作者：部分來源于菜鳥博士_懸空七少?CytoViva 超光譜納米顯微鏡-好牛耶

CytoViva 超光譜操作手冊

Scale bar 標(biāo)注方法：

尺度的定義：

10倍鏡頭下：110對應(yīng)每μm，x40倍鏡頭下，乘以4為440，。。。

定義所需要的尺度：如1μm 或XX

其他資料:

我校公共實驗平臺Cytoviva超光譜納米顯微鏡的使用培訓(xùn)通知sysysbglc.cpu.edu.cn/3a/4d/c9704a145997/page.htm

Cytoviva：CytoViva納米高光譜顯微成像系統(tǒng)應(yīng)用介紹-標(biāo)記植物組織中的納米材料

Label-Free Liposome & ExosomeHyperspectral Microscopy

納米尺度囊泡(如脂質(zhì)體和外泌體)作為藥物傳遞載體的研究取得了顯著進(jìn)展，使其在FDA的臨床試驗數(shù)量不斷增加。大多數(shù)早期臨床試驗工作都集中在攜帶藥物或基因治療負(fù)載的工程脂質(zhì)體上。然而，天然外泌體載體正在迅速取得進(jìn)展。

Research of nano-scale vesicles such as liposomes and exosomes as drug delivery vectors has progressed significantly leading to increasing numbers of FDA clinical trials. Most of these early clinical trial efforts have focused on engineered liposomes carrying a drug or gene therapy load. However, rapid progress is now being made with natural exosomal vectors.

關(guān)鍵挑戰(zhàn)仍然是了解這些納米級載體靶向腫瘤細(xì)胞的功效，這通常通過內(nèi)吞事件或直接細(xì)胞膜融合發(fā)生，最終將藥物釋放到細(xì)胞中。由于囊泡的熒光標(biāo)記會干擾其任務(wù)，因此需要一種無標(biāo)記的體外成像方法來確認(rèn)各種囊泡-藥物負(fù)載組合的這種功效。成像方法還應(yīng)有助于確定囊泡樣品中的適當(dāng)藥物負(fù)荷，并確認(rèn)其與活細(xì)胞的相互作用和攝取。

Key challenges remain in understanding the efficacy of tumor cell targeting with these nanoscale carriers, which most often happens through endocytic events or direct cell membrane fusion for eventual release of the drugs to the cell. Since fluorescent labeling of the vesicle can interfere with its task, a label-free, in-vitro imaging method is required to confirm this efficacy with a variety of vesicle-drug load combinations. The imaging method should also help

determine proper drug load across the vesicle sample and confirm its interaction and uptake with live cells.

CytoViva 的增強(qiáng)型暗場高光譜顯微鏡在幫助研究人員了解囊泡載體靶向細(xì)胞以及裝載藥物的定時釋放方面非常有效。使用這種無標(biāo)簽成像技術(shù)，可以：

CytoViva's Enhanced Darkfield Hyperspectral Microscope is proving to be highly effective in helping researchers understand both the targeting of vesicle carriers to cells as well as the timed release of their drug cargo. With this label free imaging technique it is possible to:

? 在體外和溶液中成像<100 nm 的無標(biāo)記外泌體或脂質(zhì)體

? 區(qū)分具有不同有效載荷的囊泡和空囊泡

? 使用光譜圖對囊泡藥物構(gòu)建體進(jìn)行無標(biāo)記細(xì)胞運輸，以識別與活細(xì)胞或固定細(xì)胞的相互作用

? Image label-free exosomes and liposomes < 100 nm in-vitro and in solution? Distinguish vesicles with differing payloads as well as empty vesicles? Conduct label-free cell trafficking of the vesicle-drug construct with spectral mapping to identify interaction with live or fixed cells

上面顯示了用于此應(yīng)用的 CytoViva 系統(tǒng)的具體示例。在此示例中，將前列腺癌特異性肽添加到負(fù)載多柔比星的脂質(zhì)體中。借助 CytoViva 增強(qiáng)型暗場高光譜顯微鏡系統(tǒng)，研究人員能夠創(chuàng)建負(fù)載阿霉素的脂質(zhì)體（圖 2）獨有的參考光譜庫（圖 1）。請注意，圖 1 中所示的參考光譜在 575nm 處有一個非常明顯的峰，這與阿霉素的光譜特性一致。圖 3 顯示了已與脂質(zhì)體構(gòu)建體一起孵育的無標(biāo)記腫瘤細(xì)胞。圖 4 中的紅色像素展示了參考光譜的光譜映射，確認(rèn)了脂質(zhì)體構(gòu)建體在腫瘤細(xì)胞內(nèi)的存在和位置。

A specific example of the CytoViva system being used for this application is shown above. In this example, a prostate cancer-specific peptide is added to a doxorubicin-loaded liposome. With the CytoViva Enhanced Darkfield Hyperspectral Microscope system, the researcher is able to create a reference spectral library (figure 1) unique to the doxorubicin-loaded liposome (figure 2). Note that the reference spectra illustrated in figure 1 has a very distinct peak at ~575nm that is consistent with the spectral properties of doxorubicin. Figure 3 shows a label-free tumor cell that has been incubated with the liposome construct. The red pixels in figure 4 demonstrate the spectral mapping of the reference spectrum confirming the presence and location of the liposomal construct within the tumor cell.

CytoViva 系統(tǒng)的一個主要優(yōu)點是它不需要熒光標(biāo)記或改變細(xì)胞及脂質(zhì)體的結(jié)構(gòu)即可有效的成像和分析。此外，這些樣本可以使用 SynVivo for CytoViva 活細(xì)胞微流控系統(tǒng)進(jìn)行活細(xì)胞成像。使用該系統(tǒng)，納米藥物遞送結(jié)構(gòu)可以添加到活細(xì)胞培養(yǎng)物中，并且可以隨著時間的推移捕獲圖像，記錄動態(tài)相互作用。該系統(tǒng)還可用作“模擬體內(nèi)”系統(tǒng)，用于觀察生物環(huán)境中的納米粒子命運。

A key benefit of the CytoViva system is that it does not require fluorescent labeling or other alteration of the cell structure or the liposomes for effective imaging and analysis. Additionally, these samples can be imaged as live cells using the SynVivo for CytoViva live-cell microfluidics chamber. With this system, the nano-drug delivery construct can be added to the live cell culture and images can be captured over time, recording the dynamic interaction. This system can also be used as a "simulated in-vivo" system for observing nanoparticle fate in the biological environment.

要了解高光譜顯微鏡如何促進(jìn)您的納米顆粒研究工作，請通過china@cytoviva.com聯(lián)系我們，或關(guān)注公眾號Cytoviva思拓唯沃科學(xué)技術(shù)。私信我，我們在全球納米研究實驗室提供納米高光譜顯微成像技術(shù)方面擁有十五年的經(jīng)驗。我們將很高興地討論您的研究，并安排在我們的實驗室或現(xiàn)有客戶實驗平臺對您的樣品進(jìn)行圖像測試。

納米顯微鏡其他筆記：

納米顯微鏡描述：

Dark field optical images were obtained with a CytoViva? ultra resolution imaging system, composed by a special arrangement mounted on an Olympus BX51 microscope.

HSI measurements were performed using Cytoviva hyperspectral imaging system equipped with Cytoviva? high resolution dark field condenser (oil immersion).

The CytoViva (CytoViva Inc., Auburn, AL, USA) technology in combination with dark field microscopy was used to image GNP distribution within cells. 應(yīng)用Cytoviva(Cytoviva Inc.，Auburn，AL，USA)技術(shù)與暗場顯微鏡技術(shù)相結(jié)合，對細(xì)胞內(nèi)國產(chǎn)總值的分布進(jìn)行了觀察。

(CytoViva, Inc., Auburn, AL).

The CytoViva hyperspectral imaging microscopy system (CytoViva, Inc., Auburn, AL, USA) mounted on an Olympus BX43 darkfield microscope (Olympus Corporation, Tokyo, Japan) was used to analyze the internalization of the different gold NPs by BEAS-2B cells.

CytoViva高光譜成像顯微鏡系統(tǒng)(CytoViva，Inc.，阿本，美國)安裝在奧林巴斯BX43暗場顯微鏡（奧林巴斯公司，東京，日本）上，分析BEAS-2B細(xì)胞對不同金NPs的內(nèi)化。