JP3335341B2 - Fluorescent beads for determining cell temperature and methods of using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention generally relates to a novel method for determining the temperature of a single cell or a group of cells in a tissue sample. A method using the method as a diagnostic aid is also included.

[0002]

2. Description of the Related Art Living cells require a stable rate of metabolism. Thus, in both the cell and the organism containing the cell, its regulation is under stringent control. About 50% of the total energy of carbohydrates is converted to ATP and the rest is released as heat [Alberts et al., Molecular Biology of the Cell, (19
89)]. ATP is then used for biosynthesis during growth, or otherwise for work, and its energy is released as heat. This release of heat is used to warm the body in the form of temperature. Failure to maintain stable metabolic levels at the organism level is a condition called fever, ie, an increase in the temperature of the organism. Changes at the metabolic level are found in a variety of conditions, including bacterial or viral infections, cancer, and general weakness seen in sepsis or cachexia.

[0003] The temperature of an organism is often used as an indicator of the health of the organism. Even a slight shift in temperature indicates a medical condition. However, the temperature of individual cells cannot be analyzed due to current technology limitations. That is, it is not yet known how much the cell temperature changes over time within a cell or between two different cells. However, based on experiments with large populations of cells, there is ample argument that pathology can be detected by measuring temperature at the single cell level.

[0004] Cellular metabolism is regarded as a balance sheet for all enzymatic reactions occurring within a cell. In this context, the net exothermic activity is seen as blackbody radiation, the heat that is analyzed when measuring the temperature of a cell. Previous studies of biometabolic activity used calorimetry applied at the level of whole organisms or to very small individual organs. Microcalorimetry is applied to only about 10 cells. However, there are severe limitations on the limits of current technology for breaking down heat generated from single cells [Kemp, Therma
l and Energetic Studies of Cellular Biological Sys
tems, AM James, ed. (Bristol: Wright), pp. 147-16
6 (1987)]. The most sensitive technique applied to measure metabolism in tumor cells is a Cartesian diver that can degrade hundreds of cells [Lutton & Kopac, Cancer
Res., 31: 1564-1569 (1971)]. However, this approach is still too immature to degrade a heterogeneous population of cells and requires dissociation of the cells. The physiology of many normal cells is
A dynamic process that requires the interaction of cells in a three-dimensional matrix. Many cell activities are altered by cell-cell contact. All this disappears when the cells are dissociated. Recently, techniques have been developed to measure metabolites such as ATP, glucose and lactate in living cells [Hossman et al., Acta Neuropathologica, 69: 139-147.
(1986); Okada et al., Journal of Neurosurgery, 77: 917-9.
26 (1992)]. These techniques are based on photographic film [Hossma
Okada et al., 1992] or a photon counting camera [Ta
mulevicius & Streffer, British Journal of Cancer,
72: 1102-1112 (1995)], and demonstrated considerable heterogeneity of metabolism in tumors when analyzed with millimeter spatial resolution.

[0005] Unfortunately, many important issues at present cannot be mentioned because the metabolism of individual cells cannot be determined. For example, it is not currently possible to experiment with heterogeneous populations of cells and to degrade the activity of individual cells rather than the average of a mixture. For example, tumors consist of cancer cells, normal cells, and infiltrating immune cells, each metabolizing at very different rates. In this case, the measurement of the average metabolism of the tumor cannot reflect the actual metabolism of the individual cells.

[0006] In most cells, aerobic respiration
It is involved in almost all of the production of ATP and the rest is considered anaerobic glycolysis. It was stated more than 50 years ago that anaerobic respiration was significantly increased in ascites tumor cells [Warburg et al., Th
e Metabolism, ed. O. Warburg, Constable & Company
LTD., London, pp. 129-170 (1930a); Warburg et al., The
Metabolism of Tumors, ed. O. Warburg, Constable &
Company LTD., London, pp. 254-265 (1930b)]. This led to the hypothesis that aerobic respiration is impaired in tumor cells [Warburg, Science, 123: 309-314 (1
956)]. During that time, knowledge of metabolism has shown that aerobic respiration is normal in tumor cells but anaerobic respiration increases. The mechanisms involved in this increase in anaerobic respiration are currently unknown. The main limiting factor in knowing the mechanism is the inability to analyze the relative metabolic levels of individual cells. As mentioned above,
One particular problem is that tumors consist of a mixture of normal, malignant, and immune cells. Current techniques can only measure the average metabolism of cells in this mixed population. The second problem is that it is quite heterogeneous even within tumor cells. For example, in tumors, oxygenation often limits tumor growth [Kallinowski et al., J. Ce.
l. Physiol., 138: 183-191 (1989)]. Thus, in rapidly growing tumors, growth is limited by angiogenesis, the growth of new blood vessels that deliver oxygen and nutrients.

[0007] Chemotherapy is a powerful tool used to combat tumors. However, tumor cells often resist chemotherapeutic agents. This resistance is seen as reduced sensitivity to a broad spectrum of chemotherapeutic agents, whose cells have been classified as multidrug resistant cells [Simon et al., Proc.
tl. Acad. Sci. USA, 91: 3497-3504 (1994); Schindler
Et al., Biochemistry 35, 2811-2817 (1996); May 18, 1998
U.S. Patent Application Serial No. 09 / 080,739, filed on December 22, 1998; U.S. Patent No. 5,851,789, issued December 22, 1998, the disclosures of which are incorporated herein. These cells were first seen as "supercells" that could withstand a therapeutic attack, but now there is increasing evidence that multidrug-resistant cells can escape chemotherapy by behaving like normal cells. In many ways, these cells are thought to have undergone a "reverse" transformation in nature [Biedler et al., Cancer & Metastasis Reviews,
13: 191-207 (1994)]. When the chemotherapeutic agent is removed, these cells regain their aggressive malignant nature. It is not known what happens to the metabolism of multidrug resistant cells during this time. Indirect results indicate that anaerobic respiratory levels in these cells have returned to levels found in non-cancerous cells [Miccadei et al., Oncology Research, 8: 27-3
5 (1996)]. Therefore, there is a need to measure the metabolism of individual cells that are multidrug resistant cells in order to diagnose when a tumor transitions from the quiescent phase to the malignant phase of the multidrug resistant cells. Further, there is a need for a method of measuring the temperature of individual cells to determine the metabolic changes that occur in normal or tumor cells. Further, there is a need for a method of measuring the temperature of cells from a new biopsy of living tissue in order to quickly diagnose the presence of tumor cells in the tissue.

Although the emission of almost all fluorophores is affected by temperature, the use of fluorophores that are particularly sensitive to temperature was first used by Kolodner et al. As a technique for assaying temperature [Appl. Phys. Lett. 40: 782-784 (19
82); Appl. Phys. Lett. 42: 782-784, (1983)]. Kolodne
r is a polymer, a fluorophore embedded in PMMA, Eu (TTA) ₃ to obtain a resolution of 0.07 ° K and a resolution of 10 μM spatial resolution.
Was used. Therefore, this temperature sensitive fluorophore was used to quantify temperature.

Various fluorophores, including Eu (TTA) _3, have been dissolved in various solvents and polymers and used in the laboratory of John P. Sullivan to "paint" the wings of airplanes (Perch ( Pursue) University, (Campbell et al., Temperature Sensi
tive Fluorescent Paint Systems, 18: 94-2483 (1994)]
See Table 1 modified from Changes in fluorescence are used, for example, to monitor changes in airplane wing temperature in a wind tunnel. To date, temperature-sensitive fluorophores have been used in the diagnosis of integrated circuits [Kolodner et al., Appl. Phys. Let
t., 42: 117-119 (1983)], detecting changes in the boundary layer for a two-dimensional wing and visualizing the interaction between the leading edge vortex and the surface of the delta wing [Campbel
l et al., 6th International Symposium on the Application of Laser Technology to Fluid Dynamics, Lisbon, Portugal (1992); Campbell
Et al., Temperature Measurement Using Fluorescent Pain
t Systems, 18: 94-2483 (1994); Harnner et al., A Scanning.
Laser System for Temperature and Pressure Sensiti
ve Paint, 32: 94-0728 (1994)]. Citation of a document herein shall not be construed as an admission that the document is available as "prior art" in the present application.

[0010]

SUMMARY OF THE INVENTION The present invention provides a means for measuring cell temperature by using a temperature-sensitive fluorophore and a temperature-insensitive fluorophore encapsulated in beads. Aspects of the invention provide fluorescent beads comprising at least one temperature-sensitive fluorophore and at least one temperature-insensitive fluorophore embedded in a polymeric material. In a specific embodiment, the polymeric fluorescent beads include one temperature sensitive fluorophore and one temperature insensitive fluorophore embedded in a polymeric material.

Preferably, the fluorescence emission by the temperature-sensitive fluorophore and the fluorescence emission by the temperature-insensitive fluorophore are mutually decomposed. In a specific embodiment of the polymeric fluorescent beads, the temperature sensitive fluorophore is Eu (TTA) ₃ [199
See US patent application Ser. No. 09 / 003,628 filed Jan. 7, 2008. The description of this specification shall be included in the specification of the present application. ]. In another embodiment of the polymeric fluorescent beads, the temperature-insensitive fluorophore is coumarin. In a preferred embodiment, the temperature sensitive fluorophore is Eu (TTA) ₃ and the temperature insensitive fluorophore is coumarin. In embodiments, the size of the fluorescent beads is between 40 and 150 nm.
In a preferred embodiment, the size of the fluorescent beads is between 60 and 11
0 nm. In a preferred embodiment of this type, the size of the polymeric fluorescent beads is between 80 and 90 nm.

[0012] In a specific embodiment, the polymeric fluorescent beads are comprised of polystyrene. In another embodiment, the polymeric fluorescent beads comprise a non-aromatic synthetic polymer. In such an embodiment, the polymeric fluorescent beads comprise poly (methyl methacrylate) (PMMA). The present invention also provides a method for producing the polymer fluorescent beads of the present invention.

Further, the present invention provides a method using the polymer fluorescent beads of the present invention. For example, the present invention provides a method for determining the temperature of a cell. The method includes the step of placing the polymeric fluorescent beads of the present invention into a cell.
Next, a temperature-sensitive fluorophore and a temperature-insensitive fluorophore
Upon excitation with the appropriate ultraviolet, visible, or infrared light, the temperature-sensitive fluorophore and the fluorescent signal emitted by the temperature-sensitive fluorophore are detected. When the temperature sensitive fluorophore and the temperature insensitive fluorophore are excited with the appropriate ultraviolet, visible or infrared light, they produce a detectable fluorescent signal that is individually degraded. Preferably, the fluorescent signal by the temperature-sensitive fluorophore is affected by the metabolic rate / temperature of the cell,
The fluorescence signal of the temperature-insensitive fluorophore is not affected.
Importantly, however, for certain bead constructions, the change in fluorescence of the bead's temperature-sensitive fluorophore due to changes in intracellular temperature is quite different from that of the temperature-insensitive fluorophore partner. This allows discrimination between changes in intracellular temperature and changes in fluorescence due to intracellular bead motion (eg, local bead concentration changes). Thus, the intensity of the fluorescent signal that detects a temperature-sensitive fluorophore is indicative of the temperature of the cell when local bead concentration is corrected by the emission of the corresponding temperature-insensitive fluorophore.

In a related embodiment, instead of beads containing a temperature-sensitive fluorophore and a temperature-insensitive fluorophore,
The present invention provides beads comprising two temperature sensitive fluorophores having opposite temperature dependencies. That is, one fluorophore increases fluorescence emission at high temperatures, while the other attenuates fluorescence emission at high temperatures. Over the appropriate temperature range, these temperature-sensitive fluorescence changes described herein are preferably linear and / or pre-assayed. In any case, the change in cell temperature shifts the fluorescence ratio of the two fluorophores, and the change in temperature allows to determine the cells containing the beads. On the other hand, the movement of the beads within the cell causes an absolute change in the measured fluorescence emission, but the ratio of the fluorescence emission by the two fluorophores remains unchanged.

The present invention further provides a method for detecting the metabolic activity of cells using the polymer fluorescent beads of the present invention. The embodiment includes the steps of determining the temperature of the cells with the above-described polymer fluorescent beads of the present invention, and correcting the temperature with metabolic activity. The present invention also provides a method for detecting the presence of abnormal metabolism of cells surrounded by healthy tissue in a tissue sample using the polymeric fluorescent beads of the present invention. That embodiment includes determining the temperature of the cells. Differences in cell temperature relative to control cells indicate that the cells are undergoing abnormal metabolism. In a specific embodiment of this type, the tissue sample is obtained from a tumor biopsy. In other embodiments,
The abnormal metabolism of cells is indicative of UV damage. In another embodiment, the abnormal metabolism of the cells is also indicative of cachexia. In another embodiment, also, the abnormal metabolism of the cell is indicative of apoptosis.

When the fluorophore is excited with the appropriate ultraviolet, visible, or infrared light, it emits a detectable fluorescent signal. When fluorescent beads are placed in a cell, the intensity of the fluorescent signal of the temperature-sensitive fluorophore is affected by the metabolic rate of the cell, but the fluorescent signal of the temperature-insensitive fluorophore is substantially (or preferably not) entirely fluorescent. ), Not affected by the metabolic rate of the cells.

[0017] Preferably, the fluorescent beads contain a fluorophore capable of producing a stable fluorescent signal, and the beads emit light at 37 ° C.
Adjacent to an aqueous solution of about pH 7.5 for at least one hour upon excitation with (eg, ultraviolet, visible, or infrared light). In a specific embodiment, when the fluorescent beads are placed in living cells, the fluorescence-sensitive fluorophore has a temperature change in the intensity of the fluorescent signal of 1 relative to the corresponding change in the fluorescence signal of the temperature-insensitive fluorophore. It rises by 1 to 2.5% per ° C.

In a specific embodiment of the present invention, cells containing fluorescent beads are placed on a microscope, a fluorophore is excited, and the luminescence is detected using the microscope. In that embodiment, the microscope is a laser scanning confocal microscope with an excitation ultraviolet laser. In another embodiment, the microscope is a Hg having an ultraviolet light excitation filter.
Or a fluorescence microscope equipped with a xenon lamp and a 615 nm +/- visible light emission filter. These and other aspects of the invention will be more fully understood from the following drawings and detailed description.

[0019]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method of using the fluorescent beads described herein to monitor temperature and / or metabolic changes in a single cell or group of cells. Cells are dissociated from other cells or remain part of the cell sample. For example, if the cell is a tumor cell, its monitoring allows new insight into metabolic changes in the tumor cell. It also aids in understanding the general cell biology of tumor cells.

In addition, the fluorescent beads of the present invention can be used to determine if a tumor cell is present in a new biopsy of living tissue within a time frame, thereby enabling diagnosis during a tumor biopsy. To In this way, analysis of the biopsy is performed immediately following tissue removal, allowing the physician to immediately determine whether additional tissue must be removed or to conclude that surgery is not required. Enable. Such procedures require an initial biopsy, and then typically evaluate the tissue for 7-10 days, often offering many advantages over current techniques where additional surgical procedures are continued. Thus, the present invention allows a physician to perform a single surgical procedure and to be immediately responsive to tissue analysis, thus further preventing tissue deterioration during this analysis.

The present invention also includes a method for determining the temperature of a cell using the fluorescent beads of the present invention. In a preferred embodiment, the fluorescent beads of the present invention comprise at least one temperature-sensitive fluorophore and at least one temperature-insensitive fluorophore both embedded in a bead, preferably a polymer such as polystyrene or PMMA. Contains fluorophores.

The beads are made from as many solid support materials as the material is substantially impermeable to water (eg,
Polystyrene or PMMA). Beads are prepared by standard techniques and / or obtained from many commercial sources. next,
The solid support beads previously produced are swollen in the presence of the fluorophore, so that the fluorophore is captured by the beads. It is preferable not to use solid support beads that are permeable to water, since the fluorescence from the embedded fluorophore must be independent of solution changes, eg, salt concentration, pH, etc.

Next, the fluorescent beads are filled in the cells. Fluorescent beads were used to fill cells exemplified below (Scrape) [McNei et al., J. Cell Biol. 98: 556-1564 (198
4); Altan et al., Journal of Experimental Medicine 187:
155-175 (1998)] or microinjection of beads
[Tanasugarn et al., J. Cell Biol. 2: 717-724 (1984)]. In an alternative embodiment, the fluorescent beads are loaded into the cells by syringe loading. Cells were dissociated from the substrate with trypsin and EDTA and then transferred back and forth between the two syringes in the presence of beads [Clarke & McNeil,
J. Cell Sci., Pt3: 533-541 (1992)]. Other methods require cells to endocytose the beads in hyperosmotic media and then shock the cells in hypotonic media, where the endosomes lyse the open interior of the cells [Oka
da & Rechsteiner, Cell 29: 33-41 (1982)].

The beads prevent the fluorophore from the cytosol of the cell and protect the fluorophore from being affected by changes in ions or viscosity. This is an important aspect of the present invention because most fluorophores are affected by the environment. That is, changes in the solvent, salt concentration, temperature, or viscosity of the medium can all affect the fluorescent properties of the fluorophore. In a preferred embodiment of the present invention, only heat penetrates or escapes the fluorescent beads, thus allowing the temperature of the cytosol, or cytosol compartment, to be determined almost exactly.

In an embodiment of the present invention, the fluorescent beads are
At least one temperature-sensitive fluorophore and at least one temperature-insensitive fluorophore to allow for unavoidable changes in the distribution of beads within the cell that can result in a corresponding change in the local concentration of the fluorophore Contains a fluorophore. Therefore,
By co-embedding a temperature-sensitive fluorophore with the least temperature-sensitive fluorophore, the temperature-insensitive fluorophore serves as a control for the change in fluorescence due to the distribution of beads within the cell. This allows for an accurate assay of the fluorescence of the fluorescent beads as a function of temperature. Thus, where the following terms appear herein, their definitions are set forth below.

"Metabolic probe" as used herein
Comprises a solid substrate comprising a temperature-sensitive fluorophore embedded in a polymer, wherein when the fluorophore is excited with light of an appropriate wavelength (e.g., ultraviolet, visible or infrared light), the fluorophore is When a live cell (alone or as part of a cell sample) emits a detectable fluorescent signal, it is placed on a metabolic probe.
The fluorescent signal is enhanced (ie, increased or attenuated) by the heat emitted from the cell (eg, measured as the temperature and / or metabolic state of the cell). The fluorescent beads of the present invention are a particular type of metabolic probe.

[0027] As used herein, a "solid substrate" is made from a solid material, preferably plastic, metal, or glass. The solid substrate used for the fluorescent beads herein is preferably impermeable to aqueous solutions. As used herein, “about pH 7.5” includes the pH range of pH 7.0 to pH 8.0.

As used herein, a temperature-sensitive fluorophore is a compound whose emission intensity varies monotonically as a function of temperature in the region of at least 35-40 ° C. In embodiments, the exponential or linear dependence of the fluorophore on temperature varies from 0.25 to 2.5% fluorescence emission intensity per degree Celsius. Exponential or linear dependence of preferred fluorophores on temperature varies by at least 1% fluorescence emission intensity per degree Celsius. As used herein, a "temperature-insensitive fluorophore" is a fluorophore that is substantially unaffected by the rate of metabolism of the cell, for example, has an emission intensity of less than 0.1% per ° C, preferably less than 0.025% per ° C. Only the fluorophore that fluctuates.

The “fluorescent beads” used in the present specification are:
Due to the different fluorescent properties of the fluorophores, the beads contain at least two different fluorophores embedded in the beads that are used to measure intracellular temperature changes. In embodiments, the fluorescent beads comprise at least one temperature-sensitive fluorophore and at least one temperature-insensitive fluorophore. The fluorophore is embedded in a bead (preferably a polymer), and either or both fluorophores are excited at the appropriate wavelength of light (e.g., ultraviolet, visible or infrared). If the fluorophore emits a detectable fluorescent signal, the temperature-sensitive fluorophore's fluorescent signal can be distinguished from the temperature-insensitive fluorophore when the beads are placed in living cells (single cells or cell samples). (E.g., increased or attenuated) by heat (e.g., measured as the temperature and / or metabolic state of the cell) released from the cell. In another embodiment, the beads comprise two temperature-sensitive fluorophores whose temperature dependence is opposite. That is, one fluorophore increases emission at elevated temperatures, while the other decreases emission at elevated temperatures.

In the application of the present invention, the temperature of cells obtained from a cell sample from a patient suspected of having cachexia is determined. Cachexia is a debilitating condition that often leads to death. The cachexia triggered by tumor necrosis factor is thought to be the result of activating the substrate cycling cycle between fructose 6-phosphate and fructose 1,6-bisphosphate with increased lactic acid production. This depletes cellular ATP [Zentella et al.,
Cytokine, 5: 436-447 (1993)]. Thus, analysis of elevated cell temperatures obtained from patients suspected of having cachexia is consistent with a diagnosis of cachexia.

In another embodiment of the present invention, the mechanism by which macrophage inflammatory proteins cause fever can be examined. Fever is a factor released by macrophages in animals: macrophage inflammatory proteins [Myers et al., Neurochemical
Research, 18: 667-673 (1993)]. Although this factor is known to modulate food intake by animals, it is not yet known where or how cells increase metabolism and increase temperature. Thus, determining that a particular cell (or cell type) is emitting excess heat (and thus causing heat) is accomplished by quantifying the temperature of individual cells (or cell types). You.

In a specific embodiment of the present invention, the temperature of the cultured cells is determined. In that embodiment, a population of breast tumor cells is used. In another embodiment, drug-resistant breast tumor cells are used. In another embodiment, non-cancerous breast cells are used.
In a preferred embodiment, two of these cell types are used. In a preferred embodiment, all three cells are used. In a preferred embodiment, the breast tumor cells are of human origin. In a specific embodiment, brown adipose tissue from the tumor cell line HIB 1B is used.

In another embodiment of this aspect of the invention, a biopsy of human tissue is used as a cell source. In a specific embodiment, a new biopsy of breast tissue is used as a cell source. Sections in the breast include adipocytes (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 faster than "normal" cells. Analysis of skin tumors using surface thermometry shows a temperature difference (upon irradiation) of up to 3.3 ° C. In another embodiment, the biopsy is colon tissue.

In current medical practice, excised tissue obtained during a biopsy is prepared for tissue analysis over 7-10 days. Thus, the patient is not in the operating room when results are sought. Therefore, it is often necessary to schedule subsequent surgery. The method for determining the temperature of cells in a section using the method of the present invention is to determine whether the cells of a tissue section to be analyzed immediately using a standard fluorescence microscope have local `` hot spots '' of metabolic activity as fingerprints of tumor cells. Is possible. Such an analysis allows the surgeon to immediately determine whether to ablate additional tissue.

Another embodiment of the present invention further includes determining the temperature / metabolism of individual cells to monitor the migration of quiescent multidrug resistant cells to a malignant state. In this case, the rise in temperature coincides with the movement. Such monitoring is particularly effective during chemotherapy, especially after termination of the treatment.

In a specific embodiment, the fluorescent beads of the present invention, when placed on a cell or tissue biopsy, permit substantially simultaneous luminescence measurements from temperature-sensitive and temperature-insensitive fluorophores. Is detected with a laser scanning confocal microscope equipped with an exciting laser. In other embodiments, detection is performed on a fluorescence microscope equipped with a Hg or xenon lamp with an appropriate excitation filter and an emission filter that monitors the fluorescence emission of the fluorophore. If the beads are used with a temperature-sensitive fluorophore and a temperature-insensitive fluorophore, the temperature is determined from the ratio of the emission of the temperature-sensitive fluorophore to the emission of the temperature-insensitive fluorophore. On the other hand, if the beads are used with two temperature-sensitive fluorophores of opposite temperature dependence (see above), the temperature is assayed from the ratio of the emission of the two fluorophores.

As noted above, a preferred temperature-sensitive fluorophore for fluorescent beads is Eu (TTA) ₃ , which has a maximum excitation of 355-360 nm and a maximum emission of 614 nm. In a specific embodiment of the fluorescent beads, Eu (TTA) ₃ is combined with a temperature-insensitive fluorophore and a polymer, such as poly (methyl methacrylate), (PMMA). However, other suitable combinations are envisioned by the present invention. The potential combinations are illustrated in Table 1. These and other combinations are
Tested to determine suitability to serve as a component of fluorescent beads.

In a specific embodiment of the fluorescent beads, Eu (TTA) ₃ and coumarin are embedded in polystyrene.
In a related embodiment of the fluorescent beads, Eu (TTA) ₃ and coumarin are embedded in PMMA. Other materials and their combinations
To determine suitability to serve as a component of a fluorescent bead, it is easily tested, for example, by the methods described herein.

Accordingly, the present invention provides fluorescent beads used to determine the metabolic rate of a cell. The value of a temperature-sensitive fluorophore is to detect the rate of metabolism, while the value of a temperature-insensitive fluorophore is to allow for the concentration of fluorescent beads to be assayed, e.g., to detect changes in fluorescence due to bead migration as a function of temperature. Is to be distinguished from the change in fluorescence due to the change in Thus, the proper pairing of a temperature-sensitive fluorophore and a temperature-insensitive fluorophore will depend on the particular measurement being performed.

The key criteria for selecting a temperature-sensitive and temperature-insensitive fluorophore pair for the fluorescent beads of the invention are (i) the temperature-sensitive fluorophore, preferably embedded in a polymer. (Ii) the temperature-insensitive fluorophore is preferably embedded in a polymer, but the fluorescence change at a temperature change of 37 ° C. Minimum and both fluorophores
(iii) ability to be embedded in the polymer and away from aqueous solutions; (iv) non-toxic to living cells; (v)
Act as independent fluorophores (e.g., do not undergo fluorescence energy transfer, have no Foster energy interaction, or do not act as mutual quenching agents, etc.)
And (vi) having fluorescence spectra that can be easily resolved from each other.

Fluorophores are sensitive to temperature changes at 37 ° C., stability in aqueous conditions, stability during embedding in polymers, and resistance to photobleaching (long-term observation and All tests are required). The greater the sensitivity of the temperature-sensitive fluorophore to changes in temperature at 37 ° C (and correspondingly the less insensitive the temperature-insensitive fluorophore to changes in temperature at 37 ° C), the above conditions including resistance to photobleaching The greater the stability of the fluorophore, the better the fluorophore as a component of the fluorescent beads. Similarly,
The polymer is chosen to have an optimizing effect on the fluorophore. Finally, the fluorophore / polymer combination is
Must be compatible with living cells. That is, it should not adversely affect cell viability.

[0042] In a specific embodiment, the size of the polystyrene fluorescent beads is between 80 and 90 nm. Generally, the size of the beads must be optimized to load the cells. The preferred range is about 40-150 nm. Beads smaller than 40 nm tend to aggregate (primarily from charge effects) but are acceptable. Beads larger than 150 nm are somewhat difficult to pack into cells and potentially disrupt cell structures. Large beads are loaded into cells by a number of techniques, including osmotic shock.

The surface of the fluorescent beads is preferably designed to minimize aggregation of the beads after injection into the cells (eg, the beads used in the examples below were coated with carboxyl groups). Also, the beads are coated with a specific substance to target the subcellular compartment. The hydrophobic coat promotes binding to the cell membrane. Peptide coats where proteins target mitochondria localize the beads to mitochondria.
Similarly, target signal peptides are used in chloroplasts or endoplasmic reticulum. Further, the fluorescent beads are coated with a specific antibody or antigen-binding fragment thereof. In fact, if the surface of the beads is unmodified, the beads do not move because they can aggregate or bind to the cell matrix.
Thus, different concentrations of positive or negative charges on the surface of the beads may be effective. The present invention will be more fully understood by the following non-limiting examples, given by way of illustration of the invention. The following examples are provided to more fully illustrate the preferred embodiments of the present invention. It should not, however, be construed as limiting the broad scope of the invention.

[0044] In the fluorescent beads ordinal tissue used as an example the metabolic probes, or in between tissue, that there is a change in cell activity and metabolism is recognition by intuition. However, the correlation between changes in metabolic activity changed, and cell activity was not determined. It is also unclear whether there is a metabolic gradient in metabolic level, for example, a metabolic difference between the growth cone and cell body, or between dendrites and axons. Furthermore, it is unclear whether the potential gradient is stimulated by growth factors or other hormonal factors. The present invention provides a means to solve at least some of these important metabolic problems by using fluorescent beads containing both temperature-sensitive and temperature-insensitive fluorophores. Next, the fluorescent beads are loaded into the cells as temperature / metabolism probes.

To select appropriate fluorophores and polymers for the fluorescent beads of the present invention, (i) optimal excitation and emission spectra by recording excitation and emission scans; (ii) photobleaching rate in water and Stability (tested by recording the average fluorescence signal over time); and (iii) a temperature coefficient determined by recording the change in fluorescence intensity, usually at a temperature change of 1 ° C.
Several criteria were introduced, including the analysis of (intensity / ° C change). For example, the sensitivity of a temperature-sensitive fluorophore to a change in temperature at 37 ° C. is an important criterion, the higher the sensitivity the better, but the temperature-insensitive fluorophore requires a correspondingly minimal change. Similarly, fluorophores are selected for stability in aqueous conditions because viable cells are most stable in aqueous conditions. In addition, fluorophores are selected for stability during embedding in the polymer. For beads that are 100 nm, a thin layer of polymer can further separate the fluorophore from cellular secretion of proteins, metabolites, or ions that can modify the properties of the fluorophore.

In addition, a polymer is chosen which optimizes the properties of the fluorophore, in particular the properties which allow the two fluorophores to be distinguished, for example the fluorescence emission and / or excitation spectrum, and the temperature sensitivity. . Such a polymer should be chosen for its relative resistance to photobleaching (a property that requires prolonged observation and subsequent assays). In addition, the polymer, and the fluorophore, must be compatible with living cells to ensure that the polymer and fluorophore do not adversely affect cell viability.

Materials and Methods Synthesis of fluorescent beads : temperature sensitive fluorophore, Eu (TTA)
₃ [Europium thenoyl trifluoroacetonate, abbreviation Eu (TTA) ₃ or EuTTA (Advans
ced Materials), New Hill, NC), and a temperature-insensitive fluorophore, coumarin (Sigma Chemical Company)
(Sigma Chemical Co.), or Molecular Probes (M
Two different polymers, polystyrene and PMMA, were used for the manufacture of olecular Probes)).

Beads made from polystyrene are 80-
90 size. Polystyrene beads were stable but were found to interfere with Eu (TTA) ₃ fluorescence. The fluorescence intensity in polystyrene was generally 40% of the fluorescence intensity in PMMA. The surface of the beads was coated with carboxyl groups which were found to minimize aggregation after injection into the cells.

The filling of the fluorescent beads of cell: by Scrape filling the cells put fluorescent beads to the cell [McNei
l et al., J. Cell Biol. 98: 556-1564 (1984); Altan et al., Jou.
rnal of Experimental Medicine 187: 155-175 (1998)].
In summary, 24-36 hours before filling cells with beads
Load on polystyrene with 50% confluence. The medium is aspirated from the dish and the cells are coated with 50 μl of beads. The cells are then scraped from the polystyrene with a rubber scraper and placed in pre-chilled tubes containing 1 ml of serum-free medium. The cells are then recovered by spinning 100 g for 5 minutes. Aspirate the medium,
The medium was replaced with a pre-cooled serum-free medium. The cells are then recovered by spinning. Finally, the medium was aspirated and replaced with medium containing serum. The cells are then placed on a cover glass of polylysine-coated glass.

Tissue culture cells as model system :
Human breast tumor cells, drug-resistant human breast tumor cells and non-cancerous breast cells, and brown adipocytes that produce heat in hibernating animals are used as cells in the first in situ experiments to optimize fluorescent bead temperature analysis. For example, MCF-7 / ADR cells are a cell line that resists chemotherapy from human breast cancer. 5% pCO ₂ at 37 ° C (Forma Scientific, OH)
Maintained in modified Eagle's medium containing phenol red, L-glutamine, bovine serum insulin 10 μg / ml, and 10% FBS in a humidified incubator. In addition, MCF-7 / ADR cells are continuously maintained in 0.8 μM adriamycin. Fluorescence measurement : 12-24 hours before the experiment, the cells are packed without fluorescent beads. Cells are usually scraped with beads and harvested for 24 hours. Cells are placed in phenol red-free medium to reduce background fluorescence. The cells are then loaded into the chamber of an inverted microscope (eg, a biotex closed imaging chamber).

Xenon lamp, Uniblitz
Shutter (Vincent and Associates)
Associates), Rochester, NY) and with an Olympus fluorescence microscope equipped with filter wheels on both the excitation and emission surfaces. The shuttering of the light source is controlled by a computer. Filter holders were manufactured to hold various filters including 355 nm, 450 nm and 490 nm excitation filters. Data is collected on a Hamamatsu 4972 (ORCA) cooled charge coupled device (Hamamatsu Photonics) and digitized with software written to National Instruments LabView. The cells are viewed in a temperature controlled chamber constantly perfused with medium equilibrated at 37 ° C. with 5% CO ₂ . The objective was heated to 37 ° C.

In addition, cells were treated with λex 355 nm and λem 614 nm (Eu
(TTA) excited with λex 428 nm and λem 480 nm (to record ₃ fluorescence, eg, temperature sensitive fluorophore) (to record the temperature-insensitive fluorophore) I do. No cells were detected when using the percentage of serial images. However, upon encapsulation of a proton ionophore that increases the hydrolysis of ATP because it increases the proton permeability of FCCP and mitochondria, the fluorescence emission is enhanced consistently with increasing temperature. Maximum temperature rise is about 10
0 ° mK, most consistently found in cell growth cones
(FIGS. 2B and 2D).

Results The temperature sensitive fluorophores tested change their fluorescence in response to temperature. However, the fluorescence was sensitive to certain protein, carbohydrate, pH or ionic changes. Thus, the fluorophore is embedded in the polymer used to coat the beads. Polystyrene is its supporting polymer. It is stable to many different treatments, is non-toxic, and is used as a substrate for proliferating cells. Eu vs. temperature
The response characteristics of (TTA) ₃ are not optimal for polystyrene. Eu (TTA) ₃ where benzene ring of polystyrene causes loss of fluorescence activity
It appears to interact disadvantageously with the group On the other hand, PMMA and poly (methyl methacrylate) are non-aromatic fluorophores.
This polymer was known to be sensitive to the ultraviolet wavelengths used to excite EU-TTA. However, a mixture of Eu (TTA) ₃ and PMMA is immersed in an aqueous environment without adversely affecting mechanical stability; the fluorescence signal of Eu (TTA) ₃ is stable for at least 1 hour during UV excitation .
Water does not adversely affect the fluorescent signal; the fluorescence changes by 2.5% for every 1 ° C change. Furthermore, the noise is very small
(Approximately 100: 1), 0.01 ° C changes can be resolved. A plot of the change in fluorescence intensity with temperature is shown in FIG. In Table 1, the Eu (TTA) ₃ / PMMA mixture is compared with other fluorophore / polymer sampling for maximum Log slope / ° C.

Table 1

[0055]

Fluorescent beads containing a temperature-sensitive fluorophore, Eu (TTA) ₃ , and a temperature-insensitive fluorophore, coumarin, were prepared. Eu (TTA) ₃ is optimally excited at 350 nm and emits at 614 nm (plus a peak at 595 nm). Coumarin is optimally 4
Excited at 20-460 nm and emitted at 480 nm (further peak at 510 nm). Eu (TTA) coumarin is not excited in ₃ of optimal excitation, Eu (TTA) ₃ is at the optimum excitation of coumarin is not excited. Fluorescent beads were manufactured with polystyrene or PMMA co-embedded with Eu (TTA) ₃ and coumarin. The fluorescence emission by Eu (TTA) ₃ changed by 2.5% for every 1 ° C change. The noise is very small (the ratio of signal to noise is about 100: 1), so 0.0
The resolution of the 1 ° C change was accurately determined.

As can be seen from FIG. 3, when excited at 350 nm,
Eu (TTA) ₃ emission at 614 nm representing the fluorescence of europium 1
A book peak occurred. When excited at 420 nm, there was a peak at 480 nm and the emission at 614 nm was minimal. Eu (TTA) ₃ : coumarin ratio of beads adjusted so 350nm excitation
614 nm emission seen when performed at 420 nm excitation
Is approximately the same amplitude as the emission peak due to coumarin at 480 nm when performed at (see FIG. 3).

The temperature sensitivity of the fluorescent beads was determined by incubating in a cuvette and measuring the fluorescence emission from the temperature-sensitive and temperature-insensitive fluorophores. As shown on the right of FIG. 4, the fluorescence emission of Eu (TTA) ₃ is temperature sensitive, whereas the fluorescence emission of coumarin is not temperature sensitive. Thus, the properties of the exemplified beads are suitable for use as disclosed herein.

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. Various documents are cited herein, the contents of which are incorporated herein by reference.

[Brief description of the drawings]

FIG. 1 is a graph showing a change in fluorescence intensity with respect to temperature of Eu (TTA) ₃ / PMMA. The excitation wavelength was 365 nm and the emission wavelength was 621 nm.

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

FIG. 3 shows an emission spectrum of beads containing Eu (TTA) ₃ and coumarin. When the excitation was performed at 350 nm, only a peak at 614 nm, which indicates the fluorescence of Eu (TTA) ₃ europium, was seen. 480 nm with coumarin when excitation is performed at 420 nm
, But only minimal emission was seen at 614 nm.

FIG. 4A is a diagram showing a change in amplitude of fluorescence emission at 614 nm with respect to a temperature when fluorescent beads are excited at 350 nm. Beads contained Eu (TTA) ₃ and coumarin. The measured value of the change in fluorescence is based on the temperature-sensitive fluorescence of Eu (TTA) ₃ . FIG. 4B is a graph showing the corresponding ratio of the fluorescence intensity of Eu (TTA) ₃ : coumarin to temperature. Fluorescent beads were excited at 420 nm and coumarin was monitored at 480 nm. The two sets of symbols in each experiment are from two different experiments on the same sample.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI G01K 11/20 G01K 11/20 G01N 21/64 G01N 21/64 EF 33/48 33/48 M 33/483 33/483 C // C08K 5/00 C08K 5/00 5/1545 5/1545 C08L 101/00 C08L 101/00 (C12M 1/34 C12R 1:91 C12R 1:91) (C12Q 1/02 C12R 1:91) (56 References JP-A-49-31382 (JP, A) JP-A-11-258159 (JP, A) JP-A-10-253622 (JP, A) JP-A-10-160595 (JP, A) 10-148614 (JP, A) Table 6-507022 (JP, A) WO 99/23939 (WO, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/52 G01K 11/20 G01N 21/64 G01N 33/48 G01N 33/483 JICST file (JOIS)

Claims (13)

(57) [Claims] 1. A polymer fluorescent bead comprising a temperature-sensitive fluorophore and a temperature-insensitive fluorophore embedded in a polymer material, wherein the fluorescent light is emitted by the temperature-sensitive fluorophore and the fluorescence is emitted by the temperature-insensitive fluorophore. The polymer fluorescent bead as described above, wherein the fluorescence emission is decomposed. 2. The polymer fluorescent beads according to claim 1, wherein said temperature-sensitive fluorophore is Eu (TTA) ₃ . 3. The polymeric fluorescent bead of claim 1, wherein said temperature-insensitive fluorophore is coumarin. 4. The polymeric fluorescent bead of claim 1, wherein said polymer is a non-aromatic synthetic polymer. 5. The polymer fluorescent beads according to claim 4, wherein said non-aromatic synthetic polymer is poly (methyl methacrylate). 6. The polymer material is polystyrene.
2. The polymer fluorescent bead according to claim 1. 7. The polymer fluorescent beads according to claim 1, wherein the size of the fluorescent beads is 80 to 90 nm. 8. A polymeric fluorescent bead comprising a first temperature-sensitive fluorophore and a second temperature-sensitive fluorophore, both of which are embedded in a polymeric material, wherein said polymeric fluorescent beads are embedded in a polymeric material. Although the fluorescence emission increases with temperature,
(2) The polymer fluorescent bead described above, wherein the fluorescence emitted by the temperature-sensitive fluorophore attenuates with temperature. 9. A method for determining the temperature of a cell, comprising: (a)
Placing the polymeric fluorescent beads of claim 1 into a cell; producing a detectable fluorescent signal that is mutually decomposed when the temperature-sensitive fluorophore and the temperature-insensitive fluorophore are excited with appropriate light; (B) exciting the temperature-sensitive fluorophore and the temperature-insensitive fluorophore with the appropriate light; and (c) exposing the fluorescent signal by the temperature-sensitive fluorophore to the temperature of the cell; and Measuring the intensity of the fluorescent signal emitted by the temperature-sensitive fluorophore and the temperature-insensitive fluorophore; measuring the intensity of the fluorescent signal of the temperature-sensitive fluorophore to determine the intensity of the temperature-insensitive fluorophore. Indicating the temperature of the cell when the local concentration is corrected by the corresponding intensity; determining the temperature. 10. A method for detecting the metabolic activity of a cell, comprising determining the temperature of the cell by the method of claim 9, and correlating the temperature with the metabolic activity of the cell. 11. A method for detecting the presence of abnormal metabolism of cells surrounded by healthy tissue in a tissue sample, wherein the temperature of the cells is determined by the method of claim 9 and the cells are compared to control cells. The above method, wherein the difference in temperature indicates that the cell is undergoing abnormal metabolism. 12. The tissue sample is obtained from a tumor biopsy.
The method of claim 11. 13. The method of claim 11, wherein said abnormal metabolism of said cells is indicative of ultraviolet damage, cachexia, or apoptosis.