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我们上次介绍了多功能成像是生物或医学实验室最基本的仪器设备之一，可以满足多种类型样品的成像及分析，如斑点印迹杂交，蛋白芯片，多色荧光，细胞培养皿成像，荧光抑菌圈以及动植物活体成像等。除此之外，多功能成像还可以实现更多类型样品的成像及分析吗？让我们跟着已发表的文章，看看还能实现哪些应用, 本期我们将重点介绍一下In gel荧光及微孔板荧光成像。
案例一 In-Gel Fluorescence/近红外
PAGE胶及Western blot是生命科学研究最常用的实验方法。来自德国的研究人员报道了一种基于荧光超级螯合剂探针进行快速in-gel蛋白检测的方法。该方法可直接，快速，超灵敏的in-gel检测及分析可溶性蛋白，完整的膜蛋白复合物，大分子核糖核蛋白颗粒。基于Alexa647标记，使用FUSION FX 光源Spectra Capsule 640 与滤光片F710组合进行荧光成像。
Figure1. HisQuick-PAGE for selective and ultrafast in-gel fluorescence detection and Sensitivity of fluorescent chelator probes for HisQuick-PAGE detection.
Stefan Bruchert et al. Ultrafast in-gel detection by fluorescentsuper-chelator probes with HisQuick-PAGE.Communications Biology, 2020,3:138
案例二 FRET & In-Gel Fluorescence/可见光
进行blotting实验前，胶中包含构建的FRET表达蛋白，采用mVenus-和mTurquoise2-分别标记受体和供体，mTurquoise2: 激发440nm/发射535nm, mVenus: 激发480nm/发射535nm。
Figure2. Expression of FRET test proteins was confirmed by immunodetection and in-gel fluorescence monitoring of mVenus and mTurquoise2 fluorescence.
Tatjana Kleinow, et al. Phosphorylations of the Abutilon Mosaic VirusMovement Protein Affect Its Self-Interaction, Symptom Development, Viral DANAccumulation, and Host Range.Frontiers in Plant Science. 2020,doi:10.3389/fpls.2020.01155
案例三 In-Gel多重荧光检测
除上述案例中的基于不同激发光和同一发射滤光片的FRET分析外，还可以进行多通道荧光不同组合的方式进行荧光FRET检测，从而进行多区域蛋白特定位点标记的物化分析。如下图基于Fusion Green&Red激发光及F595&F695滤光片组合进行的荧光成像。
Figure3. Physicochemical analysis of fluorescently labelled mFAS. (E) SDS-PAGE (NuPAGE Bis-Tris 4–12%) of the same three variants: 1: wild type mFAS, 2: mFAS (Gly2113AzPhe and Ser2150Ala) and 3: mFAS (Gly2113AzPhe) after enzymatic labelling with CoA-547 and clicking of the AzPhe containing variants with BCN-649. In-gel fluorescence was detected with three different settings: excitation with green light and filter F595 (for 595 nm); excitation with red light and filter F695 (695 nm) and excitation with green light and filter F695. The gel after Coomassie-staining is attached. All samples show specific fluorescent bands in the respective channels with little unspecific binding due to denaturing conditions. The doubly labelled sample Gly2113AzPhe shows FRET signal.
参考文献：
Christian S. Heil, et al. Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression. Scientific Reports. 2018, 8: 14864
案例四 微孔板&荧光成像
T6SSs是革兰氏阴性菌中广泛存在，被严密监管的蛋白传递工具，用于竞争生存。细菌竞争荧光（BaCoF）方法是依靠检测荧光信号作为T6SS敏感的细菌存活和生长的标示。图示BaCoF (细菌竞争荧光)方法学，Erm红霉素，ON 过夜。
Figure4. BaCoFscreen reveals V. parahaemolyticus mutants defective in T6SS1-mediatedbacterial killing.
Rotem Ben-Yaakovand Dor Salomon, The regulatory network of Vibrion parahaemlyticus type VIsecretion system 1. Environmental Microbiology，2019，21(7), 2248-2260
案例五 Western blot & 新冠肺炎研究
疫情是近期大家最最关心的问题之一，而Fusion多功能成像也参与了近期新冠肺炎研究以及辉瑞公司mRNA疫苗的研发。
中国科学院微生物所施一和高福研究组解析了SARS-CoV-2核心聚合酶复合物的近原子分辨率结构。由FUSION成像系统完成了SARS-CoV-2及SARS-CoV核心聚合酶复合物RNA合成活性的检测，研究表明，与SARS-CoV病毒相比，SARS-CoV-2病毒的核心聚合酶复合物的活性降低。
Figure5. In Vitro PolymeraseActivity of nsp12 and Regulatory Effects of Cofactors
Qi Peng et al. Structural and Biochemical Characterization of the nsp12-nsp7-nsp8 Core Polymerase Complex from SARS-CoV-2. Cell Reports, 2020, 31, 107774
此外，FUSION多功能成像系统还助力德国生物新技术公司BioNTech和美国辉瑞共同研制新冠肺炎mRNA疫苗BNT162b。
Figure6. Western blot ofdenatured and non-denatured samples of size exclusion chromatography fractionsof concentrated medium from HEK293T cell transfected with BNT12b1 RNA.
Annette B. Vogel, et al. BNT162b vaccines are immunogenic and protect non-human primates against SARS-CoV-2. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.11.421008
FUSION多功能成像系统，多至7通道荧光光源，覆盖400~800nm，满足市面上几乎所有常见荧光标记或荧光染料的成像需求。采用模块化的样品台设计，从而可实现多种不同类型样品的成像。满足但不限于PAGE胶，膜，微孔板，细胞培养板，培养皿，植物幼苗，植株，小鼠，大鼠......
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