微型模式動(dòng)物、模式植物、大體積細(xì)胞等在生命科學(xué)研究中有著非常廣泛的應(yīng)用但是由于這些對(duì)象體積太大普通的流式細(xì)胞儀的粒徑尺寸一般在幾十微米難以對(duì)秀麗線蟲、斑馬魚這樣的大粒徑模式生物進(jìn)行分析和分選；手工鏡下分選非常耗時(shí)耗力、效率低下、準(zhǔn)確性難以保證。大粒徑流式分選系統(tǒng)是一款特殊設(shè)計(jì)的分選型流式細(xì)胞儀流通室徑向尺寸可達(dá)2000微米可以對(duì)秀麗線蟲、蠶卵、斑馬魚卵及幼魚、果蠅等模式生物進(jìn)行高通量、自動(dòng)化的分析和分選。

大粒徑流式分選系統(tǒng)能夠很大程度上提高了分選效率完成大規(guī)模的樣品分選工作實(shí)現(xiàn)高通量分選。還可對(duì)多種數(shù)據(jù)進(jìn)行分析可以開展用傳統(tǒng)實(shí)驗(yàn)手段難以開展的課題實(shí)驗(yàn)。

大粒徑流式分選系統(tǒng)在國(guó)際上有數(shù)百家實(shí)驗(yàn)室的成功應(yīng)用案例在很多期刊也有很多引用文獻(xiàn)。該設(shè)備的應(yīng)用能夠幫助實(shí)驗(yàn)室在很大程度上提高實(shí)驗(yàn)效率、拓展科研方法、縮短科研產(chǎn)出時(shí)間。 

產(chǎn)品型號(hào)BioSorter

主要特點(diǎn)

• ? 多種規(guī)格的流動(dòng)室可選分選粒徑20-1500um的模式生物、細(xì)胞群、大粒徑生物微粒、微珠

• ? 高通量分選平臺(tái)可集成2-3個(gè)流動(dòng)室單元具備更寬泛的粒徑尺寸分選范圍兼顧多個(gè)不同的模式生物和研究方向

• ? 全自動(dòng)、快速、精確分選和分析
• ? 提供分選顆粒數(shù)字化定量數(shù)據(jù)可用于對(duì)目標(biāo)群體的統(tǒng)計(jì)分析
• ? 無菌環(huán)境及溫和氣流分選方式能夠有效保持分選樣品的潔凈度和存活率

主要功能
? 高通量分選能夠很大程度上提高分選效率同時(shí)可以開展用傳統(tǒng)試驗(yàn)方法難以開展的實(shí)驗(yàn)
? 設(shè)備通過檢測(cè)樣品的多種光學(xué)信號(hào)依據(jù)檢測(cè)到的信號(hào)進(jìn)行自動(dòng)化分選排除了人為主觀因素分選更客觀更準(zhǔn)確
? 設(shè)備可以把生物樣本自動(dòng)分選到244896或384孔板中也可以自定義實(shí)現(xiàn)分選的自動(dòng)化
? 大粒徑流式分選系統(tǒng)是在常規(guī)分選型流式細(xì)胞儀基礎(chǔ)上研發(fā)和改進(jìn)而成流動(dòng)室內(nèi)壓力小采用溫和的氣流分選方式可以確保生物體的活性和完整性對(duì)后續(xù)培養(yǎng)、分析甚至是再次分選不會(huì)產(chǎn)生負(fù)面影響
? 系統(tǒng)自動(dòng)存儲(chǔ)每個(gè)模式生物、微粒的光學(xué)數(shù)據(jù)可用于進(jìn)一步的統(tǒng)計(jì)和分析

? 可根據(jù)需要選配專業(yè)化分析軟件能夠在樣本徑向長(zhǎng)度方向上進(jìn)行多達(dá)8000個(gè)斷層的光學(xué)監(jiān)測(cè)給出細(xì)胞-器官水平的光學(xué)參數(shù)信息包括尺寸、光密度和熒光強(qiáng)度（例如斑馬魚心臟的神經(jīng)元上的熒光信號(hào)）并以峰值曲線圖的形式導(dǎo)出可提供峰的個(gè)數(shù)、峰寬、峰高和相對(duì)位置等各種參數(shù)

? 儀器操作簡(jiǎn)單一個(gè)人便可完成大規(guī)模的樣品分選工作實(shí)現(xiàn)高通量分選

主要應(yīng)用

? 種群差異分析（Population differences analysis）

 ·成長(zhǎng)階段分選

 ·藥物篩選、化學(xué)誘導(dǎo)
? 熒光表達(dá)篩選（Fluorescence expression sorting）

 ·單熒光篩選

 ·多熒光篩選
? 基因表達(dá)定位分析（Gene expression localization analysis）

 ·熒光峰值篩選

 ·樣品亞群分析
? 熒光定量分析（Fluorescent quantitative analysis）

工作原理及主要配置

工作原理

通過獲取多種光學(xué)參數(shù)徑向尺寸、光密度、三色熒光強(qiáng)度對(duì)微粒進(jìn)行分選

分選方式

采用溫和的氣流分選方式能夠很大程度上保障分選后生物微粒的生物活性和完整性（可對(duì)同一個(gè)樣品多次分選）

光學(xué)部件布局圖

COPAS高通量分選系統(tǒng)的光學(xué)原件布局俯視圖

流動(dòng)室

可集成2-3個(gè)流動(dòng)室單元具備更寬泛的粒徑尺寸分選范圍；

設(shè)備可作為一個(gè)多用途的分選平臺(tái)兼顧多個(gè)不同的模式生物和研究方向；

能夠根據(jù)需要在多個(gè)流動(dòng)室單元之間快速、靈活切換；

激光器

配備488nm激光器用于測(cè)量物體的軸向長(zhǎng)度和光學(xué)密度還可以用來激發(fā)熒光

根據(jù)研究需要選擇合適的激光器可調(diào)整激光器的配置多種型號(hào)可選375nm405nm488nm561nm或640nm

分選參數(shù)

通過檢測(cè)和分析生物微粒的多種參數(shù)進(jìn)行分選

飛行時(shí)間（TOF）、透光度（EXT）、熒光強(qiáng)度（FLU） 同時(shí)檢測(cè)多達(dá)三種顏色的熒光

基礎(chǔ)分析軟件

分選參數(shù)設(shè)置、分選控制與數(shù)據(jù)分析

Profile高階分析軟件

具備多種參數(shù)峰值特性分選功能

生物微粒通過流動(dòng)室的過程中系統(tǒng)對(duì)生物微粒軸向方向的光密度和熒光強(qiáng)度進(jìn)行量化；

下圖是使用Profile軟件Profiling GFP-expressing seam cells in nematodes（牛津大學(xué)伍拉德實(shí)驗(yàn)室）

LP Sampler自動(dòng)進(jìn)樣器

能夠?qū)崿F(xiàn)對(duì)多孔板樣品的自動(dòng)化加樣；

完整的高通量分選平臺(tái)

BioSorter大粒徑流式分選系統(tǒng)能可配合LP Sampler組成一套更加完整的高通量分選平臺(tái)；

能夠?qū)崿F(xiàn)從多孔板進(jìn)樣到多孔板分選的全自動(dòng)化操作；

主要技術(shù)參數(shù)

· 用于做所有發(fā)育階段的秀麗線蟲、10-150um的細(xì)胞與細(xì)胞集群、海膽胚胎、生物微珠、花粉、腎導(dǎo)管細(xì)胞等

· 可自動(dòng)檢測(cè)流經(jīng)系統(tǒng)的每個(gè)樣品的各種檢測(cè)參數(shù)并記錄與統(tǒng)計(jì)

· 流通室內(nèi)徑尺寸250um

· 可以檢測(cè)多種參數(shù)飛行時(shí)間（TOF）、透光度（EXT）、熒光強(qiáng)度（FLU） 同時(shí)檢測(cè)多達(dá)三種顏色的熒光

· 包含系列高精度的檢測(cè)元件如光電二極管和光電倍增管、系列光柵可同時(shí)檢測(cè)綠色黃色和紅色熒光

· 配備488nm固體激光器用于測(cè)量物體的軸向長(zhǎng)度和光學(xué)密度還可以用來激發(fā)熒光

· 熒光檢測(cè)靈敏度:FITC<100MESF

· 全峰寬變異系數(shù):CV<2%

· 濾光片熒光通道濾光片包括530/30nm、585/42nm、670nm LP

· 樣品檢測(cè)處理速度1000個(gè)樣品/秒；分選時(shí)樣品處理速度為50個(gè)樣品/秒

· 自動(dòng)分選精度＞95%

· 樣品接收器可選用24孔板48孔板96孔板

· 每個(gè)孔板樣品數(shù)量可自由定義最多每個(gè)孔可加載570個(gè)樣品（標(biāo)準(zhǔn)96孔板）

· 具有自動(dòng)化的X-Y移動(dòng)臺(tái)可放置微孔板或其它收集器自動(dòng)加載定位

· 最小分選體積0.4ul

· 采用通用型樣品杯容量為50ml

· 具有內(nèi)置攪拌混勻功能通過軟件設(shè)置混勻參數(shù)

· 采用的氣流分選機(jī)制減少分選對(duì)樣品的影響分選過程對(duì)樣品的生物活性無不良影響

· 具有樣品回收功能用于收集沒被分選的樣本使樣本可以重復(fù)使用

· 可設(shè)置分選樣品時(shí)的液滴長(zhǎng)度和延遲時(shí)間

· 具有大容量鞘液容器容量4L可支持系統(tǒng)連續(xù)運(yùn)行6小時(shí)

· 具備壓力實(shí)時(shí)監(jiān)測(cè)功能可實(shí)時(shí)測(cè)量鞘液壓力、樣品壓力、分選壓力、清洗壓力

· 具備熒光補(bǔ)償功能高精度自動(dòng)補(bǔ)償裝置在線或脫機(jī)補(bǔ)償

· 配備計(jì)算機(jī)工作站并預(yù)裝操作系統(tǒng)和分析軟件

· 基于PC機(jī)和WINDOWS操作平臺(tái)的軟件系統(tǒng)具備實(shí)時(shí)分析功能

· 數(shù)據(jù)可追溯系統(tǒng)自動(dòng)保存每個(gè)樣品的檢測(cè)數(shù)據(jù)可保存為CSV格式可用于后期分析

· 自動(dòng)統(tǒng)計(jì)分選樣品占總樣品的比例并實(shí)時(shí)自動(dòng)顯示

The BioSorter instrument is designed for laboratories where a single instrument needs to address the requirements of multiple researchers and sample types; hence it is ideal for departmental shared equipment (core laboratory) facilities.

The BioSorter® is a continuous flow system capable of analyzing, sorting and dispensing objects ranging in size from 10 to 1,500 µm. Relative axial size is measured by an axial light-loss detector and optical density is determined by the total integrated signal of the light blocked.The fluorescence intensity can be simultaneously measured at three different wavelengths as determined by the excitation and emission filters in the system.

The system is controlled by a Windows® based workstation utilizing FlowPilot™ or FlowPilot-Pro™ software with real-time data acquisition via on-board customized electronics. Additional copies of the software are available for off-line post-run data analysis.

Using FlowPilot software, real-time analysis of the measured parameters is used to make sort decisions. Only those objects meeting the user-selected sort criteria are dispensed into multiwell plates, tubes or stationary receptacles. Those objects not meeting the sort criteria are gently deflected to a separate container where they may be recovered, unharmed and viable.

Instrument parameters and sample data are displayed on a large format LCD display; a second monitor is optional.

Each BioSorter system includes a fluidics caddy which organizes the reservoirs, waste container and pumps into a single, roll-around module.

Samples are introduced via a 50 ml conical tube (40 ml working volume) with suspended stirrer. For larger samples we offer optional 750ml and 1.5L stirred cups. For handling extremely delicate samples (adipocytes, plant protoplasts, etc.) and for samples that tend to either float or sink in a standard sample cup, a flow-through 5 or 10 ml rotating sample introduction chamber with integrated agitator is also optional.

An X-Y-Z stage allows dispensing into 24-, 48-, 96-, or 384-well multiwell plates, tubes and bulk receptacles. The z-direction allows adjustment for different height plates (deep well plates) and tubes.

High Throughput Screening features include liquid level sensors as well as automatic sample and pressure controls for walk-away operation.

參考文獻(xiàn)

1) Da Silveira Cavalcante L, Tessier SN. Zebrafish as a New Tool in Heart Preservation Research. J Cardiovasc Dev Dis. 2021;8(4):39. Published 2021 Apr 8. doi:10.3390/jcdd8040039

2) Howe K, Clark MD, Torroja CF, et al. The zebrafish reference genome sequence and its relationship to the human genome [published correction appears in Nature. 2014 Jan 9;505(7482):248. Cooper, James [corrected to Cooper, James D]; Eliott, David [corrected to Elliot, David]; Mortimer, Beverly [corrected to Mortimore, Beverley]; Begum, Sharmin [added]; Lloyd, Christine [added]; Lanz, Christa [added]; Raddatz, Günter [added]; Schuster, Ste]. Nature. 2013;496(7446):498-503. doi:10.1038/nature12111

3) Echeazarra L, Hortigón-Vinagre MP, Casis O, Gallego M. Adult and Developing Zebrafish as Suitable Models for Cardiac Electrophysiology and Pathology in Research and Industry. Front Physiol. 2021;11:607860. Published 2021 Jan 13. doi:10.3389/fphys.2020.607860

4) Santoso F, Farhan A, Castillo AL, et al. An Overview of Methods for Cardiac Rhythm Detection in Zebrafish. Biomedicines. 2020;8(9):329. Published 2020 Sep 4. doi:10.3390/medicines8090329

5) Ling D, Chen H, Chan G, Lee SM. Quantitative measurements of zebrafish heartrate and heart rate variabilityA survey between 1990-2020. Comput Biol Med. 2022;142:105045. doi:10.1016/pmed.2021.105045

6) Patton EE, Zon LI, Langenau DM. Zebrafish disease models in drug discoveryfrom preclinical modelling to clinical trials. Nat Rev Drug Discov. 2021;20(8):611-628. doi:10.1038/s41573-021-00210-8

7) Yan J, Li H, Bu H, et al. Aging-associated sinus arrest and sick sinus syndrome in adult zebrafish. PLoS One. 2020;15(5):e0232457. Published 2020 May 13. doi:10.1371/journal.pone.0232457

8) Liu H, Chen X, Zhao X, et al. Screening and Identification of Cardioprotective Compounds From Wenxin Keli by Activity Index Approach and in vivo Zebrafish Model. Front Pharmacol. 2018;9:1288. Published 2018 Nov 13. doi:10.3389/fphar.2018.01288

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