JP2012177549A - Fluorescent temperature probe and temperature measuring instrument using the same - Google Patents

Fluorescent temperature probe and temperature measuring instrument using the same Download PDF

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JP2012177549A
JP2012177549A JP2009149338A JP2009149338A JP2012177549A JP 2012177549 A JP2012177549 A JP 2012177549A JP 2009149338 A JP2009149338 A JP 2009149338A JP 2009149338 A JP2009149338 A JP 2009149338A JP 2012177549 A JP2012177549 A JP 2012177549A
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Kenji Nagai
健治 永井
Ippei Kodera
一平 小寺
Takuya Iwasaki
卓也 岩崎
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Hokkaido University NUC
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Abstract

PROBLEM TO BE SOLVED: To provide a temperature measuring instrument capable of performing temperature measurement of the insides of an individual living being, tissue, cell and organelle in a cell, a water solution, or a solid body such as metal and plastic, and performing quantitative imaging of temperature without being affected by a proteinous fluorescent temperature probe suitable to imaging by spatial solution (<1 μm) having a high absolute temperature distribution nor by fluctuations of probe concentration in a measuring object.SOLUTION: A fluorescent temperature probe is configured by connecting a temperature sensitivity fluorescent protein in which relative fluorescence intensity greatly changes within the range of 20 to 50°C and a reference fluorescent substance in which a change in relative fluorescent intensity is small at a fixed ratio.

Description

本発明は、蛍光温度プローブおよびそれを用いた温度測定装置に関する。   The present invention relates to a fluorescent temperature probe and a temperature measuring device using the same.

従来から温度の測定には、熱電対温度計、抵抗温度計または水銀温度計等が用いられている。熱電対温度計は熱起電力値から温度を算出し、抵抗温度計は電気抵抗値から温度を算出する。しかし、熱電対温度計および抵抗温度計においては、測定対象物あるいはその外部からの無関係な電磁誘導ノイズが発生しやすく、また高電圧下では漏電するおそれがあるため、電界下または磁界下での温度測定には不適であるという問題があった。また、水銀温度計は、水銀の熱膨張を利用することにより温度を算出する。しかし、水銀温度計においては、その形状および熱容量の大きさに問題があるため測定対象物が限られるという問題があった。   Conventionally, a thermocouple thermometer, a resistance thermometer, a mercury thermometer, or the like is used for temperature measurement. The thermocouple thermometer calculates the temperature from the thermoelectromotive force value, and the resistance thermometer calculates the temperature from the electric resistance value. However, thermocouple thermometers and resistance thermometers tend to generate irrelevant electromagnetic induction noise from the object to be measured or from the outside, and there is a risk of leakage under high voltage. There was a problem that it was unsuitable for temperature measurement. The mercury thermometer calculates the temperature by utilizing the thermal expansion of mercury. However, the mercury thermometer has a problem in that the object to be measured is limited because of its shape and heat capacity.

これらの温度計を用いることのできない領域における温度測定には、放射温度計、蛍光体または半導体を用いた温度センサが用いられている。放射温度計は、測定対象物の放出する熱放射のエネルギを利用して温度を算出する。しかし、放射温度計は 測定対象物表面の温度しか測定することができず、また低温域において測定物固有のスペクトルまたは反射スペクトルが存在する場合には正確な温度測定をすることができないという問題があった。   A temperature sensor using a radiation thermometer, a phosphor or a semiconductor is used for temperature measurement in an area where these thermometers cannot be used. A radiation thermometer calculates temperature using the energy of the thermal radiation which a measurement object discharge | releases. However, the radiation thermometer can only measure the temperature of the surface of the object to be measured, and there is a problem that accurate temperature measurement cannot be performed when there is a spectrum or reflection spectrum unique to the object in a low temperature range. there were.

特許文献１には、ポリピリジン金属錯体またはその誘導体を含む蛍光体を用いた温度センサが開示されている。この蛍光体を用いた温度センサは、蛍光強度と温度との関係を調べることにより作成された温度特性曲線により温度を算出する。しかし、この蛍光体を用いた温度センサにおいては、蛍光強度と温度との関係が必ずしも比例関係等の明確な数式に従うものではないため、蛍光強度と温度との関係を細かい間隔で多数点調べて正確な温度特性曲線を作成しなければならないという問題があった。また、この蛍光体を用いた温度センサにおいては、温度センサの劣化等の経時変化に対応するために温度特性曲線の校正を頻繁に行なう必要があるが、その校正のたびに蛍光強度と温度との関係を細かい間隔で多数点調べて温度特性曲線の校正をしなければならないという問題もあった。   Patent Document 1 discloses a temperature sensor using a phosphor containing a polypyridine metal complex or a derivative thereof. The temperature sensor using this phosphor calculates the temperature from the temperature characteristic curve created by examining the relationship between the fluorescence intensity and the temperature. However, in the temperature sensor using this phosphor, the relationship between the fluorescence intensity and the temperature does not always follow a clear mathematical expression such as a proportional relationship. There was a problem that an accurate temperature characteristic curve had to be created. In addition, in a temperature sensor using this phosphor, it is necessary to frequently calibrate the temperature characteristic curve in order to cope with changes over time such as deterioration of the temperature sensor. There was also a problem that the temperature characteristic curve had to be calibrated by examining many points at a fine interval.

特許文献２には、半導体を光ファイバの先端に取り付けた温度センサが開示されている。この半導体を用いた温度センサは、温度により光の透過率が変わることを利用して温度を算出する。しかし、この半導体を用いた温度センサにおいては、温度と光の透過率との関係を示す温度特性曲線を作成する際に標準透過率を示す標準体が必要となるという問題があった。   Patent Document 2 discloses a temperature sensor in which a semiconductor is attached to the tip of an optical fiber. This temperature sensor using a semiconductor calculates the temperature by utilizing the fact that the light transmittance changes depending on the temperature. However, this temperature sensor using a semiconductor has a problem that a standard body indicating standard transmittance is required when creating a temperature characteristic curve indicating the relationship between temperature and light transmittance.

特許文献３には、電界下および磁界下での温度計測に適し、かつ温度特性曲線の作成および校正が容易な温度センサが開示されている。この発明では塩化物からなるマトリックスにドープされたエルビウムイオンまたはツリウムイオンの蛍光スペクトルが、特定の波長の前後において蛍光強度と温度との関係が逆転する性質を利用して温度を算出する。しかし、この温度センサは公報に記載の図1によれば25℃から50℃への温度変化に対して520-540nmの蛍光強度と540-560nmの蛍光強度の比の変化が数％に過ぎない。従って、生理条件下である10−50℃の範囲を感度良く測定することができない。また、塩化物マトリクス構造からなる温度センサであるため、水溶液に溶かして使用する事が困難である。さらに、温度センサが遺伝子にコードされていないため、温度センサを恒常的に発現する遺伝子導入動植物の作成は不可能であるという問題があった。   Patent Document 3 discloses a temperature sensor that is suitable for temperature measurement under an electric field and a magnetic field and that can easily create and calibrate a temperature characteristic curve. In the present invention, the temperature is calculated using the property that the fluorescence spectrum of erbium ions or thulium ions doped in a matrix made of chloride reverses the relationship between the fluorescence intensity and the temperature before and after a specific wavelength. However, according to FIG. 1 described in the publication, this temperature sensor has only a few percent change in the ratio of the fluorescence intensity at 520-540 nm and the fluorescence intensity at 540-560 nm with respect to the temperature change from 25 ° C. to 50 ° C. . Therefore, the range of 10-50 ° C. under physiological conditions cannot be measured with high sensitivity. Further, since it is a temperature sensor having a chloride matrix structure, it is difficult to use it dissolved in an aqueous solution. Furthermore, since the temperature sensor is not encoded by a gene, there is a problem that it is impossible to create a transgenic animal or plant that constantly expresses the temperature sensor.

特開平５−１３３８１９号公報Japanese Patent Laid-Open No. 5-133819 特開昭５８−３９９１７号公報JP 58-39917 A 特開２００４−２８６２９JP 2004-28629

本発明は、生物個体内、組織内、細胞内、細胞内小器官内および、水溶液、または金属やプラスチックなどの固体の温度計測ならびに絶対温度分布の高い空間分解能(＜1μm)による画像化に適したタンパク質性の蛍光温度プローブを提供することを目的とする。   The present invention is suitable for temperature measurement of living organisms, tissues, cells, organelles, and aqueous solutions, or solids such as metals and plastics, and imaging with high spatial resolution (<1 μm) of absolute temperature distribution. It is an object to provide a proteinaceous fluorescent temperature probe.

また、本発明は、測定対象内のプローブ濃度の変動に左右されずに温度の定量的画像化を可能にする温度測定装置を提供することを目的とする。   Another object of the present invention is to provide a temperature measuring apparatus that enables quantitative imaging of temperature without being influenced by fluctuations in probe concentration within the measurement target.

本発明者は、上記課題に鑑み検討を重ねた結果、温度感受性が異なる２種以上の蛍光物質を融合した温度プローブを用いることにより、測定対象内のプローブ濃度の変動に左右されずに温度の定量的画像化を可能にすることを見出した。   As a result of repeated examinations in view of the above problems, the present inventor uses a temperature probe in which two or more kinds of fluorescent substances having different temperature sensitivities are fused, so that the temperature of the measurement target is not affected by fluctuations in the probe concentration in the measurement target. It was found that quantitative imaging is possible.

本発明は、以下の蛍光温度プローブおよびそれを用いた温度測定装置を提供するものである。
項１． 20〜50℃の範囲で相対蛍光強度が大きく変化する温度感受性蛍光タンパク質と、相対蛍光強度の変化が小さい基準蛍光物質を一定の比率で連結してなる蛍光温度プローブ。
項２． 前記基準蛍光物質が、相対蛍光強度の変化が小さい蛍光タンパク質である、項１に記載の蛍光温度プローブ。
項３． 温度感受性蛍光タンパク質と基準蛍光物質の相対蛍光強度の差が0.2〜0.7程度である、項１または２に記載の蛍光温度プローブ。
項４． 温度感受性蛍光タンパク質が、mSEGFP、mOrange、TagRFP、mCherry、EBFP、SECFPまたはSiriusである、項１〜３のいずれかに記載の蛍光温度プローブ。
項５． 基準蛍光物質が、Topaz、EYFP、Venus、DsRed、Cy3、Cy5、FITC、ローダミン、FAM、TxR、ペリジニンクロロフィリンタンパク質、カスケードブルー、ＡＭＣＡ、反応性インドカルボシアニン、TRITC、アロフィコシアニン(APC)、フィコシアニン(PC)、DAPI、HEX(4,5,2',4',5',7'-ヘキサクロロ-6-カルボキシフルオレセイン)、5-IAF、TAMRA(6-カルボキシテトラメチルローダミン)、TET(4,7,2',7'-テトラクロロ-6-カルボキシフルオレセイン)からなる群から選ばれる、項１〜４のいずれかに記載の蛍光温度プローブ。
項６． 項１〜５のいずれかに記載の蛍光温度プローブを含む温度測定装置。
項７． 前記蛍光温度プローブと、前記蛍光温度プローブを励起させる手段と、温度プローブの励起によって生じた蛍光スペクトルを検出する手段と、検出されたスペクトルから温度を演算する手段と、演算された温度を表示する手段とを備えた、項６に記載の温度測定装置。 The present invention provides the following fluorescent temperature probe and a temperature measuring device using the same.
Item 1. A fluorescent temperature probe obtained by linking a temperature-sensitive fluorescent protein whose relative fluorescent intensity changes greatly in a range of 20 to 50 ° C. and a reference fluorescent substance whose relative fluorescent intensity changes little at a fixed ratio.
Item 2. Item 2. The fluorescent temperature probe according to Item 1, wherein the reference fluorescent material is a fluorescent protein having a small change in relative fluorescence intensity.
Item 3. Item 3. The fluorescent temperature probe according to Item 1 or 2, wherein the difference in relative fluorescence intensity between the temperature-sensitive fluorescent protein and the reference fluorescent material is about 0.2 to 0.7.
Item 4. Item 4. The fluorescent temperature probe according to any one of Items 1 to 3, wherein the temperature-sensitive fluorescent protein is mSEGFP, mOrange, TagRFP, mCherry, EBFP, SECFP, or Sirius.
Item 5. Reference fluorescent substances are Topaz, EYFP, Venus, DsRed, Cy3, Cy5, FITC, rhodamine, FAM, TxR, peridinin chlorophyllin protein, cascade blue, AMCA, reactive indocarbocyanine, TRITC, allophycocyanin (APC), phycocyanin (PC), DAPI, HEX (4,5,2 ′, 4 ′, 5 ′, 7′-hexachloro-6-carboxyfluorescein), 5-IAF, TAMRA (6-carboxytetramethylrhodamine), TET (4, Item 7. The fluorescent temperature probe according to any one of Items 1 to 4, which is selected from the group consisting of 7,2 ′, 7′-tetrachloro-6-carboxyfluorescein).
Item 6. Item 6. A temperature measuring device including the fluorescent temperature probe according to any one of Items 1 to 5.
Item 7. The fluorescence temperature probe, means for exciting the fluorescence temperature probe, means for detecting a fluorescence spectrum generated by excitation of the temperature probe, means for calculating the temperature from the detected spectrum, and displaying the calculated temperature Item 7. The temperature measuring device according to Item 6, comprising means.

本発明によれば、電界下および磁界下でも高精度に温度を測定することができ、かつ温度特性曲線の作成および温度校正を容易に行なうことができる蛍光温度プローブおよびそれを用いた温度測定装置を提供することができる。   According to the present invention, a fluorescent temperature probe capable of measuring a temperature with high accuracy even under an electric field and a magnetic field, and easily creating a temperature characteristic curve and calibrating the temperature, and a temperature measuring device using the same Can be provided.

温度の影響を受けやすい青色蛍光タンパク質。左側：AはSirius、BはSECFP、CはEBFPの蛍光スペクトルを示す。右側：低温→高温、高温→低温の蛍光強度の変化を示す(●が低温→高温、○が高温→低温)。Blue fluorescent protein that is sensitive to temperature. Left: A shows the fluorescence spectrum of Sirius, B shows the SECFP, and C shows the EBFP fluorescence spectrum. Right side: Changes in fluorescence intensity from low temperature to high temperature and high temperature to low temperature (● indicates low temperature → high temperature, ○ indicates high temperature → low temperature). 温度の影響を受けにくく安定している黄色蛍光タンパク質。左側：AはTopaz、BはEYFP、CはVenusの蛍光スペクトルを示す。右側：低温→高温、高温→低温の蛍光強度の変化を示す(●が低温→高温、○が高温→低温)。A yellow fluorescent protein that is not affected by temperature and is stable. Left: A is the topaz, B is the EYFP, and C is the Venus fluorescence spectrum. Right side: Changes in fluorescence intensity from low temperature to high temperature and high temperature to low temperature (● indicates low temperature → high temperature, ○ indicates high temperature → low temperature). 青色蛍光タンパク質と黄色蛍光タンパク質の変化量の中間に位置している蛍光タンパク質。左側：AはDsRed、BはmSEGFP、CはmOrange、DはTagRFP、EはmCherryの蛍光スペクトルを示す。右側：低温→高温、高温→低温の蛍光強度の変化を示す(●が低温→高温、○が高温→低温)。Fluorescent protein located between the amount of change between blue and yellow fluorescent proteins. Left: A is DsRed, B is mSEGFP, C is mOrange, D is TagRFP, E is mCherry fluorescence spectrum. Right side: Changes in fluorescence intensity from low temperature to high temperature and high temperature to low temperature (● indicates low temperature → high temperature, ○ indicates high temperature → low temperature). Fluorescein (Ex: 479 nm/Em: 500-608 nm) の温度感受性。左側：Fluoresceinの蛍光スペクトルを示す。右側：低温→高温、高温→低温の蛍光強度の変化を示す(●が低温→高温、○が高温→低温)。Temperature sensitivity of Fluorescein (Ex: 479 nm / Em: 500-608 nm). Left side: Fluorescein fluorescence spectrum. Right side: Changes in fluorescence intensity from low temperature to high temperature and high temperature to low temperature (● indicates low temperature → high temperature, ○ indicates high temperature → low temperature). 円順列変異体Venus (Ex: 500 nm/Em: 515-650 nm) の温度感受性。左側：Aはcp147 Venus、Bはcp 148 Venus、Cはcp173 Venusの蛍光スペクトルを示す。右側：低温→高温、高温→低温の蛍光強度の変化を示す(●が低温→高温、○が高温→低温)。Temperature sensitivity of circular permutation Venus (Ex: 500 nm / Em: 515-650 nm). Left: A shows the fluorescence spectrum of cp147 Venus, B shows the fluorescence spectrum of cp148 Venus, and C shows the fluorescence spectrum of cp173 Venus. Right side: Changes in fluorescence intensity from low temperature to high temperature and high temperature to low temperature (● indicates low temperature → high temperature, ○ indicates high temperature → low temperature). 融合蛍光タンパク質の規格化蛍光強度レシオの変化。Changes in the normalized fluorescence intensity ratio of the fusion fluorescent protein. 遺伝子コードされた蛍光温度センサを用いた細胞内温度イメージング。Intracellular temperature imaging using gene-encoded fluorescence temperature sensors.

１つの好ましい実施形態において、本発明は蛍光強度の温度感受性が異なる少なくとも１種の蛍光タンパク質（温度感受性蛍光タンパク質）と少なくとも１種の基準蛍光物質を共有結合又は複合体化（金属錯体、或いはアビジン-ビオチンなどにより結合）したものが挙げられる。   In one preferred embodiment, the present invention provides a covalent bond or complex (metal complex or avidin) of at least one fluorescent protein (temperature-sensitive fluorescent protein) having different temperature intensities of fluorescence intensity and at least one reference fluorescent substance. -Bonded by biotin etc.).

「温度感受性蛍光タンパク質」とは、20℃を基準にしたときの50℃の相対蛍光強度が、0.8以下、好ましくは0.7以下、より好ましくは0.6以下、さらに好ましくは0.5以下、特に好ましくは0.4以下、特に0.2〜0.4である。温度感受性蛍光タンパク質の相対蛍光強度は、小さいほど好ましい。   “Temperature sensitive fluorescent protein” means relative fluorescence intensity at 50 ° C. relative to 20 ° C. of 0.8 or less, preferably 0.7 or less, more preferably 0.6 or less, further preferably 0.5 or less, particularly preferably 0.4 or less. In particular, it is 0.2 to 0.4. The smaller the relative fluorescence intensity of the temperature sensitive fluorescent protein, the better.

「基準蛍光物質」とは、20℃を基準にしたときの50℃の相対蛍光強度が、1に近いか、1よりも大きい蛍光物質をいう。一般の蛍光性の化学物質、例えばTopaz、EYFP、Venus、DsRed、Cy3、Cy5、FITC、ローダミン、FAM、TxR、ペリジニンクロロフィリンタンパク質、カスケードブルー、AMCA、反応性インドカルボシアニン、TRITC、アロフィコシアニン(APC)、フィコシアニン(PC)、DAPI、HEX(4,5,2',4',5',7'-ヘキサクロロ-6-カルボキシフルオレセイン)、5-IAF、TAMRA(6-カルボキシテトラメチルローダミン)、TET(4,7,2',7'-テトラクロロ-6-カルボキシフルオレセイン)などは、温度変化による蛍光強度の変化は小さいかほとんど或いは全く無く、基準蛍光物質として使用できる。さらに、Topaz、EYFP、Venus、DsRedなどは、相対蛍光強度が１に近いため、「基準蛍光物質」として使用することができる。「基準蛍光物質」の20℃を基準にしたときの50℃の相対蛍光強度は、0.6〜1.5、好ましくは0.7〜1.4、より好ましくは0.8〜1.3、特に好ましくは0.85〜1.2である。   The “reference fluorescent substance” refers to a fluorescent substance having a relative fluorescent intensity at 50 ° C. close to 1 or larger than 1 with 20 ° C. as a reference. Common fluorescent chemicals such as Topaz, EYFP, Venus, DsRed, Cy3, Cy5, FITC, rhodamine, FAM, TxR, peridinin chlorophyllin protein, cascade blue, AMCA, reactive indocarbocyanine, TRITC, allophycocyanin ( APC), phycocyanin (PC), DAPI, HEX (4,5,2 ′, 4 ′, 5 ′, 7′-hexachloro-6-carboxyfluorescein), 5-IAF, TAMRA (6-carboxytetramethylrhodamine), TET (4,7,2 ′, 7′-tetrachloro-6-carboxyfluorescein) or the like can be used as a reference fluorescent material with little or no change in fluorescence intensity due to temperature change. Furthermore, Topaz, EYFP, Venus, DsRed, and the like can be used as “reference fluorescent substances” because the relative fluorescence intensity is close to 1. The relative fluorescence intensity at 50 ° C. with respect to 20 ° C. of the “reference fluorescent substance” is 0.6 to 1.5, preferably 0.7 to 1.4, more preferably 0.8 to 1.3, and particularly preferably 0.85 to 1.2.

本発明の蛍光温度プローブにおいて、温度感受性蛍光タンパク質と基準蛍光物質の相対強度の差は、好ましくは0.2以上、より好ましくは0.3以上、さらに好ましくは0.4以上、特に好ましくは0.5以上、特に0.6〜0.8である。相対蛍光強度の差は、大きいほど温度変化が鋭敏に測定できるので好ましい。   In the fluorescent temperature probe of the present invention, the difference in relative intensity between the temperature sensitive fluorescent protein and the reference fluorescent material is preferably 0.2 or more, more preferably 0.3 or more, still more preferably 0.4 or more, particularly preferably 0.5 or more, particularly 0.6 to 0.8. It is. The larger the difference in relative fluorescence intensity, the better because the temperature change can be measured more sensitively.

温度感受性蛍光タンパク質と基準蛍光物質の好ましい組み合わせとしては、以下のものが挙げられる。
Sirius-Venus、Venus-Sirius、Sirius-DsRed、DsRed-Sirius、Topaz-Sirius、Venus-mCherry、mCherry-Venus
SECFP-Venus、Venus-SECFP、SECFP-DsRed、DsRed-SECFP、Topaz-SECFP
EBFP-Venus、Venus-EBFP、EBFP-DsRed、DsRed-EBFP、Topaz-EBFP
Sirius-EYFP、EYFP-Sirius、EYFP-mCherry、mCherry-EYFP、SECFP-EYFP、EYFP-SECF、EBFP-EYFP、EYFP-EBFP
Sirius: ACCESSION AB444952
mseCFP: ACCESSION AB435576
Venus: Nature Biotechnology 20, 87 - 90 (2002)
mCherry, mOrange: PNAS June 11, 2002 vol. 99 no. 12 7877-7882
EGFP, EFYP: Science Vol. 273. no. 5280, pp. 1392 - 1395
本発明の蛍光温度プローブは、従来提供されていなかった20〜50℃の範囲において、相対蛍光強度の差により温度を測定できるものであるが、0〜100℃程度、好ましくは5〜80℃、より好ましくは10〜60℃の範囲において温度を良好に測定できる。より高温で温度を測定する場合、他の温度プローブと組み合わせることが可能である。100℃以上のような高温での温度測定に好適なセンサとしては、例えば温度により伸縮する網目状のポリマー内に蛍光分子を入れておき、温度が高くなると水が放出されて光るシステムなどが挙げられる。 Preferred combinations of temperature sensitive fluorescent protein and reference fluorescent material include the following.
Sirius-Venus, Venus-Sirius, Sirius-DsRed, DsRed-Sirius, Topaz-Sirius, Venus-mCherry, mCherry-Venus
SECFP-Venus, Venus-SECFP, SECFP-DsRed, DsRed-SECFP, Topaz-SECFP
EBFP-Venus, Venus-EBFP, EBFP-DsRed, DsRed-EBFP, Topaz-EBFP
Sirius-EYFP, EYFP-Sirius, EYFP-mCherry, mCherry-EYFP, SECFP-EYFP, EYFP-SECF, EBFP-EYFP, EYFP-EBFP
Sirius: ACCESSION AB444952
mseCFP: ACCESSION AB435576
Venus: Nature Biotechnology 20, 87-90 (2002)
mCherry, mOrange: PNAS June 11, 2002 vol. 99 no. 12 7877-7882
EGFP, EFYP: Science Vol. 273.no.5280, pp. 1392-1395
The fluorescent temperature probe of the present invention can measure the temperature by the difference in relative fluorescence intensity in the range of 20 to 50 ° C., which has not been provided conventionally, but about 0 to 100 ° C., preferably 5 to 80 ° C., More preferably, the temperature can be measured well in the range of 10 to 60 ° C. When measuring temperature at higher temperatures, it can be combined with other temperature probes. As a sensor suitable for temperature measurement at a high temperature such as 100 ° C. or more, for example, a system in which fluorescent molecules are placed in a network polymer that expands and contracts depending on temperature, and water is emitted when the temperature rises, etc. It is done.

基準蛍光物質が蛍光タンパク質の場合には、少なくとも１つの温度感受性蛍光タンパク質と少なくとも１つの基準蛍光物質を直接或いは適当な長さのリンカーペプチドを介して結合することにより、相対蛍光強度の異なる２つの蛍光タンパク質を１：１、２：１、１：２などの所定の比率（特に１：１）で連結することができる。本発明では、基準蛍光物質と温度感受性蛍光タンパク質の相対蛍光強度の違いにより温度を測定するため、これらの相対比率が一定であることが望ましい。なお、基準蛍光物質と温度感受性蛍光タンパク質を化学的に二価の連結基を介して結合したり、ビオチン-アビジン系を用いて複合体化する場合、基準蛍光物質と温度感受性蛍光タンパク質の比率が確定しない場合があり得るが、このような場合でも、所定の温度における相対蛍光強度を測定することにより、両者の比率を推定でき、蛍光温度プローブとして使用できる。   When the reference fluorescent substance is a fluorescent protein, at least one temperature-sensitive fluorescent protein and at least one reference fluorescent substance are bound directly or via a linker peptide having an appropriate length, so that two different fluorescent intensities can be obtained. Fluorescent proteins can be linked at a predetermined ratio (particularly 1: 1) such as 1: 1, 2: 1, 1: 2. In the present invention, since the temperature is measured based on the difference in relative fluorescence intensity between the reference fluorescent substance and the temperature-sensitive fluorescent protein, it is desirable that these relative ratios are constant. When the reference fluorescent substance and temperature-sensitive fluorescent protein are chemically bound via a divalent linking group or complexed using a biotin-avidin system, the ratio between the reference fluorescent substance and the temperature-sensitive fluorescent protein is Even in such a case, the ratio between the two can be estimated by measuring the relative fluorescence intensity at a predetermined temperature, and can be used as a fluorescence temperature probe.

蛍光タンパク質が酵母などの真核細胞で発現されて糖鎖を有する場合、これを過ヨウ素酸塩などの酸化剤でアルデヒドに導き、アミノ基を有する基準蛍光物質と反応させてもよい。或いは、ＮＨＳ基とマレイミド基を有する二価の連結基を用いて基準蛍光物質と温度感受性蛍光タンパク質を連結してもよい。   When the fluorescent protein is expressed in a eukaryotic cell such as yeast and has a sugar chain, it may be led to an aldehyde with an oxidizing agent such as periodate and reacted with a reference fluorescent substance having an amino group. Alternatively, the reference fluorescent substance and the temperature sensitive fluorescent protein may be linked using a divalent linking group having an NHS group and a maleimide group.

本発明の特に好ましい実施形態は、20℃を基準にしたときの50℃の相対蛍光強度の差が大きい温度感受性蛍光タンパク質と、基準蛍光物質となる蛍光タンパク質を直接又は適当な長さのペプチドリンカーで連結し、大腸菌等の微生物で発現させて得た温度プローブであり、測定対象内のプローブ濃度の変動に左右されずに温度の定量的画像化を可能にすることを特徴とする。   A particularly preferred embodiment of the present invention is a peptide linker of a temperature-sensitive fluorescent protein having a large difference in relative fluorescent intensity at 50 ° C. relative to 20 ° C. and a fluorescent protein serving as a reference fluorescent substance directly or of an appropriate length. The temperature probe obtained by ligation and expression in microorganisms such as Escherichia coli is characterized in that it enables quantitative imaging of the temperature without being influenced by fluctuations in the probe concentration in the measurement target.

本発明は、さらに、上記温度プローブを励起させる手段と、温度プローブの励起によって生じた蛍光スペクトルを検出する手段と、検出されたスペクトルから温度を演算する手段と、演算された温度を表示する手段とを備えた温度測定装置であることを特徴とする。   The present invention further includes means for exciting the temperature probe, means for detecting a fluorescence spectrum generated by excitation of the temperature probe, means for calculating the temperature from the detected spectrum, and means for displaying the calculated temperature. And a temperature measuring device.

上記プローブの励起波長は、図１〜４に示す。   The excitation wavelength of the probe is shown in FIGS.

本発明で使用できる温度プローブを励起させる手段としては、蛍光タンパク質ないし蛍光分子を励起可能な光源であれば特に限定されず、レーザー光源が好ましく使用される。蛍光スペクトルを検出する手段としては、CCDカメラ、CMOSカメラ、フォトマルチプライヤーなどが挙げられる。   The means for exciting the temperature probe that can be used in the present invention is not particularly limited as long as it is a light source capable of exciting fluorescent proteins or fluorescent molecules, and a laser light source is preferably used. Examples of means for detecting the fluorescence spectrum include a CCD camera, a CMOS camera, and a photomultiplier.

本発明の温度プローブは、例えば２つの蛍光タンパク質を連結した融合タンパク質タイプのものは、細胞内に組み込むことで、細胞の温度を測定することができる。融合タンパク質をミトコンドリアなどの特定の器官内で発現させることにより、特定の器官（オルガネラ）の温度を測定することもできる。例えば２つの蛍光タンパク質を連結した融合タンパク質を発現させたトランスジェニック非ヒト哺乳動物は、表面もしくは深部の細胞の温度を測定することができる。なお、トランスジェニック非ヒト哺乳動物の深部の細胞の温度は、例えば二光子顕微鏡を用いて測定できる。   For example, when the temperature probe of the present invention is of a fusion protein type in which two fluorescent proteins are linked, the temperature of the cell can be measured by incorporating it into the cell. The temperature of a specific organ (organelle) can also be measured by expressing the fusion protein in a specific organ such as mitochondria. For example, a transgenic non-human mammal that expresses a fusion protein in which two fluorescent proteins are linked can measure the temperature of a surface or deep cell. In addition, the temperature of the cell of the deep part of a transgenic non-human mammal can be measured using a two-photon microscope, for example.

或いは、本発明の温度プローブは、光透過性の樹脂に練り込み、フィルム、シート、或いは成形体としてもよい。   Alternatively, the temperature probe of the present invention may be kneaded into a light transmissive resin to form a film, a sheet, or a molded body.

以下、本発明を実施例を用いてより詳細に説明するが、本発明がこれら実施例に限定されないことはいうまでもない。
略号
本明細書では、以下の略号が用いられる。
BFP blue fluorescent protein
cAMP cyclic adenosine monophosphate
CaM calmodulin
CFP cyan fluorescent protein
DNA deoxyribonucleic acid
EGTA ethylene glycol tetraacetic acid
EGFP enhanced green fluorescent protein
FRET Forster resonance energy transfer
FASTR fully automated single-tube recombination
GFP green fluorescent protein
MDCK Madin Darby canine kidney
MOPS 3-morpholinopropane sulfuric acid
mono monomeric
LB Luria Bertani
PBS phosphate buffered saline
PCR polymerase chain reaction
TN-XL troponinC-based biosensor
Wt wild-type
YC3.60 yellow cameleon 3.60
mYFP monomeric yellow fluorescence protein

実施例１：GFP変異体の温度感受性
蛍光タンパク質の様々な波長変異体における蛍光強度の温度感受性を測定した。その結果、蛍光タンパク質の種類により、蛍光強度の温度感受性が大きく異なることを見出した (表１) 。 EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, it cannot be overemphasized that this invention is not limited to these Examples.
Abbreviations In this specification, the following abbreviations are used.
BFP blue fluorescent protein
cAMP cyclic adenosine monophosphate
CaM calmodulin
CFP cyan fluorescent protein
DNA deoxyribonucleic acid
EGTA ethylene glycol tetraacetic acid
EGFP enhanced green fluorescent protein
FRET Forster resonance energy transfer
FASTR fully automated single-tube recombination
GFP green fluorescent protein
MDCK Madin Darby canine kidney
MOPS 3-morpholinopropane sulfuric acid
mono monomeric
LB Luria Bertani
PBS phosphate buffered saline
PCR polymerase chain reaction
TN-XL troponinC-based biosensor
Wt wild-type
YC3.60 yellow cameleon 3.60
mYFP monomeric yellow fluorescence protein

Example 1: Temperature sensitivity of GFP mutants The temperature sensitivity of fluorescence intensity in various wavelength mutants of fluorescent proteins was measured. As a result, it was found that the temperature sensitivity of the fluorescence intensity varies greatly depending on the type of fluorescent protein (Table 1).

Figure 2012177549
Figure 2012177549

全体的な傾向として、青色系の蛍光タンパク質（Sirius、SECFP、EBFP）は、温度の影響を受けやすいことが分かった。   Overall, blue fluorescent proteins (Sirius, SECFP, EBFP) were found to be sensitive to temperature.

Siriusの場合は、20℃から50℃に温度を上げた時、蛍光強度が1から0.36へ下がった。本研究で測定した蛍光タンパク質の中でSiriusは最も温度感受性の高い蛍光タンパク質である (図１Ａ)。SECFP (図１Ｂ) やEBFP (図１Ｃ)も同様に高い温度感受性を示し、20℃の時の蛍光強度を1とした場合の50℃での蛍光強度はそれぞれ0.37と0.39であった。   In the case of Sirius, the fluorescence intensity decreased from 1 to 0.36 when the temperature was raised from 20 ° C to 50 ° C. Among the fluorescent proteins measured in this study, Sirius is the most temperature sensitive fluorescent protein (FIG. 1A). Similarly, SECFP (FIG. 1B) and EBFP (FIG. 1C) showed high temperature sensitivity, and the fluorescence intensity at 50 ° C. when the fluorescence intensity at 20 ° C. was 1 was 0.37 and 0.39, respectively.

黄色系の蛍光タンパク質（Topaz、EYFP、Venus）は、温度の影響を受けにくく、温度に対して蛍光強度が安定していることが明らかになった (表１)。   It was revealed that yellow fluorescent proteins (Topaz, EYFP, Venus) are less affected by temperature and the fluorescence intensity is stable with respect to temperature (Table 1).

Topazは、本発明者が測定した中では温度の変化に対して蛍光強度が最も安定している蛍光タンパク質であった。20℃で測定したTopazの蛍光強度を1とすると、50℃では蛍光強度は1.09となった (図2A)。EYFPとVenusは、20℃の時の蛍光強度で規格化した50℃の時の蛍光強度がそれぞれ0.89 (図2B)と0.83 (図2C)であった。   Topaz was a fluorescent protein whose fluorescence intensity was most stable with respect to changes in temperature as measured by the present inventors. When the fluorescence intensity of Topaz measured at 20 ° C. is 1, the fluorescence intensity is 1.09 at 50 ° C. (FIG. 2A). EYFP and Venus had 0.89 (FIG. 2B) and 0.83 (FIG. 2C) fluorescence intensity at 50 ° C. normalized by the fluorescence intensity at 20 ° C., respectively.

また、赤色系の蛍光タンパク質（DsRed、mOrange、TagRFP、mCherry）と緑色蛍光タンパク質（mSEGFP）では、これまでの計測したタンパク質の中間的な値を示した。20℃で測定した蛍光強度を1とした時の50℃で蛍光強度は、それぞれ、0.79 (図3A)、0.60 (図3B)、0.59 (図3C)、0.54 (図3D)、0.65 (図3E)であった。   In addition, red fluorescent proteins (DsRed, mOrange, TagRFP, mCherry) and green fluorescent proteins (mSEGFP) showed intermediate values of proteins measured so far. The fluorescence intensity at 50 ° C. when the fluorescence intensity measured at 20 ° C. is 1, respectively, is 0.79 (FIG. 3A), 0.60 (FIG. 3B), 0.59 (FIG. 3C), 0.54 (FIG. 3D), and 0.65 (FIG. 3E). )Met.

一方で、蛍光タンパク質以外の蛍光物質の代表例としてフルオレセインの温度感受性も解析した。20℃で測定したフルオレセインの蛍光強度を1とした時、50℃での蛍光強度は0.91であり、ほとんど変化しなかった (図4)。   On the other hand, the temperature sensitivity of fluorescein was also analyzed as a representative example of fluorescent substances other than fluorescent proteins. When the fluorescence intensity of fluorescein measured at 20 ° C. was 1, the fluorescence intensity at 50 ° C. was 0.91, which was almost unchanged (FIG. 4).

また、比較的温度に対する蛍光強度の変化が少なかった黄色蛍光タンパク質について、円順列変異体の温度感受性について測定を行った。cp147 Venus、cp148 Venus、cp173 Venusについて、20℃の時の蛍光強度を1とした時に、50℃の時の蛍光強度は、それぞれ、0.69、0.76、0.90であった (図5)。   In addition, the yellow fluorescent protein having a relatively small change in fluorescence intensity with respect to temperature was measured for the temperature sensitivity of the circular permutation mutant. For cp147 Venus, cp148 Venus, and cp173 Venus, the fluorescence intensity at 50 ° C. was 0.69, 0.76, and 0.90, respectively, assuming that the fluorescence intensity at 20 ° C. was 1 (FIG. 5).

なお、円順列変異体とは、タンパク質を任意の部位で切断し、N末端断片とC末端断片とを入れ替えてつなげる変異体である。このようにして作成された変異体は、元のタンパク質とほぼ同等の機能を有することが多いが、今回の実験結果は、数種類の円順列変異体において異なる温度感受性を示すものであった。このような例は報告が無く、今後温度センサを開発する上で重要な知見となるものである。   The circular permutant is a mutant that cuts a protein at an arbitrary site and connects the N-terminal fragment and the C-terminal fragment. The mutants thus prepared often have almost the same function as the original protein, but the results of this experiment showed different temperature sensitivities in several types of circular permutants. Such an example has not been reported, and will be an important finding in the future development of temperature sensors.

ここまで得られた結果から、温度の影響を受けやすい蛍光タンパク質(Sirius、mCherry)と温度の影響を受けにくく安定している蛍光タンパク質(Venus、Topaz、mOrange)を組み合わせることで、温度に対して蛍光強度がレシオメトリックに変化するプローブが実現可能か検討した。温度感受性の高い蛍光タンパク質と温度感受性の低い蛍光タンパク質をフレキシブルなリンカーアミノ酸（Gly-Gly-Ser）でつなぎ、以下の組合せの温度センサーを作成した（Topaz-Sirius、Sirius-Venus、Venus-Sirius、Sirius-mOrange、mOrange-Sirius、Venus-mCherry、mCherry-Venus）。これらの温度センサーを、分光高度計の中で20℃から50℃に温度を変化させ、プローブのそれぞれの蛍光タンパク質の蛍光強度の変化を測定した(図6)。その結果、最も大きな変化率を見せたTopaz-Siriusにおいて、128％のダイナミクレンジを達成した(表2)。   From the results obtained so far, by combining fluorescent proteins that are susceptible to temperature (Sirius, mCherry) and fluorescent proteins that are stable and resistant to temperature (Venus, Topaz, mOrange) We investigated whether a probe whose fluorescence intensity changed ratiometrically was feasible. A temperature sensor with the following combinations (Topaz-Sirius, Sirius-Venus, Venus-Sirius) was created by connecting a fluorescent protein with high temperature sensitivity and a fluorescent protein with low temperature sensitivity using a flexible linker amino acid (Gly-Gly-Ser). Sirius-mOrange, mOrange-Sirius, Venus-mCherry, mCherry-Venus). The temperature of these temperature sensors was changed from 20 ° C. to 50 ° C. in the spectrophotometer, and the change in the fluorescence intensity of each fluorescent protein of the probe was measured (FIG. 6). As a result, the topaz-Sirius that showed the greatest rate of change achieved a dynamic range of 128% (Table 2).

Figure 2012177549
Figure 2012177549

実施例2
温度の影響を受けやすい蛍光タンパク質(Sirius, mCherry)と温度の影響を受けにくく安定している蛍光タンパク質(Venus, Topaz, mOrange)をアミノ酸（Gly-Gly-Ser）で繋げ、2波長励起で蛍光強度を測定した。縦軸は規格化蛍光強度レシオ、横軸は温度を示す。 Example 2
A fluorescent protein (Sirius, mCherry) that is susceptible to temperature and a stable fluorescent protein (Venus, Topaz, mOrange) that is less susceptible to temperature are linked by an amino acid (Gly-Gly-Ser), and fluorescent when excited by two wavelengths. The strength was measured. The vertical axis represents the normalized fluorescence intensity ratio, and the horizontal axis represents the temperature.

細胞内(HeLa細胞)で2つの蛍光タンパク質を発現させて、細胞周囲の温度を変化させながらそれぞれの蛍光タンパク質からの蛍光強度を測定後、蛍光強度比でイメージングを行った。   Two fluorescent proteins were expressed in the cells (HeLa cells), and the fluorescence intensity from each of the fluorescent proteins was measured while changing the temperature around the cells, followed by imaging at the fluorescence intensity ratio.

図６で得られた結果に比べて、温度変化による蛍光強度比の変化は小さかったが、２５℃〜３５℃の範囲内で、ＨｅＬａ細胞の温度を測定できることが明らかになった
以上の実施例から、蛍光タンパク質の温度に対する感受性は、蛍光タンパク質の種類により大きく異なることが明らかになった。そこで、最も感受性の高いタンパク質と低いタンパク質を組み合わせることで、感度の高い温度プローブを開発することに成功した。この温度プローブは20℃から50℃へ温度を変化させたときに128%ものダイナミックレンジを示し、温度プローブとして十分な性能を有していることが確認された。 Compared to the results obtained in FIG. 6, the change in the fluorescence intensity ratio due to the temperature change was small, but it became clear that the temperature of the HeLa cells could be measured within the range of 25 ° C. to 35 ° C. Thus, it became clear that the sensitivity of fluorescent proteins to temperature varies greatly depending on the type of fluorescent protein. Therefore, we have succeeded in developing a highly sensitive temperature probe by combining the most sensitive and low proteins. This temperature probe showed a dynamic range of 128% when the temperature was changed from 20 ° C. to 50 ° C., and it was confirmed that the temperature probe has sufficient performance as a temperature probe.

本発明の温度プローブは、細胞で用いる際の指標となるダイナミックレンジにおいて、十分な数値を達成している。この温度プローブを用いて、褐色脂肪細胞のミトコンドリアにおける熱産生のメカニズムを検討することができる。   The temperature probe of the present invention achieves a sufficient value in the dynamic range that is an index when used in cells. This temperature probe can be used to investigate the mechanism of heat production in brown adipocyte mitochondria.

またこれ以外にも、例えば、生体の温度プローブである温度依存性チャネル（TRPV）の動態を細胞内で可視化したり、ミトコンドリアのUCP以外での熱産生を探索したりするなど、様々な応用例が考えられる。   In addition to this, various applications such as visualizing the dynamics of temperature-dependent channels (TRPV), which are biological temperature probes, in the cell, and searching for heat production other than mitochondrial UCP, etc. Can be considered.

Claims (7)

20〜50℃の範囲で相対蛍光強度が大きく変化する温度感受性蛍光タンパク質と、相対蛍光強度の変化が小さい基準蛍光物質を一定の比率で連結してなる蛍光温度プローブ。 A fluorescent temperature probe obtained by linking a temperature-sensitive fluorescent protein whose relative fluorescent intensity changes greatly in a range of 20 to 50 ° C. and a reference fluorescent substance whose relative fluorescent intensity changes little at a fixed ratio. 前記基準蛍光物質が、相対蛍光強度の変化が小さい蛍光タンパク質である、請求項1に記載の蛍光温度プローブ。 2. The fluorescent temperature probe according to claim 1, wherein the reference fluorescent substance is a fluorescent protein having a small change in relative fluorescent intensity. 温度感受性蛍光タンパク質と基準蛍光物質の相対蛍光強度の差が0.2〜0.7程度である、請求項１または２に記載の蛍光温度プローブ。 The fluorescence temperature probe according to claim 1 or 2, wherein the difference in relative fluorescence intensity between the temperature sensitive fluorescent protein and the reference fluorescent substance is about 0.2 to 0.7. 温度感受性蛍光タンパク質が、mSEGFP、mOrange、TagRFP、mCherry、EBFP、SECFPまたはSiriusである、請求項１〜３のいずれかに記載の蛍光温度プローブ。 The fluorescent temperature probe according to any one of claims 1 to 3, wherein the temperature-sensitive fluorescent protein is mSEGFP, mOrange, TagRFP, mCherry, EBFP, SECFP, or Sirius. 基準蛍光物質が、Topaz、EYFP、Venus、DsRed、Cy3、Cy5、FITC、ローダミン、FAM、TxR、ペリジニンクロロフィリンタンパク質、カスケードブルー、ＡＭＣＡ、反応性インドカルボシアニン、TRITC、アロフィコシアニン(APC)、フィコシアニン(PC)、DAPI、HEX(4,5,2',4',5',7'-ヘキサクロロ-6-カルボキシフルオレセイン)、5-IAF、TAMRA(6-カルボキシテトラメチルローダミン)、TET(4,7,2',7'-テトラクロロ-6-カルボキシフルオレセイン)からなる群から選ばれる、請求項１〜４のいずれかに記載の蛍光温度プローブ。 Reference fluorescent substances are Topaz, EYFP, Venus, DsRed, Cy3, Cy5, FITC, rhodamine, FAM, TxR, peridinin chlorophyllin protein, cascade blue, AMCA, reactive indocarbocyanine, TRITC, allophycocyanin (APC), phycocyanin (PC), DAPI, HEX (4,5,2 ′, 4 ′, 5 ′, 7′-hexachloro-6-carboxyfluorescein), 5-IAF, TAMRA (6-carboxytetramethylrhodamine), TET (4, The fluorescent temperature probe according to any one of claims 1 to 4, which is selected from the group consisting of 7,2 ', 7'-tetrachloro-6-carboxyfluorescein). 請求項１〜５のいずれかに記載の蛍光温度プローブを含む温度測定装置。 A temperature measuring device including the fluorescent temperature probe according to claim 1. 前記蛍光温度プローブと、前記蛍光温度プローブを励起させる手段と、温度プローブの励起によって生じた蛍光スペクトルを検出する手段と、検出されたスペクトルから温度を演算する手段と、演算された温度を表示する手段とを備えた、請求項６に記載の温度測定装置。 The fluorescence temperature probe, means for exciting the fluorescence temperature probe, means for detecting a fluorescence spectrum generated by excitation of the temperature probe, means for calculating the temperature from the detected spectrum, and displaying the calculated temperature The temperature measuring device according to claim 6, comprising: means.
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