超高分辨活细胞荧光红外显微成像系统

超高分辨活细胞荧光红外显微成像系统

荧光作为生物学特异性识别的主要手段,一直以来在生命科学中发挥着重要作用。但是这需要被分析的物质具有荧光或者可以被荧光所标记。

振动光谱(IR & Raman)是成熟无标记的技术,能够直接提供物质本身的结构信息,能够为生命科学提供广泛的大分子、药物、材料、脂质体等无标记物质的表征能力,在生命科学研究中具备重大潜力。

具有亚微米和同步拉曼能力的O-PTIR克服了传统红外显微镜分辨率不足和在不平整表面米氏散射严重的问题,使得这种广泛的大分子表征现在可以在<500 nm的生物相关空间尺度上进行,实现红外与拉曼和荧光成像分辨率相匹配,具备真正意义上的共定位能力。 现在,mIRage-LS将这些技术完全集成到一个系统上,仅需一台设备即可实现样品的全方位红外、拉曼、荧光信号分析,获得任意一种单一技术本身都无法获得的额外信息和见解。

产品特点:


☆  荧光红外共定位成像分析

☆  亚微米尺度红外拉曼分辨率

☆  红外拉曼同步测量

☆  非接触式测量,同时支持透射、反射模式并且无米氏散射问题

☆  可测试活细胞(液体环境)


优势领域:


单细胞分析:

☆  正常/患病细胞分化

☆  药物-细胞相互作用

☆  细胞内(脂滴) 成像研究


组织分析:

☆  细胞分型

☆  钙化、疾病状态区分

☆  胶原蛋白取向


细菌观测:

☆  单细菌鉴定

☆  细菌代谢研究



光学光热红外O-PTIR在生命科学领域应用的显著优势:


☆  亚微米级的空间分辨率;

☆  可直接获取液体中活细胞的红外成像;

☆  灵敏度高,可直接观测单细胞 (如细菌、哺乳动物细胞等);

☆  无米氏散射干扰,即使在细胞边缘也不受影响;

☆  超高光谱分辨率;

☆  无需直接接触即可测量软组织的红外光谱;

☆  可实现红外和拉曼同步测量;

☆  可实现超过10 μm厚的样品测试,直接置于载玻片上观察分析;

☆  可配置极化的红外光源


超分辨红外技术O-PTIR

理想空间分辨率横向对比 (FTIR, QCL and O-PTIR microscopes)


专为生物样本设计的新型双区(C-H/FP)” QCL

新型“双区(C-H/FP)”QCL能够在在一台设备中同时涵盖了C-H拉伸和指纹区 (3000-2700、1800-950cm-1) 反射模式下收集的O-PTIR光谱在数据库(Wiley KnowItAll)搜索结果,匹配率超过95%。


1. 荧光成像与O-PTIR联合表征


荧光成像对于分子生物学机制的研究具有十分重要的意义,而传统红外很难原位测量细胞的红外图谱,因此无法将蛋白定位与原位细胞的红外图谱进行原位叠合,这对于红外在生物学的机制研究中的应用十分不利。而O-PTIR能够直接在不损伤细胞的情况下测量不同区域的红外图谱,与荧光图像相结合探究蛋白结构与分布上的变化。

 

图1. 阿尔兹海默症脑组织切片样品,左侧白光图,中间荧光图,右侧O-PTIR在中图中的红色与蓝色区域的采集的红外图谱

 

2. 感染疟原虫的红细胞表征


疟原虫属寄生虫引起的疟疾是威胁生命的主要疾病之一,而疟原虫引发的感染周期十分复杂,因此在细胞和分子水平观察疟原虫的变化对于研究疟原虫的致病有着重要意义。Agnieszka M. Banas等人通过使用O-PTIR对疟原虫感染的红细胞在亚微米尺度的分子特征变化进行了表征,结果显示正常红细胞的蛋白呈现环状分布,而感染后的红细胞蛋白质则呈现无规则分布。通过对比传统FTIR与基于O-PTIR技术能够发现,O-PTIR能够提供更为详细的图像分辨率并且能够测量红细胞不同位置的光谱信息。而传统FTIR受制于米氏散射限制,效果较差。

图2. 对比FTIR与O-PTIR对红细胞成像的结果:(a)红细胞的白光图;(b)图a中红色方块放大的区域;(c,e)FTIR的蛋白/脂质空间分布的红外成像;(d,f)O-PTIR的蛋白/脂质空间分布的红外成像;(g)红细胞的FTIR红外光谱;(h)红细胞的O-PTIR红外光谱;(g,i)疟原虫感染红细胞和正常红细胞的PCA(PC1&PC2,PC1&PC3)得分;(h,j)疟原虫感染红细胞和正常红细胞的PCA(PC1&PC2,PC1&PC3)得分

 

参考文献:B. [Malaria] “Comparing infrared spectroscopic methods for the characterization of Plasmodium falciparum-infected human erythrocytes” (Nature Communication Chemistry, https://doi.org/10.1038/s42004-021-00567-2). Advantages: 1, 3, 4, 5, 6

 

3. 单个病毒红外成像


受制于红外极限分辨率的限制,单个病毒的红外光谱成像一直以来都是十分困难的,对于只有100 nm左右的病毒进行红外光谱成像显得十分无力。Yi Zhang等人使用O-PTIR技术成功实现对单个痘病毒进行了检测,并成功观测到了病毒的外形,同时对病毒表面的蛋白的光谱进行了表征。

图3. 单个痘病毒的光谱和成像表征。(a)痘病毒的干涉散射图像;(b)痘病毒1550cm-1波数下的MIP图像;(c)痘病毒1650cm-1波数下的MIP图像;(d)随机选取病毒上4个点的光谱

 

参考文献:“Vibrational Spectroscopic Detection of a Single Virus by Mid-Infrared Photothermal Microscopy” (Analytical Chemistry,  https://dx.doi.org/10.1021/acs.analchem.0c05333). Advantages: 1, 3, 4, 5, 6

 

4. 光学光热红外O-PTIR与Raman光谱协同分析固定或活的单细胞


英国曼彻斯特大学的Peter Gardner教授近期发表了他们关于活(和固定)细胞振动光谱分析的研究结果。作者使用光学光热红外O-PTIR与Raman光谱,并借助于两个激发源(QCL和OPO激光器),对细胞进行了宽光谱范围的覆盖,从而使所有与生物学相关的分子振动都能被检测到,且保持一致的亚微米的空间分辨率。此外,红外光谱采集与拉曼光谱有效的结合起来,在相同的激发位置,形成振动互补,得到一套完整的振动光谱信息。如下图所示,该红外和拉曼的组合方式可以用来分析液体环境中固定或活细胞的亚细胞结构,其中的蛋白质二次结构及富脂体均可以在亚微米尺度上被有效地识别出来。

图4. O-PTIR观测固定未染色MIA PaCa-2细胞成像。(a)固定的未染色的MIA PaCa-2细胞的光学图像;(b)红色方块区域的放大图像;(c)OPO波束段的O-PTIR红外光谱;(d)QCL波束段O-PTIR的红外光谱;(e)黑色区域的拉曼和红外光谱

 

参考文献:D. [Mammalian cancer cell] “Analysis of Fixed and Live Single Cells Using Optical Photothermal Infrared with Concomitant Raman Spectroscopy” (Analytical Chemistry, https://dx.doi.org/10.1021/acs.analchem.0c04846). Advantages: 1, 2, 3, 4, 5, 6, 7

 

5. O-PTIR与S-XRF联用探究阿尔兹海默症


阿尔兹海默症(AD)是老年痴呆症常见的病症之一,而淀粉样β蛋白沉淀是引发AD的重要病因之一,因此对于淀粉样β蛋白分布的研究就显得十分重要。Nadja Gustavsson等人通过O-PTIR成功观测到了神经中的淀粉样β蛋白分布,并且结合S-XRF分析发现铁簇与淀粉样β-折叠结构和氧化的脂质存在共定位关系。这项研究充分预示了O-PTIR/S-XRF联合技术可在AD疾病的研究中发挥重要作用。

图5. 单个神经元的O-PTIR与X光荧光成像。(a)单个神经元的光学(左)与O-PTIR图像(中和右);(b)神经元上铜、铁的分布;(c)铁与蛋白叠合图;(d)铁与脂质的叠合图


单细胞分析:

☆  正常/患病细胞分化

☆  药物-细胞相互作用

☆  细胞内(脂滴) 成像研究

 

细胞内的荧光+红外共定位分析

                                               

利用荧光同时观测细胞结构和细胞中的脂滴分布,研究脂滴在细胞中的共定位分析,提供潜在活体无标记相互作用分析数据。

磷脂成像 (2856cm-1 (CH2) / 2874cm-1 (CH3) 100 nm pixel size. ~5 mins. 荧光染色细胞核(蓝色),蛋白(红色))

 

活体细胞的组分分布分析

磷脂成像,可观测活细胞内的脂滴的分布并且基本不会受到水的干扰,这是传统红外所难以达到的。 (2856cm-1 (CH2)/ 2874cm-1 (CH3) 100 nm pixel size. ~5 mins.)

 

固定细胞的组分分布分析

磷脂成像没可观测到细胞内的脂滴分布情况。 (2856cm-1 (CH2)/ 2874cm-1 (CH3100 nm pixel size. ~5 mins.)

 

组织分析:

☆  细胞分型

☆  钙化、疾病状态区分

☆  胶原蛋白取向

 

组织切片分析观测

肿瘤组织钙化分析1050cm-1,传统的FTIR只有大约12微米的空间分辨率,这往往比实际特征大得多,这就是为什么以前没有看到如此小的局部钙化。


细菌观测:

☆  单细菌鉴定

☆  细菌代谢研究


红外拉曼联合细菌表征,可以同时观测到细菌的红外和拉曼图谱

发表文章应用领域
Optical photothermal infrared spectroscopy for nanochemical analysis of pharmaceutical dry powder aerosols. Khanal, D. et al.International Journal of Pharmaceutics, 2023Pharmaceuticals
Fluorescently Guided Optical Photothermal Infrared Microspectroscopy for Protein-Specific Bioimaging at Subcellular Level. Prater, C et al.Journal of Medicinal Chemistry, 2023Life Science
Innovative Vibrational Spectroscopy Research for Forensic Application. Weberm A. et al.Analytical Chemistry, 2023Forensic
High-Throughput Antimicrobial Susceptibility Testing of Escherichia coli by Wide-Field Mid-Infrared Photothermal Imaging of Protein Synthesis. Guo, Z. et al.Analytical Chemistry, 2023Life Science
Prebiotic-Based Nanoamorphous Atorvastatin Attenuates Nonalcoholic Fatty Liver Disease by Retrieving Gut and Liver Health. Cui, J, et al.Small Structures, 2023Life Science
Optical photothermal infrared spectroscopy: A novel solution for rapid identification of antimicrobial resistance at the single-cell level via deuterium isotope labeling. Shams, S. et al.Front. Microbiol., 2023Life Science
Mapping ancient sedimentary organic matter molecular structure at nanoscales using optical photothermal infrared spectroscopy. Jubb, A. et al.Organic Geochemistry, 2023Paleontology
A review on analytical performance of micro- and nanoplastics analysis methods. Thaiba, B.M. et al.Arabian Journal of Chemistry, 2023Microplastics
Video-rate Mid-infrared Photothermal Imaging by Single Pulse Photothermal Detection per Pixel. Xin, J. et al.bioRxiv, 2023Life Science
Microfluidics as a Ray of Hope for Microplastic Pollution. Ece, E. et al.biosensors, 2023Microplastics
Critical assessment of approach towards estimation of microplastics in environmental matrices. Raj, D. et al.Land Degradationa and Development, 2023Microplastics
Development of a Binary Digestion System for Extraction Microplastics in Fish and Detection Method by Optical Photothermal Infrared. Yan, F. et al.Frontiers in Marine Science, 2022Microplastics
Automated analysis of microplastics based on vibrational spectroscopy: are we measuring the same metrics?. Dong, M. et al.Analytical and Bioanalytical Chemistry, 2022Microplastics
Vitamin D and Calcium Supplementation Accelerate Vascular Calcification in a Model of Pseudoxanthoma Elasticum. Bouderlique, E. et al.International Journal of   Molecular Sciences, 2022Pharmaceuticals
Polarization Sensitive Photothermal Mid-Infrared Spectroscopic Imaging of Human Bone Marrow Tissue. Mankar, R. et al.Applied Spectroscopy, 2022Biomedical and life science
Identification of spectral features differentiating fungal strains in infrared absorption spectroscopic images. Stancevic, D. et al.Lund Univ, Ugrad Thesis, 2022Bio and environmental
Optical photothermal infrared spectroscopy can differentiate equine osteoarthritic plasma extracellular vesicles from healthy controls. Clarke, E. et al.BioXvid, 2022BioXvid
Correlative imaging to resolve molecular structures in individual cells: substrate validation study for super-resolution infrared microspectroscopy. Paulus, A.  et al.Nanomedicine: Nanotechnology, Biology, and Medicine, 2022Biomedical and life science
Leveraging high-resolution spatial features in mid-infrared spectroscopic imaging to classify tissue subtypes in ovarian cancer. Gajjela, C. et al.BioarXiv, 2022Biomedical and life science
APPLICATION OF OPTICAL PHOTOTHERMAL INFRARED (O-PTIR) SPECTROSCOPY TO ASSESS BONE COMPOSITION AT THE SUBMICRON SCALE. Reiner, E. et al.Temple Univ, Master thesis, 2022Biomedical and life science
Matrix/Mineral Ratio and Domain Size Variation with Bone Tissue Age: a Photothermal Infrared Study. Ahn, T. et al.Journal of Structural Biology, 2022Journal of Structural Biology
Simultaneous Raman and infrared spectroscopy: a novel combination for studying bacterial infections at the single cell level. Lime, C. et al.Chemical Science, 2022Biomedical and life science
Phase separation in surfactant-containing amorphous solid dispersions: Orthogonal analytical methods to probe the effects of surfactants on morphology and phase composition. Yang, R. et al.International Journal of Pharmaceutics, 2022Pharmaceuticals
Synovial joint cavitation initiates with microcavities in interzone and is coupled to skeletal flexion and elongation in developing mouse embryo limbs. Kim, M. et al.Biology Open, 2022Biomedical and life science
Steam disinfection enhances bioaccessibility of metallic nanoparticles in   nano-enabled silicone-rubber baby bottle teats, pacifiers, and teethers. Su, Y. et al.Journal of Environmental Science, 2022Microplastics
NOVEL SPECTROSCOPY TECHNIQUES USED TO INTERROGATE EQUINE OSTEOARTHRITIC EXTRACELLULAR VESICLES. Clarke, E. et al.Osteoarthritis and Cartilage, 2022Biomedical and life science
Using   mid infrared to perform investigations beyond the diffraction limits of microcristalline pathologies: advantages and limitation of Optical PhotoThermal IR spectroscopy. Bazin, D. et al.Comptes Rendus. Chimie, 2022Biomedical and life science
Optical   photothermal infrared spectroscopy can differentiate equine osteoarthritic plasma extracellular vesicles from healthy controls. Clarke, E. et al.Analytical Methods, 2022Biomedical and life science
Probing Individual Particles Generated at the Freshwater–Seawater Interface through Combined Raman, Photothermal Infrared, and X-ray Spectroscopic Characterization. Mirrielees, J. et al.ACS Meas. Sci. Au, 2022Environmental and Microplastics
Parts-per-Million Detection of Trace Crystal Forms Using AF-PTIR Microscopy. Razumtcev, A. et al.Analytical Chemistry, 2022Pharmaceuticals
Ultrafast   Widefield Mid-Infrared Photothermal Heterodyne Imaging. Paiva, E. et al.Analytical Chemistry, 2022Photonics, bio
Chapter 8 - Raman-integrated optical photothermal infrared microscopy: technology and applications. Li, X. et al.Molecular and Laser Spectroscopy, 2022Photonics, bio
Chapter 9 - Optical photothermal infrared spectroscopic applications in microplastics—comparison with Fourier transform infrared and Raman   spectroscopy. Krafft, C. et al.Molecular and Laser Spectroscopy, 2022Microplastics
Contribution of Infrared Spectroscopy to the Understanding of Amyloid Protein Aggregation in Complex Systems. Ami, D. et al.Front. Mol. Biosci., 2022Bio and life science review
Novel Submicron Spatial Resolution Infrared Microspectroscopy for Failure Analysis of Semiconductor Components. Zulkifli, S. et al.IPFA 2022 Proceedings, 2022FA/contamination
Overcoming challenging Failure Analysis sample types on a single IR/Raman platform. Anderson, J. et al.ISTFA 2022 Proceedings, 2022FA/contamination
Optical photothermal infrared spectroscopy with simultaneously acquired Raman spectroscopy for two-dimensional microplastic identification. Boeke, J. et   al.Scientific Report, 2022Microplastics
Super-resolution infrared microspectroscopy reveals heterogeneous distribution of photosensitive lipids in human hair medulla. Sandt, C. et al.Talanta, 2022Life science, hair



BTV专访:非接触式亚微米分辨红外拉曼同步测量新技术如何解决微塑料监测难题


mIRage Demo演示-Microplastics


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