Flipper-TR細(xì)胞膜膜張力探針

Flipper-TR ® 是一個(gè)活細(xì)胞熒光膜張力探針，實(shí)時(shí)地測量活細(xì)胞膜張力

Flipper-TR® fluorescent cell membrane tension probe

Flipper-TR®膜張力探針介紹：

熒光膜張力探針Flipper-TR®(Spirochrome, Ltd.)的面世解決了這些挑戰(zhàn)，它可以搭配FLIM(熒光壽命成像顯微鏡)可以實(shí)現(xiàn)對活細(xì)胞膜的可視化染色。Flipper-TR®是一種活細(xì)胞熒光膜張力探針，是di一個(gè)為機(jī)械生物學(xué)領(lǐng)域開發(fā)的熒光膜張力探針。Flipper-TR®的熒光壽命與膜張力密切相關(guān)，利用FLIM可將不同壽命的熒光轉(zhuǎn)換成不同的顏色顯現(xiàn)出來，因此可以通過熒光和顏色及年度即可直觀的測量膜內(nèi)張力的時(shí)空分布。Flipper-TR®開辟了一個(gè)quan新的領(lǐng)域，它提供了一種靈敏、可靠和無創(chuàng)的方法，通過這種方法可以快速、實(shí)時(shí)地測量活細(xì)胞的膜張力(圖1)。

圖1. Flipper-TR感知高滲休克處理后的細(xì)胞膜張力變化

左側(cè)：與 Flipper -TR染色的細(xì)胞的圖像®   高滲休克之前（上圖）和后（下圖）。

灰色程度代表熒光強(qiáng)度，不同顏色代表不同熒光壽命（藍(lán)-紅：0-6納秒）

右側(cè)：直方圖顯示高滲休克后的壽命變化，圖片由Colom等人提供。

熒光Flipper -TR ®探針的工作原理是特異性地靶向細(xì)胞質(zhì)膜，通過報(bào)告熒光壽命變化來反映膜張力變化情況。它是Flipper探針家族中的成員，它通過機(jī)械載體上的兩個(gè)扭曲的二硫噻吩的扭轉(zhuǎn)角和極化來感知脂質(zhì)雙層膜結(jié)構(gòu)的變化。Flipper -TR ®自發(fā)插入到細(xì)胞的質(zhì)膜中，只有插入到脂質(zhì)膜中才會發(fā)出熒光，通過FLIM(熒光壽命成像顯微鏡)檢測熒光的強(qiáng)度及顏色即可判斷膜張力的大小，F(xiàn)lipper -TR ®探針具有廣泛的吸收和發(fā)射光譜; 通常在488nm處進(jìn)行激發(fā)，而發(fā)射光譜則在575和625nm之間（圖2）。

圖2. Flipper-TR的工作原理及吸收/發(fā)射光譜

A.Flipper-TR機(jī)制及其與不同張力膜相互作用的示意圖

左側(cè)：Flipper-TR的基本分子結(jié)構(gòu)

右側(cè)：Flipper-TR在低張力扭曲（綠色）和高張力平行排列（紅色）

B.對乙酸乙酯中的Flipper-TR溶液進(jìn)行掃描和發(fā)射掃描，并將結(jié)果繪制在同一張圖上。

點(diǎn)狀橙色線激發(fā)光，實(shí)心橙色線為發(fā)射光。

染色和FLIM成像的詳細(xì)步驟可以在參考文獻(xiàn)中找到（點(diǎn)擊了解）。

圖3 . Flipper-TR在酵母細(xì)胞中的染色

Flipper-TR ® 適用于范圍廣泛的種屬：包括細(xì)菌，哺乳動物， 植物和酵母。

對Flipper-TR染色的酵母細(xì)胞進(jìn)行低滲透性choc（左）或高滲性choc（右）處理。

Flipper-TR的熒光壽命不同處理?xiàng)l件下高低變化，不同顏色表示不同的熒光壽命。

圖片由UNIGE的Roux集團(tuán)的M. Riggi提供。

Flipper -TR探針的常見問題解答® 

1. 什么是FLIM顯微鏡以及它如何用于Flipper-TR？

FLIM顯微鏡全稱熒光壽命成像顯微鏡（Fluorescent Lifetime Imaging Microscopy）。膜張力研究的重要性在于，在Flipper-TR之前，膜張力測量需要耗費(fèi)巨大的人力和昂貴的設(shè)備，但現(xiàn)在Flipper-TR卻可以簡單的適配當(dāng)前顯微鏡來實(shí)現(xiàn)高靈敏度的張力測量，其設(shè)備可從許多供應(yīng)商處獲得，它原理是基于記錄熒光團(tuán)激發(fā)后發(fā)射的時(shí)間來反映膜張力的變化，其通常非常快，大約1-10納秒（ns），在FLIM成像過程中，可將熒光壽命的差異通過時(shí)間來展示，一般來說，較短的壽命為綠色，中等壽命為黃色，較長的壽命為橙色和紅色。FLIM還可以與其他高分辨率顯微技術(shù)（如全內(nèi)反射熒光（TIRF）或受激發(fā)射耗盡（STED）顯微鏡）相結(jié)合，用于高空間分辨率檢測。

FLIM顯微鏡需要配備許多科學(xué)顯微鏡供應(yīng)商提供的時(shí)間分辨光檢測器，例如PicoQuant的升級套件，參考文獻(xiàn)1描述了有關(guān)FLIM顯微鏡實(shí)驗(yàn)裝置的更多細(xì)節(jié)。

圖4 . FLIM結(jié)構(gòu)和時(shí)間分辨彩色編碼細(xì)胞圖像示意圖。

左側(cè)：標(biāo)準(zhǔn)FLIM顯微鏡結(jié)構(gòu)圖

右側(cè)：Flipper -TR®染色的細(xì)胞 ，灰度表示熒光強(qiáng)度，不同顏色代表熒光壽命。

圖片由Colom等人提供     Flipper-TR是瑞士Spirochrome SA的注冊商標(biāo)。

2. Flipper-TR探針如何檢測熒光壽命變化？

熒光Flipper-TR®探針通過特異性靶向細(xì)胞質(zhì)膜，并通過其熒光壽命變化來反映膜張力變化。Flipper-TR®自發(fā)地插入細(xì)胞的質(zhì)膜中，并且僅在插入脂質(zhì)膜時(shí)才會激發(fā)熒光。探針通過機(jī)械載體上的兩個(gè)扭曲的二硫噻吩之間的扭轉(zhuǎn)角和極化改變來感知脂質(zhì)雙層膜結(jié)構(gòu)的變化（參見圖5）。當(dāng)處于緊張狀態(tài)時(shí)（二硫噻吩并排），發(fā)射壽命短（2-4ns），而在松弛狀態(tài)下（二硫噻吩扭曲），發(fā)射壽命則更長（4.1-8.0ns）。方差（cv）約為0.3ns（cv = 4-15％），這允許在分辨率細(xì)微變化的情況下進(jìn)行高分辨率分析，在FLIM成像過程中，可將熒光壽命的差異通過時(shí)間來展示，一般來說，較短的壽命為綠色，中等壽命為黃色，較長的壽命為橙色和紅色（見圖5）

圖5. Flipper-TR的結(jié)構(gòu)和張力檢測機(jī)制示意圖

左邊：Flipper-TR的基本分子結(jié)構(gòu)

右邊：Flipper-TR低張力時(shí)扭曲（綠色）和高張力時(shí)并列排布（紅色）

3. 這些探針的濾光片組件是什么？

Flipper-TR探針使用長分離濾光片組進(jìn)行可視化，因?yàn)槠浼ぐl(fā)峰值比發(fā)射峰值短100 nm以上。因此，理想的濾光片組是488 +/- 20nm的激發(fā)波長和575-675 +/- 40nm的發(fā)射波長（圖3）。時(shí)間分辨測量方法允許非常低的背景，它在水性環(huán)境中具有低熒光，詳情參見下面的問題4。

4. 為什么Flipper-TR探針與其他質(zhì)膜探針相比具有低背景？

Flipper-TR探針在諸如組織培養(yǎng)基或固定緩沖液等水環(huán)境中的背景非常低，因?yàn)樗?扭曲的狀態(tài)，容易形成膠束并自我淬滅（參考文獻(xiàn)3）。插入膜后，其扭曲度變小，開始發(fā)出高熒光（見圖2）。

5. Flipper-TR探頭在室溫下是否穩(wěn)定？

探針在室溫下以粉末形式穩(wěn)定幾天。在無水DMSO中溶解后后（不要使用舊的打開過的DMSO瓶子，可以使用Sigma或Spectrum Chemicals的干燥DMSO瓶子。在-20°C下冷凍和解凍是穩(wěn)定的，但不建議將其分成小份進(jìn)行儲存，因?yàn)樗谶@些條件下會降解。

6. Flipper-TR對細(xì)胞有毒嗎？

在數(shù)據(jù)表中給定的條件下操作，探針對細(xì)胞無毒的。根據(jù)細(xì)胞類型和培養(yǎng)條件，細(xì)胞一般可存活2-4天, 且熒光亮度不會有太大變化。

7. Flipper-TR探針染色哪些生物和組織？

目前所有已知的生物都可用Flipper-TR染色，包括組織培養(yǎng)細(xì)胞，活的/固定的組織切片，哺乳動物細(xì)胞，昆蟲細(xì)胞，植物細(xì)胞，酵母和細(xì)菌。

8. Flipper-TR探針是否適用于3D細(xì)胞培養(yǎng)？

探針能夠在3D培養(yǎng)條件下進(jìn)行細(xì)胞染色。

9. 膜中的量子產(chǎn)率和消光系數(shù)是多少？

乙酸乙酯中的量子產(chǎn)率= 0.30。

Flipper-TR® fluorescent cell membrane tension probe

Flipper-TR^®: A Revolutionary New Fluorescent Probe for Measuring Membrane Tension in Cells and Tissues

Flipper-TR^® is a live cell fluorescent membrane tension probe which breaks down the technical barriers hitherto circumvented by technical feats known only to biophysicists with custom equipment. The Flipper-TR membrane tension probe simplifies the methodology by using standard fluorescence lifetime measurements (see Flipper-TR FAQ for practical details). Here we describe the background of Flipper-TR in more detail.

Lipid membranes are dynamic, fluid structures (~4 nm thick) which is a biological necessity as they must change shape and tension for a cell to engage in basic cellular and subcellular physiological functions such as migration, cell spreading, phagocytosis, cell division, endocytosis, mechano-transduction, and metabolism, to name but a few. Consequently, membrane tension is under constant regulation due to its required dynamicity, and in turn, membrane tension regulates cell growth, development, motility, endocytosis, and metabolism. As the membrane is remodeled during these cellular processes, bending, tearing, and stretching of the membrane is common. These changes in membrane shape and tension occur over time and in different locations around and inside the cell and are important parameters to measure in order to understand how membrane tension is regulated and how it regulates these various basic, essential cellular processes. Understanding how membrane tension regulates cellular physiology is relevant in the study of healthy and diseased cells.

Membrane tension measurements usually relied on low resolution and slow physical methods to determine forces and tension within the plasma membrane. For example, a standard technique for measuring membrane tension involves pulling on membrane tubes from the plasma membrane with a bead trapped in an optical tweezer – a technique fraught with several methodological and technical limitations. For these reasons, novel, sensitive, reliable, and non-invasive research tools capable of rapidly measuring in vivo changes in membrane tension in real-time are in great demand. The fluorescent membrane tension probe Flipper-TR^® (Spirochrome, Ltd.) answers these challenges as it has achieved unparalleled membrane tension sensitivity and temporal resolution through the use of FLIM (fluorescence lifetime imaging microscopy) to visualize Flipper-TR^® staining of membranes in living cells. Flipper-TR^® is a live cell fluorescent membrane tension probe and the first fluorescent membrane tension reporter developed for the field of mechanobiology. The fluorescence lifetime of Flipper-TR^® is strongly dependent on the membrane tension. Using FLIM, the precise measurement of the spatio-temporal distribution of tension in membranes is now possible. Flipper-TR^® opens up a whole new field by providing a sensitive, reliable, and non-invasive means by which rapid, real-time membrane tension in live cells is measured (Figure 1).

Figure 1 - Flipper-TR sensing membrane tension in cell undergoing hyperosmotic shock.

Legend Figure 1: Flipper-TR^® staining of cells. Left side: Image of cells stained with Flipper-TR^®  before (top) and after (bottom) hyperosmotic shock. Greyscale represents fluorescence intensity, and color codes represent fluorescence lifetime. On the right the histogram shows the lifetime shift after osmotic shock. Images courtesy of Colom et al. 2018 (Ref. 1). Flipper-TR is a registered trademark of UNIGEM, Switzerland.

The fluorescent Flipper-TR^® probe works by specifically targeting the plasma membrane of cells and reports membrane tension changes through its fluorescence lifetime changes. It is the most advanced member of the Flipper probes family^2,3, which sense changes in the organization of lipid bilayer membranes through changes of the twist angle and polarization between the two twisted dithienothiophenes of the mechanophore. Flipper-TR^®spontaneously inserts into the plasma membrane of cells and is only fluorescent when inserted into a lipid membrane. It has a broad absorption and emission spectrum; excitation can be commonly performed with a 488 nm laser, while emission is collected between 575 and 625 nm (Figure 2).

Figure 2 - Absorbance and emission spectra of Flipper-TR

A

B

Legend Figure 2 - A. Schematic diagram of the mechanism of Flipper-TR and its interaction with membranes with different tension. On the left, basic molecular structure of Flipper-TR. On the right, low tension green twisted Flipper-TR and on the right high tension planar structure. B. Flipper-TR solution in ethyl acetate was subject to absornace and emission scans and the results plotted on the same graph. Absorbance is dotted orange line, and emission is a solid orange line. 

A detailed protocol for staining and FLIM imaging can be found below the References (click here, and Ref.5). Flipper-TR^® works on a wide range of organisms including bacteria, mammalians, plants, and yeast^1,4 (Figure 3).

Figure 3 - Flipper-TR staining in yeast cells

A
B

Legend for Figure 3: Flipper-TR^® staining in yeast cells. Yeast cells stained with Flipper-TR and treated with either hypo-osmotic choc (left) or hyper-osmotic choc (right). The fluorescence lifetime of Flipper-TR shifts to high or low values depending on the applied conditions. The color codes for fluorescence lifetime. Images courtesy of M. Riggi from Roux group at UNIGE (Ref. 4). Note the large pseudo-color change which is proprotional to the tension in the membrane.

Data analysis

The photon histograms from ROI or single pixels are fitted with a double-exponential, and two decay times (τ1 and τ2) are extracted. The longest lifetime with the higher fit amplitude τ1 is used to report membrane tension and varies between 2.8 and 7.0 ns. Longer lifetime means more tension in the membrane. τ2 with a smaller value (between 0.5 and 2 ns) and a small fit amplitude is less suited to study membrane tension. The lifetime can be correlated to absolute membrane tension using the calibration procedure given in Ref. 1 (Colom et al. 2018).

Figure 4 - Schematic diagram of short and long lifetime photon release.
                                                 

Legend Figure 4 - Short lifetime photons represented by the red dashes. Long lifetime photons represented by the blue dashes. Note - these data are not derived from Flipper-TR but represents the effect of two types of fluorescent lifetime decay.

References

1. Colom A et al. 2018. A fluorescent membrane tension probe. Nat. Chem. 10, 1118–1125.

2. Dal Molin M. et al. 2015. Fluorescent flippers for mechanosensitive membrane probes. J. Am. Chem. Soc. 137, 568-571.

3. Soleimanpour S. et al. 2016. Headgroup engineering in mechanosensitive membrane probes. Chem. Commun. (Camb). 52, 14450-14453.

4. Riggi M et al. 2018. Decrease in plasma membrane tension triggers PtdIns(4,5)P2 phase separation to inactivate TORC2. Nat. Cell Biol. 20, 1043–1051.

5. FLIM microscopy: Lakowicz JR et al. 1994. Emerging biomedical and advanced applications of time-resolved fluorescence spectroscopy. J Fluoresc. 4(1):117-36. doi: 10.1007/BF01876666. 

Protocol 1:  General Labeling Protocol (consult the datasheet and published papers for detailed protocols)

1. Grow cells on coverslips, glass-bottom dishes, or glass-bottom multi-well plates based on standard laboratory cell culture protocols. When cells have reached the desired density, reconstitute Flipper-TR. Optimal labeling conditions for each cell type should be empirically determined.

2. Reconstitute and prepare a 1 mM master stock solution of Flipper-TR. Store as directed on the datasheet.

3. Prepare a working solution from the 1 mM master stock for staining of the cultured cells. Start with 1 μM stain in cell culture medium. Replace the culture medium with the staining solution.

4. Return the cells to the incubator at 37°C in a humidified atmosphere containing 5% CO₂ for 15 minutes before imaging.

5. Image stained cells with standard FLIM techniques using a 485 or 488 nm pulsed laser for excitation and collecting photons through a 600/50 nm bandpass filter. Optimization of the labeling procedure and image acquisition settings is recommended so that photodamage is minimized. NOTE: Membrane tension measurements can only be performed by FLIM (fluorescence intensity or wavelength are not reliable).

Data Analysis

For extraction of lifetime information, the photon histograms from ROI or single pixels are fitted with a double-exponential, and two decay times (τ1 and τ2) are extracted. The longest lifetime with the higher fit amplitude τ1 is used to report membrane tension and varies between 2.8 and 7.0 ns. Longer lifetime means more tension in the membrane. τ2 with a smaller value (between 0.5 and 2 ns) and a small fit amplitude is less suited to study membrane tension. The lifetime can be correlated to absolute membrane tension using the calibration procedure given in reference # 1 (Colom et al. 2018).