下载:
-
光学显微术作为一门古老的成像技术，为人们打开了一扇通往微观世界的大门，极大地丰富了人们的视界，拓展了人类的认知边界。随着科学技术的不断发展，人们对超高分辨率的显微图像的要求也越来越高，尤其是像生物医学、材料科学等需要对亚百纳米尺度微观结构进行观测和分析的领域更是如此[1]。然而由于阿贝衍射极限的存在，即便是共焦荧光显微技术，其空间分辨率也只能达到半波长左右，无法满足人们对于亚百纳米结构成像的要求[2]。20世纪90年代，Stefan.W.Hell提出了一种全新的可突破衍射极限的显微方法---荧光受激辐射损耗(Stimulated Emission Depletion, STED)显微成像技术。该方法可以将原先的极限分辨率提高4.5倍。STED技术虽然可以极大地提高显微成像分辨率，但是其过高的损耗调制光容易发生荧光漂白，损坏生物样品[3-4]。2012年，浙江大学匡翠方教授的超分辨显微研究组提出了一种新的超分辨显微技术---荧光辐射差分(Fluorescence Emission Difference, FED)显微术。该方法是将实心光斑扫描得到的共焦图像减去空心光斑扫描得到的负共焦图像实现的超分辨显微[5-6]，其优势在于仅利用单色光便可实现突破衍射极限实现超分辨显微，另外由于其只需要激发荧光样品发射荧光而不用进行受激辐射损耗，其光毒性和荧光漂白相比STED技术要弱很多，有利于对样品的长期观测。为了拓展FED显微成像的应用能力，本文在单色FED的基础上进一步研究并设计出了双色FED显微系统，在荧光颗粒上分别实现了488 nm激发光和640 nm激发光下135 nm和160 nm的空间分辨率，于此同时也对生物样品进行了多色同时成像，满足了同时观测和研究生物样品不同组织结构的应用要求。
-
荧光辐射差分显微术的基本原理是将两幅由不同照明光斑获得的共焦荧光显微图像进行相减，即将一幅由实心激发光斑扫描得到的共焦荧光图像减去一幅由空心激发光斑扫描得到的负共焦荧光图像[7-11]，图 1为FED原理图解。
图 1 FED原理图解：(a)，(b)分别为激发的实心光斑和空心光斑的PSF三维图；(c)，(d)为探测的实心光斑和空心光斑的PSF三维图；(e)为FED的PSF三维图；(f)，(g)，(h)分别为(c)，(d)，(e)的平面图；(i)，(j)，(k)分别为(f)，(g)，(h)在虚线处的截面图
Figure 1. Illustration of FED theory. (a)PSF of confocal excited pattern; (b)PSF of negative confocal excited pattern; (c)PSF of confocal image; (d)PSF of negative confocal image; (e) PSF of FED image; (f), (g), (h) the plane graph of (c), (d) and (e); (i), (j), (k) the sectional drawing along the dash line of (f), (g) and (h)
空心光斑由0 ~2π涡旋相位板调制产生，其调制函数如公式(1)所示[6]：
(1) 式中，φ为入射光束上一点位置矢量与水平方向的夹角。当调制后光束经二分之一波片和四分之一波片形成圆偏光后聚焦到焦平面时便会产生面包圈型空心光斑。最终的FED超分辨显微图像由公式(2)所示[5]：
(2) 式中，Ic、Id和IFED为归一化的强度分布图，Ic为归一化共焦强度图，Id为归一化负共焦强度图，IFED为归一化的FED图像强度图，γ为小于等于1的FED校准系数。共焦图像的点扩散函数可有公式(3)得出[5, 12]：
(3) 式中，PSFc表示共焦图像的PSF，如图 1(c)所示，PSFe表示激发光经过物镜的PSF，如图 1(a)所示，PSFf表示荧光经过物镜的PSF，p(x, y)表示小孔的传递函数。同理亦可得到图 1(d)的负共焦图像的PSFd。两幅图像相减后便可得到一幅FED显微图像，其PSF如图 1(e)所示。
从图 1的原理解释中可以看出，理论上，γ值越接近1，PSFFED则会越小，这就意味着其分辨率越高，然而这也就意味着通过相减得到的FED图像的有用信息就会损失的越多，即信噪比越低。此外，目前在FED系数选择上并没有一个行之有效的评价标准，一般通过实际图像效果进行主观评判，通过多次实验得知，γ值在0.7~0.85之间可以比较好的保证分辨率与信噪比。
-
从图 2(a)双色FED系统图中可以看出，激发光需先经过二分之一波片，保证了通过PBS后的分光比尽量达到1:1，而未经过位相板调制的光路由于其光程相对较长，因此需要一对焦距为50 mm的透镜组成的4F成像系统做中继透镜，保证了出射光为平行光。在调制光路部分，我们选用了Vortex Phase Plate RPC Photonics公司的VPP-1a和VPP-m633两种涡旋位相板来调制488 nm和640 nm激发光。为了形成圆偏光，调制光和未调制光经过一个PBS合束后仍需经过1/2波片和1/4波片。需要注意的是，激发光在经过4F扫描系统之前需要用一片1/4波片进行圆偏振矫正，因为激发光通过了多片二色镜和反射镜，其偏振状态会受到影响，为了保证激发光的圆偏振状态，在入射4F扫描系统前需进行圆偏振矫正。在该系统的显微镜架和物镜镜头选择上，我们采用了国产永新光学有限公司提供的N-900科研级显微镜架和Nikon公司提供的100×/1.49油浸显微物镜。为了满足系统实时扫描成像的要求，本系统使用了自主设计的反射式4f电流计式振镜扫描模块，该模块由1块焦距为50 mm的扫描透镜，2块焦距分别为50 mm和100 mm的凹面反射镜以及Cambridge Technology公司的8310k振镜模块构成，不同焦距的凹面镜不仅可以消除色差的影响，也可在一定程度上缩小量程，使得该扫描模块更加紧凑。
图 2 双色FED系统图与时序图(a)双色FED系统图，其中PBS为偏振分光镜，PM为涡旋位相板，RM为反射镜，DC为二色镜，4F SM为4F扫描模块，AL为消色差透镜，SMF为保偏光纤；(b)系统CAD设计图；(c)扫描时序图
Fi
-
摘要: 为了拓展荧光辐射差分（Fluorescence Emission Difference，FED）显微术的应用，使得该方法可以同时对生物样品的不同组织结构进行超分辨成像，本文对双色FED显微系统展开了研究。FED的基本原理是将实心光斑扫描得到的共焦显微图像减去空心光斑扫描得到的负共焦图像，以此获得超分辨显微图像。在对单色FED显微系统进行研究后，本文提出了一种可行的双色FED显微成像系统方案。实验结果表明，在488 nm和640 nm激发光下，该系统在荧光颗粒上分别实现了135 nm和160 nm的空间分辨率，另外也能对生物样品的不同组织进行多色同时超分辨显微成像，满足了实际应用的要求。Abstract: To perform super-resolution imaging of different tissue structures of biological samples using fluorescence radiation differential microscopy simultaneously, a dual-color FED microscopy system is studied in this paper. The basic principle of the FED is to remove the confocal microscopy image obtained by scanning the solid spot from the confocal microscopy image obtained by scanning the hollow spot to obtain a super-resolution microscopy image. Based on the study of the monochromatic FED microscopy system, a feasible dual-color FED microscopy imaging system is proposed and imaging experiments are performed on fluorescent particles in this paper. The experimental results indicate that under excitation light of 488nm and 640nm, the system realizes spatial resolution of 135 nm and 160 nm of the fluorescent particles respectively. In addition, this system can also perform multi-color super-resolution microscopy imaging simultaneously on different tissues of biological samples, which meets the requirements of practical applications.
-
图 1 FED原理图解：(a)，(b)分别为激发的实心光斑和空心光斑的PSF三维图；(c)，(d)为探测的实心光斑和空心光斑的PSF三维图；(e)为FED的PSF三维图；(f)，(g)，(h)分别为(c)，(d)，(e)的平面图；(i)，(j)，(k)分别为(f)，(g)，(h)在虚线处的截面图
Figure 1. Illustration of FED theory. (a)PSF of confocal excited pattern; (b)PSF of negative confocal excited pattern; (c)PSF of confocal image; (d)PSF of negative confocal image; (e) PSF of FED image; (f), (g), (h) the plane graph of (c), (d) and (e); (i), (j), (k) the sectional drawing along the dash line of (f), (g) and (h)
图 2 双色FED系统图与时序图(a)双色FED系统图，其中PBS为偏振分光镜，PM为涡旋位相板，RM为反射镜，DC为二色镜，4F SM为4F扫描模块，AL为消色差透镜，SMF为保偏光纤；(b)系统CAD设计图；(c)扫描时序图
Figure 2. Scheme and sequence chart of dual color FED system. (a)Dual color FED system, PBS:polarizing beam splitter, PM:vortex phase mask, RM:reflect mirror, DC:dichroic beam splitter, 4F SM:4F Scanning Module, AL:achromatic lens, SMF:single-mode polarization maintain fiber; (b)CAD of the system; (c)scan sequence chart
图 5 488 nm激发的荧光样品共焦和FED图像。(a)共焦图像；(b)FED图像；(c)共焦图像虚框内局部放大图像；(d)FED图像虚框内局部放大图像；(e)箭头部分归一化后的轮廓图
Figure 5. Confocal image and FED image of fluorescent beads excited at 488 nm. (a)Confocal image; (b)FED image. (c, d)Magnified view of dashed region at (a) and (b). (e)Normalized outline of arrow section of (c) and (d)
图 6 640 nm激发的荧光样品共焦和FED图像。(a)共焦图像；(b)FED图像；(c)共焦图像虚框内局部放大图像；(d)FED图像虚框内局部放大图像；(e)箭头部分归一化后轮廓图
Figure 6. Confocal image and FED image of fluorescent beads excited at 640 nm. (a)Confocal image; (b)FED image; (c, d)Magnified view of dashed region at (a) and (b); (e)Normalized outline of arrow section of (c) and (d)
图 7 生物样品多色成像图。(a)共焦图像；(b)FED图像；(c)共焦图像虚框内局部放大图；(d)FED图像虚框内局部放大图；(e)箭头指向部分轮廓图
Figure 7. Confocal image and FED image of biological structures excited at 488nm and 640nm simultaneously. (a)Confocal image; (b)FED image; (c, d)Magnified view of dashed region at (a) and (b); (e)Normalized outline of arrow section of (c) and (d)
-
[1] 李帅, 匡翠方, 丁志华, 等.受激发射损耗显微术(STED)的机理及进展研究[J].激光生物学报, 2013, 2:103-113. doi: 10.3969/j.issn.1007-7146.2013.02.002 LI SH, KUANG C F, DING ZH H, et al.. A review on concept and development of stimulated emission depletion microscopy(STED)[J]. Acta Laser Biology Sinica, 2013, 2:103-113.(in Chinese) doi: 10.3969/j.issn.1007-7146.2013.02.002 [2] HUANG B, BATES M, ZHUANG X. Super-resolution fluorescence microscopy[J]. Annual Review of Biochemistry, 2009, 78:993-1016. doi: 10.1146/annurev.biochem.77.061906.092014 [3] HELL S W, WICHMANN J. Breaking the diffraction resolution limit by stimulated emission:stimulated-emission-depletion fluorescence microscopy[J]. Optics Letters, 1994, 19(11):780-782. doi: 10.1364/OL.19.000780 [4] KLAR T A, JAKOBS S, DYBA M, et al.. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission[J]. Proceedings of the National Academy of Sciences, 2000, 97(15):8206-8210. doi: 10.1073/pnas.97.15.8206 [5] KUANG C, LI S, LIU W, et al.. Breaking the diffraction barrier using fluorescence emission difference microscopy[J]. Scientific Reports, 2013, 3:1441. doi: 10.1038/srep01441 [6] 李帅. 超分辨荧光显微方法与系统研究[D]. 杭州: 浙江大学, 2014. http://cdmd.cnki.com.cn/Article/CDMD-10335-1014269194.htm LI SH. Research on super-resolution fluorescence microscopy method and system[D]. Huangzhou: Zhejiang University, 2014. (in Chinese) http://cdmd.cnki.com.cn/Article/CDMD-10335-1014269194.htm [7] MEYER L, WILDANGER D, MEDDA R, et al.. Dual-color STED microscopy at 30-nm focal-plane resolution[J]. Small, 2008, 4(8):1095-1100. doi: 10.1002/smll.v4:8 [8] LI S, KUANG C, HAO X, et al.. Enhancing the performance of fluorescence emission difference microscopy using beam modulation[J]. Journal of Optics, 2013, 15(12):125708. doi: 10.1088/2040-8978/15/12/125708 [9] YOU S, KUANG C, RONG Z, et al.. Eliminating deformations in fluorescence emission difference microscopy[J]. Optics Express, 2014, 22(21):26375-26385. doi: 10.1364/OE.22.026375 [10] RONG Z, LI S, KUANG C, et al.. Real-time super-resolution imaging by high-speed fluorescence emission difference microscopy[J]. Journal of Modern Optics, 2014, 61(16):1364-1371. doi: 10.1080/09500340.2014.933272 [11] HAO X, KUANG C F, WANG T T, et al.. Effects of polarization on the de-excitation dark focal spot in STED microscopy[J]. Journal of Optics, 010, 12(11):115707. http://cn.bing.com/academic/profile?id=076fc5db3c528172e3bafe865a738aff&encoded=0&v=paper_preview&mkt=zh-cn [12] WILSON T. Resolution and optical sectioning in the confocal microscope[J]. Journal of Microscopy, 2011, 244(2):113-121. doi: 10.1111/jmi.2011.244.issue-2 [13] BINGEN P, REUSS M, ENGELHARDT J, et al.. Parallelized STED fluorescence nanoscopy[J]. Optics Express, 2011, 19(24):23716-23726. doi: 10.1364/OE.19.023716 [14] RANKIN B R, KELLNER R R, HELL S W. Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source[J]. Optics Letters, 2008, 33(21):2491-2493. doi: 10.1364/OL.33.002491 [15] STAUDT T M. Strategies to reduce photobleaching, dark state transitions and phototoxicity in subdiffraction optical microscopy[D]. Ruperto-Carola University of Heidelberg, Germany, 2009. https://www.researchgate.net/publication/33429512_Strategies_to_reduce_photobleaching_dark_state_transitions_and_phototoxicity_in_subdiffraction_optical_microscopy [16] HAO X, ALLGEYER E S, BOOTH M J, et al.. Point-spread function optimization in isoSTED nanoscopy[J]. Optics Letters, 2015, 40(15):3627-3630. doi: 10.1364/OL.40.003627 [17] HIGDON P D, T R K P, WILSON T. Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes[J]. Journal of Microscopy, 1999, 193(2):127-141. doi: 10.1046/j.1365-2818.1999.00448.x [18] RUST M J, BATES M, ZHUANG X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy(STORM)[J]. Nature Methods, 2006, 3(10):793. doi: 10.1038/nmeth929 [19] BRODEHL A, HEDDE P N, DIEDING M, et al.. Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants[J]. Journal of Biological Chemistry, 2012, 287(19):16047-16057. doi: 10.1074/jbc.M111.313841 [20] CARRINGTON W A, LYNCH R M, MOORE E D, et al.. Superresolution three-dimensional images of fluorescence in cells with minimal light exposure[J]. Science, 1995, 268(5216):1483-1487. doi: 10.1126/science.7770772 -
点击查看大图
计量
- 文章访问数: 506
- HTML全文浏览量: 141
- PDF下载量: 108
- 被引次数: 0
