] (A) placing a cell on a metabolic probe containing a solid substrate containing a temperature sensitive phosphor encapsulated in a polymer (when the phosphor is excited with appropriate ultraviolet, visible or infrared light, it is detected (B) emits a possible fluorescent signal, and when live cells are placed on metabolic probes, the fluorescent signal is affected by the metabolic rate of the cell); (b) exciting the fluorophore with the appropriate ultraviolet, visible or infrared light And (c) detecting a fluorescent signal (the intensity of the detected fluorescent signal is an indicator of the cell temperature). 2. The method according to claim 1, wherein the polymer is a non-aromatic synthetic polymer. 3. The method of claim 1 wherein the phosphor is capable of emitting a stable fluorescent signal while encapsulated in a polymer adjacent to the aqueous solution for at least one hour after excitation by ultraviolet light at 37 ° C., pH 7.5. the method of. 4. The method according to claim 2, wherein the polymer is poly (methyl methacrylate). 5. The method according to claim 4, wherein the phosphor is Eu (TTA) ₃ . 6. A method for detecting the metabolic activity of a cell, comprising detecting the temperature of the cell by the method according to claim 1, and correlating the temperature with the metabolic activity. 7. The method according to claim 6, wherein the cells are tumor cells. 8. A method for detecting the presence of abnormal metabolism of a cell surrounded by healthy tissue, comprising detecting the temperature of a cell according to the method of claim 1, wherein the temperature of the cell relative to a control cell is reduced. A detection method, wherein the difference indicates that the cell is undergoing abnormal metabolism. 9. The method of claim 8, wherein the live tissue is obtained from a tumor biopsy. 10. The method of claim 8, wherein the abnormal metabolism of the cell is an indicator of ultraviolet damage, cachexia, or apoptosis.