四通道動(dòng)態(tài)LED陣列近紅外光譜儀
DUAL-KLAS-NIR
同步測(cè)量PSII活性（葉綠素?zé)晒猓?/span>和PSI活性（P700）
PC（質(zhì)體藍(lán)素）Fd（鐵氧還蛋白）的氧化還原變化
2016年2月Photosynthesis Research雜志發(fā)表了Schreiber博士團(tuán)隊(duì)的研究文章Deconvolution of ferredoxin, plastocyanin, and P700 transmittance changes in intact leaves with a new type of kinetic LED array spectrophotometer，隆重介紹了DUAL-KLAS-NIR四通道動(dòng)態(tài)LED陣列近紅外光譜儀。之后2016年4月，2017年3月Schreiber博士團(tuán)隊(duì)再次發(fā)表文章，進(jìn)一步闡述DUAL-KLAS-NIR的實(shí)際應(yīng)用。
作為PSI的電子供體和電子受體，PC（質(zhì)體藍(lán)素）和Fd（鐵氧還蛋白）對(duì)PSI的氧化還原起著至關(guān)重要的調(diào)控作用。但一直缺乏科學(xué)便捷的手段對(duì)其運(yùn)轉(zhuǎn)狀態(tài)進(jìn)行檢測(cè)。集成以DUALl-PAM-100為標(biāo)志的第二代PAM的基本功能，采用先進(jìn)的去卷積技術(shù)（一種根據(jù)來(lái)源不同對(duì)信號(hào)進(jìn)行分離的技術(shù)），WALZ公司推出了可以測(cè)量PC和Fd氧化還原狀態(tài)的新一代PAM熒光儀—DUAL-KLAS-NIR四通道動(dòng)態(tài)LED陣列近紅外光譜儀。
DUAL-KLAS-NIR不但集成了Dual-PAM-100的基本功能，可以同時(shí)測(cè)量PSP和PSI，而且能夠測(cè)量4組不同波段（780-820nm，820-870nm，840-965nm，870-965nm）的信號(hào)，實(shí)現(xiàn)對(duì)P700（PSI反應(yīng)中心）、PC和Fd的氧化還原狀態(tài)分別測(cè)量。另外，它還可以測(cè)量由540nm和460nm光化光激發(fā)的葉綠素?zé)晒狻＠肈UAL-KLAS-NIR四通道動(dòng)態(tài)LED陣列近紅外光譜儀，可以準(zhǔn)同步地測(cè)量各種不同的信號(hào)，不僅在馳豫動(dòng)力下，還可持續(xù)地在自然穩(wěn)態(tài)下同時(shí)獲取各組分的信息。
突出特點(diǎn)
? 可測(cè)量活體葉片或懸浮液，對(duì)P700、PC和Fd分別進(jìn)行連續(xù)的實(shí)時(shí)的去卷積分析。
? 同時(shí)測(cè)量分別由540nm（整個(gè)葉片）和460nm（表層細(xì)胞層）波段激發(fā)的兩種葉綠素?zé)晒狻?/span>
? 通過集成發(fā)光二極管技術(shù)，獨(dú)創(chuàng)高度緊湊的固態(tài)照明系統(tǒng)，提供635nm，460nm的光化光和740nm波段遠(yuǎn)紅光，以及635nm單周轉(zhuǎn)和多周轉(zhuǎn)飽和閃光。
? 擁有和DUAL-PAM-100相似的光學(xué)部件幾何結(jié)構(gòu)，可與3010-DUAL兼容，結(jié)合GFS-3000光合儀，在可控條件（光照，溫度，濕度，CO2濃度）下，同步測(cè)量氣體交換和電子傳遞相關(guān)的氧化還原。
? 測(cè)量光頻率范圍廣（1 - 400 kHz），允許連續(xù)評(píng)估Fo，可以在高時(shí)間分辨率下記錄快速動(dòng)態(tài)瞬變（如多相熒光上升動(dòng)力學(xué)或脈沖弛豫動(dòng)力學(xué)）。
主要功能
? 測(cè)定質(zhì)體藍(lán)素（PC），PS I反應(yīng)中心（P700）和鐵氧還蛋白（Fd）的氧化還原變化。
? 通過應(yīng)用創(chuàng)新的分析方法獲得PC，P700和Fd光譜特征。在線監(jiān)測(cè)P700，PC和Fd的氧化還原變化，并確定PC / P700和Fd / P700的比值。
? 可以通過綠色或藍(lán)色PAM測(cè)量光來(lái)激發(fā)熒光。綠光比藍(lán)光更深入到葉子中。因此，綠色激發(fā)的熒光包括來(lái)自更深葉層的信息，因此非常適合與整個(gè)葉子的NIR吸收測(cè)量進(jìn)行對(duì)比分析。
? 專業(yè)數(shù)據(jù)記錄軟件，入門特別簡(jiǎn)單?？墒褂肈UAL-KLAS-NIR軟件的自動(dòng)測(cè)量程序?qū)嶒?yàn)，也可以編輯腳本（Script）或者保存手動(dòng)測(cè)量程序（Trigger），輕松執(zhí)行復(fù)雜的測(cè)量協(xié)議?？勺远x測(cè)量動(dòng)作用于特殊誘導(dǎo)過程動(dòng)力學(xué)曲線數(shù)據(jù)獲取和分析。
? 兼具慢速動(dòng)力學(xué)曲線（飽和脈沖分析、誘導(dǎo)曲線和光響應(yīng)曲線）和快速動(dòng)力學(xué)曲線（飽和脈沖動(dòng)力學(xué)曲線、高達(dá)30μs分辨率的馳豫動(dòng)力學(xué)曲線）。
DUAL-KLAS-NIR軟件近紅外測(cè)量光設(shè)置 |
同步測(cè)量Fluo, P700, PC, Fd慢速誘導(dǎo)動(dòng)力學(xué)曲線 |
應(yīng)用領(lǐng)域
光合作用電子傳遞過程各復(fù)合體的氧化還原狀態(tài)深入剖析，類囊體膜蛋白組分功能研究。
可廣泛應(yīng)用于光合合成生物學(xué)研究相關(guān)的植物學(xué)，植物生理學(xué)，分子生物學(xué)，農(nóng)學(xué)，林學(xué)的領(lǐng)域。
應(yīng)用案例
DUAL-KLAS-NIR為光合作用開辟了一個(gè)全新的研究領(lǐng)域，實(shí)時(shí)顯示P700，PC和Fd在活體材料中的氧化還原狀態(tài)，在線解卷積氧化還原信號(hào)。完美實(shí)現(xiàn)PS I及其供體側(cè)和受體側(cè)氧化還原動(dòng)力學(xué)的同步測(cè)量，從而了解它們圍繞光系統(tǒng)I的復(fù)雜相互作用，另外還可以探究PS I周圍的循環(huán)電子傳遞的信息。
在DUAL-KLAS-NIR出現(xiàn)之前，測(cè)量光系統(tǒng)I的有效量子產(chǎn)量，P700信號(hào)總是會(huì)摻雜Fd的貢獻(xiàn)和PC的變量。上圖中圖C顯示了不同光強(qiáng)梯度下甘藍(lán)型油菜葉片PSI的有效PSI量子產(chǎn)量Y(I)，PSII的有效量子產(chǎn)量Y(II)和經(jīng)PSI熒光修正后的PSII的有效量子產(chǎn)率Y(II)corr。經(jīng)過修正后，Y(II)corr和Y(I)在低光強(qiáng)下相似（小于500μmol m-2 s-1）。然而，當(dāng)光強(qiáng)大于500μmol m-2 s-1時(shí)，Y(I)明顯高于Y(II)，Y(I)/Y(II)最高可達(dá)1.45.
光系統(tǒng)I的有效天線尺寸測(cè)量。植物樣品從在黑暗條件轉(zhuǎn)移到光下時(shí)，在PSI附近，首先PC被氧化，開始積累，之后才是P700被氧化。單純的PC信號(hào)變化的初始斜率可以用作PS I的有效天線尺寸的度量。
| 右圖是放大后的PC（紅色）和P700（藍(lán)色）初始吸光度變化，顯示了他們初始斜率的巨大差異。對(duì)于黑暗適應(yīng)的葉子，轉(zhuǎn)到光下的短時(shí)間內(nèi)，光系統(tǒng)I受體側(cè)未活化，F(xiàn)d還原的初始斜率也也說明了這一點(diǎn)。 |  |
DUAL-KLAS-NIR軟件設(shè)有一個(gè)窗口顯示P700和PC氧化還原狀態(tài)的相對(duì)變化。該功能可以用來(lái)計(jì)算PC和P700之間的表觀平衡常數(shù)。這對(duì)研究P700與其供體側(cè)的相互關(guān)系是非常重要的。 |  |
對(duì)暗適應(yīng)的葉子施加飽和脈沖，測(cè)量Fd氧化還原動(dòng)力學(xué)。我們不難發(fā)現(xiàn)，飽和脈沖產(chǎn)生的電子將Fd還原，飽和脈沖之后的黑暗中，F(xiàn)d被緩慢再氧化。之后，PSI的受體側(cè)的電子流被激活，再氧化動(dòng)力學(xué)變得更快。在激活PSI的受體側(cè)之后，可以通過監(jiān)測(cè)脈沖后Fd再氧化的速率來(lái)研究Fd的暗滅活。這些動(dòng)力學(xué)變化可以通過指數(shù)擬合程序擬合。圖A給出了Fd再氧化動(dòng)力學(xué)曲線指數(shù)擬合程序擬合的實(shí)例，圖B顯示了常春藤葉片不同暗適應(yīng)時(shí)間后的PSI受體側(cè)的暗滅活動(dòng)力學(xué)差異。
PC，P700和Fd的最大NIR透射率變化與這些復(fù)合物的在樣品中的含量成比例，并且PC，P700和Fd的消光系數(shù)的比率是恒定的。這可以用于探究不同物種或不同生長(zhǎng)條件下（例如陽(yáng)生/陰生，脅迫/非脅迫）樣品的PC / P700和Fd / P700比率，以及PC和Fd庫(kù)的相對(duì)大小?，F(xiàn)已觀察到高PC / P700比率與高電子傳遞速率（ETR）值相關(guān)。上圖顯示，在常春藤陽(yáng)生和陰生葉片中，相對(duì)于P700，它們PC和Fd含量有著顯著的不同。
主要測(cè)量參數(shù)：
? 葉綠素?zé)晒鉁y(cè)量：Fo, Fm, Fm’, F, Fo’, Fv/Fm, Y(II), qP, qL, qN, NPQ, Y(NO), Y(NPQ) , ETR(II)等參數(shù)，以及各種熒光動(dòng)力學(xué)曲線。
? P700測(cè)量：必須能夠測(cè)量Pm, Pm’, Y(I), ETR(I)， Y(ND)和Y(NA)等參數(shù)，以及各種P700動(dòng)力學(xué)曲線。
? PC測(cè)量：PCm, PCm’, PCox, Rel PCox
? Fd測(cè)量：Fdm, Fdm’, Fdred, Rel Fdred, Fd/PC
? 實(shí)時(shí)顯示數(shù)據(jù)采集，可以連續(xù)顯示數(shù)據(jù)采集過程即完整的動(dòng)力學(xué)曲線過程
? 軟件程序：慢速動(dòng)力學(xué)曲線，快速動(dòng)動(dòng)力學(xué)曲線，曲線擬合
產(chǎn)地：德國(guó)WALZ
代表文獻(xiàn)
數(shù)據(jù)來(lái)源：光合作用文獻(xiàn)Endnote數(shù)據(jù)庫(kù)
原始數(shù)據(jù)來(lái)源：Google Scholar
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Schreiber U: Redox changes of ferredoxin, P700, and plastocyanin measured simultaneously in intact leaves. Photosynthesis Research 134: 343–360.
https://doi.org/10.1007/s11120-017-0394-7
2016
Klughammer C, Schreiber U: Deconvolution of ferredoxin, plastocyanin, and P700 transmittance changes in intact leaves with a new type of kinetic LED array spectrophotometer.
Photosynthesis Research 128: 195–214.
https://doi.org/10.1007/s11120-016-0219-0
Schreiber U, Klughammer C: Analysis of photosystem I donor and acceptor sides with a new type of online-deconvoluting kinetic LED-array spectrophotometer. Plant and Cell Physiology 57: 1454–1467
https://doi.org/10.1093/pcp/pcw044
