CN117129455A - 一种基于微流控的大视场荧光检测光学系统 - Google Patents
一种基于微流控的大视场荧光检测光学系统 Download PDFInfo
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Abstract
本发明提供一种基于微流控的大视场荧光检测光学系统,包括:沿第一光路依次布置的光源、微阵列透镜组、激发滤光片、二向色镜,以及沿第二光路依次布置的样本平台、发射滤光片、大视场消像差物镜、相机设备,二向色镜与样本平台成45度角摆放并布置于样本平台与发射滤光片之间,第二光路与第一光路相垂直;其中,微阵列透镜组由依次排列的准直透镜、第一微阵列透镜、第二微阵列透镜、正透镜组成。根据本发明,通过特别的结构设计实现了大视场荧光液滴检测,能在10秒之内获得20mm*15mm荧光液滴图像,无需小视场荧光成像图像拼接技术,能够对病毒中的核酸进行快速的荧光检测,大大提高检测效率,推动了大视场细菌病毒荧光检测的开发。
Description
技术领域
本发明涉及生物检测领域,更具体地涉及一种基于微流控的大视场荧光检测光学系统。
背景技术
目前COVID-2019检测代表性技术是基于实时定量PCR,该方法只能显示定量结果。并且该检测方法都需要对患者人工采集再送到实验室中才能完成检测,通常检测时间需要24小时到48小时,因此该方法只能显示定量结果,无法提供核酸浓度的绝对定量。
针对上述现象,基于微流控技术核酸检测可以作为绝对定量的方法。检测样品被分散到一个包含许多独立的纳米、皮升尺度微腔的基因芯片中,因此每个微腔中理论上就包含多量和少量的目标序列,接着进行每个单元的荧光信号检测和计算。然而,目前商用或报道的用微流控基因芯片的检测大部分都是利用小视场的荧光显微成像系统对微腔阵列进行多次捕获,从而降低了检测效率,后期子图像的拼接也要花费大约5分钟的时间,并且存在微腔因拼接偏差而被区分为异常的可能会直接影响核酸浓度的准确性。
目前商用普通显微镜检测方式存在的不足常为一体化集成检测难、操作方式繁琐,视场小,因此为了实现病毒中核酸的快速检测,探索一种具有高分辨率以及超大视场的生物荧光检测显微镜在生物检测领域的应用具有重要意义。
发明内容
本发明的目的是提供一种基于微流控的大视场荧光检测光学系统,从而解决现有技术中的荧光显微成像系统存在视场小、需要多次拼接、检测效率低、检测准确度低的问题。
根据本发明,提供一种基于微流控的大视场荧光检测光学系统,包括:沿第一光路依次布置的光源、微阵列透镜组、激发滤光片、二向色镜,以及沿第二光路依次布置的样本平台、发射滤光片、大视场消像差物镜、相机设备,所述二向色镜与样本平台成45度角摆放并布置于所述样本平台与所述发射滤光片之间,所述第二光路与所述第一光路相垂直;其中,所述微阵列透镜组由依次排列的准直透镜、第一微阵列透镜、第二微阵列透镜、以及正透镜组成;其中,所述微阵列透镜组对所述光源发出的激发光进行准直和均匀化处理,获得均匀的激发光速,所述激发滤光片过滤掉光源以外的杂光,所述二向色镜将激发光反射到所述样本平台上,样本中的荧光染料受到激发发出荧光,所述二向色镜允许荧光透过,所述发射滤光片过滤掉荧光以外的杂光,所述大视场消像差透镜组对视场范围的微腔阵列进行荧光成像,最后通过所述相机设备成图像。
优选地,所述光源可发出四种不同波段的激发光以匹配不同的荧光染料和荧光底物,所述四种不同波段包括365nm、460nm、550nm、625nm。
根据光源的不同,选择不同的激发滤光片和发射滤光片。
优选地,所述大视场消像差物镜采用-0.69X的倍率。
优选地,所述第一微阵列透镜和所述第二微阵列透镜分别由多个小透镜组成,二者共同组成双排复眼透镜。
根据该优选实施例,所述大视场荧光检测光学系统的视场达到20mm*15mm,可一次性成像辨别4万只核酸反应阴阳性液滴。
根据本发明,光源可以发出四种不同波段的激发光,即中心波长分别是365nm、460nm、550nm、625nm的激发光,以代替传统的小功率单通道LED光源。其波长与典型的荧光染料和荧光底物(如FAM、4MUG)匹配良好,对信号微弱的荧光增加强度,提高了检测效率。例如,FAM荧光通道所需激发光波长为498nm,4MUG荧光通道所需激发光波长为375nmm。
根据本发明,提供了一种基于微流控大视场一次性成像辨别4万只核酸反应阴阳性液滴的检测装置,不同于市面上主流的病毒荧光检测,本发明提供了一种集检测时间快,在线高灵敏,成像视场大为一体的荧光显微镜,视场达到了20mm*15mm。应当理解的是,要实现大视场荧光成像,那么物镜设计的倍率要足够小,本发明采用物镜的倍率为-0.69X,工作距离110mm,实现大视场的同时也要具高分辨率,当荧光波长为定量时,光学系统分辨率与数值孔径(NA)成反比,当NA越大时,分辨率越精密。视场越大,必然会导致像差,通过物镜的多透镜组合可以很大程度上消除大视场成像产生的像差,确保成像质量符合要求。本发明所要解决的技术难点主要在于在实现大视场的同时还需要实现对检测样本的均匀照明,本发明的关键发明点则主要在于通过-0.69X远心物镜以及微阵列透镜解决视场和均匀照明的问题,从而首次实现了20mm*15mm的检测视场,提供了一种大视场荧光液滴检测。
根据本发明提供的一种基于微流控的大视场荧光检测光学系统,其相对现有技术的有益效果在于:
1)采用多波段的激发光源代替传统的小功率单通道LED光源,其波长与典型的荧光染料和荧光底物匹配良好,对信号微弱的荧光增加强度,提高了检测效率;
2)通过特别的结构设计实现了大视场荧光液滴检测,能在10秒之内获得20mm*15mm荧光液滴图像,可以实现大视场一次性成像辨别4万只核酸反应阴阳性液滴图,无需小视场荧光成像图像拼接技术,在很大程度上缩短了成像和DNA浓度测定所需的时间;
3)能够对病毒中的核酸进行快速的荧光检测,大大提高检测效率,本发明可以推动大视场细菌病毒荧光检测的开发。
附图说明
图1示出了根据本发明提供的一种大视场荧光检测光学系统的示意图;
图2示出了如图1所示大视场荧光检测光学系统的立体图;
图3示出了如图1所示大视场荧光检测光学系统的几何光路图;
图4示出了如图1所示大视场荧光检测光学系统中的微阵列透镜组图;
图5示出了根据本发明一个优选实施例的FAM荧光通道图;
其中,附图标记含义如下:
110:光源;120:微阵列透镜;121:准直透镜;122:第一微阵列透镜;123:第二微阵列透镜;124:正透镜;130:激发滤光片;140:二向色镜;150:样本平台;160:发射滤光片;170:大视场消像差物镜;180:相机设备。
具体实施方式
以下结合具体实施例,对本发明做进一步说明。应理解,以下实施例仅用于说明本发明而非用于限制本发明的范围。
结合图1、图2所示,是根据本发明一个优选实施例提供的大视场荧光检测光学系统100,可一次性成像辨别4万只核酸反应阴阳性液滴。该大视场荧光检测光学系统100包括光源110、微阵列透镜组120、激发滤光片130、二向色镜140、样本平台150、发射滤光片160、大视场消像差物镜170、相机设备180。其中,光源110、微阵列透镜组120、激发滤光片130、二向色镜140沿第一光路依次布置,样本平台150、二向色镜140、发射滤光片160、大视场消像差物镜170、相机设备180沿第二光路依次布置,二向色镜140与样本平台150成45度角摆放并布置于样本平台150与发射滤光片160之间,第二光路与第一光路相垂直。
结合图3、图4所示,微阵列透镜组120由依次排列的准直透镜121、第一微阵列透镜122、第二微阵列透镜123、以及正透镜124组成。其中,第一微阵列透镜122和第二微阵列透镜123分别由多个小透镜组合而成,共同组成了双排复眼透镜,通过将该双排复眼透镜应用于本发明的照明系统,可以提高光源的光能利用率以及对样本充分的均匀照明。
根据本发明的一个优选实施例,该大视场荧光检测光学系统100的使用方法如下:
首先,将微流控芯片(图未示出)置于样本平台150上,接着选择与荧光波长匹配的激发光源110,本实施例中,样品中的荧光染料为FAM,所以我们选择中心波长495nm的光去激发;接着,微透镜组120对激发光进行准直和均匀化处理,获得均匀的激发光速,使得样本能够被足够的激光均匀激发,准直透镜121对光线准直为平行光,接着,第一微阵列透镜122将准直透镜121发出的平行光束进行偏折,第二微阵列透镜123将第一微阵列透镜122发出的光束夹角进行适当收缩之后发射到正透镜124,激发滤光片130过滤掉光源以外的杂光,由于二向色镜140相对样本平台150成45度角摆放,因此可将激发光反射到样本平台150上,样本中的荧光染料受到激发发出荧光,二向色镜140允许荧光透过,发射滤光片160过滤掉荧光以外的杂光。大视场消像差透镜组170对视场范围的微腔阵列进行荧光成像,最后通过CMOS相机180成图像,后期通过算法编写即可确定核酸阴阳性个数。
如图5所示,本发明可以实现大视场一次性成像辨别4万只核酸反应阴阳性液滴图,无需小视场荧光成像图像拼接技术,当采用荧光染料FAM标记核酸时,可以在不到10秒的时间内获得FAM通道核酸液滴荧光图像,获得20mm*15mm的大视场。
以上所述的,仅为本发明的较佳实施例,并非用以限定本发明的范围,本发明的上述实施例还可以做出各种变化。凡是依据本发明申请的权利要求书及说明书内容所作的简单、等效变化与修饰,皆落入本发明专利的权利要求保护范围。本发明未详尽描述的均为常规技术内容。
Claims (6)
1.一种基于微流控的大视场荧光检测光学系统,其特征在于,包括:沿第一光路依次布置的光源(110)、微阵列透镜组(120)、激发滤光片(130)、二向色镜(140),以及沿第二光路依次布置的样本平台(150)、发射滤光片(160)、大视场消像差物镜(170)、相机设备(180),所述二向色镜(140)与样本平台(150)成45度角摆放并布置于所述样本平台(150)与所述发射滤光片(160)之间,所述第二光路与所述第一光路相垂直;
其中,所述微阵列透镜组(120)由依次排列的准直透镜(121)、第一微阵列透镜(122)、第二微阵列透镜(123)、以及正透镜(124)组成;
其中,所述微阵列透镜组(120)对所述光源(110)发出的激发光进行准直和均匀化处理,获得均匀的激发光速,所述激发滤光片(130)过滤掉光源以外的杂光,所述二向色镜(140)将激发光反射到所述样本平台(150)上,样本中的荧光染料受到激发发出荧光,所述二向色镜(140)允许荧光透过,所述发射滤光片(160)过滤掉荧光以外的杂光,所述大视场消像差透镜组(170)对视场范围的微腔阵列进行荧光成像,最后通过所述相机设备(180)成图像。
2.根据权利要求1所述的大视场荧光检测光学系统,其特征在于,所述光源(110)可发出四种不同波段的激发光以匹配不同的荧光染料和荧光底物,所述四种不同波段包括365nm、460nm、550nm、625nm。
3.根据权利要求2所述的大视场荧光检测光学系统,其特征在于,根据光源(110)的不同,选择不同的激发滤光片(130)和发射滤光片(160)。
4.根据权利要求1所述的大视场荧光检测光学系统,其特征在于,所述大视场消像差物镜(170)采用-0.69X的倍率。
5.根据权利要求1所述的大视场荧光检测光学系统,其特征在于,所述第一微阵列透镜(122)和所述第二微阵列透镜(123)分别由多个小透镜组成,二者共同组成双排复眼透镜。
6.根据权利要求1所述的大视场荧光检测光学系统,其特征在于,所述大视场荧光检测光学系统的视场达到20mm*15mm,可一次性成像辨别4万只核酸反应阴阳性液滴。
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