测绘在细胞膜分子扩散，多目标跟踪（MTT）
Summary
多目标跟踪是一个自制的算法跟踪单独标记分子在活细胞的细胞膜。有效地检测，估计和跟踪在高密度的分子随着时间的推移，提供一个用户友好，全面的工具来研究纳米膜动力学。
Abstract
我们的目标是获得细胞膜发生在不同的生物功能的分子过程的全面描述。我们的目标是在描述复杂的组织和细胞膜的动态，在单分子水平，开发的分析工具，专门在高密度单粒子追踪 （SPT）： 多目标跟踪 （MTT）1。电视显微镜，单分子提供毫秒级和纳米级分辨率1-11，允许膜组织12-14详细表示，通过精确映射，如细 胞受体的定位，流动性，禁闭或相互作用的描述。
我们重新审视六方会谈，实验和算法。实验方面包括：优化设置和细胞标记，以达到尽可能高的标签密度特别强调，为了提供一个分子动力学动态快照就这样发生在膜。算法的问题有关的每一步都用于重建的轨迹：峰值检测，估计和重新从15,16图像分析处理，通过特定的工具。实施后，检测通缩允许抢救峰最初隐藏相邻，峰强。值得注意的是，提高检测直接影响重联，减少内轨迹的差距。已评估使用各种标签密度和噪声值，这通常代表两个主要局限在高时空分辨率的并行测量的蒙特卡罗模拟表演。
17纳米的精度得到单分子，无论是连续使用开/关photoswitching或非线性光学，可以提供详尽的意见。这是19,20风暴18日 ，棕榈，RESOLFT 21 STED 22,23，WHI等17 nanoscopy方法的基础上CH可能常常需要固定的样品成像。中心任务是有限的单分子所产生的衍射峰的检测和估计。因此，提供足够的假设，如恒定的定位精度，而不是处理布朗运动，MTT法是直截了当地适用于纳米级的分析。此外，MTT法可以从根本上被使用在任何规模：不仅为分子，同时也为细胞或动物，例如。因此，MTT法是一个功能强大的跟踪算法，发现在细胞和分子尺度的应用。
Protocol
Discussion
旁边的细胞和显微镜的各个方面，在单粒子跟踪，分析代表了相当一部分的工作。这解决了算法用于执行三大任务：检测，评估和重新连接了每帧的山峰。但随之而来的这项工作方面驻留在拟订算法本身，这可能需要适应任何新的专门调查，基本上最后，额外的步骤（如破译运动模式，相互作用或化学计量学）。
然而，一旦该算法充分开发，运行，它是简单的，特别是因为?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢我们的团队成员提供技术援助，特别是管委会Blache，以及M Irla和乙Imhof，对他们的支持和富有成效的讨论。通缩和分娩的数字再现自然方法提供。支持这个项目是由国家科学研究中心（INSERM）和马赛大学，体制补助和特定地区研究所国家杜癌症，法新社国立德拉Recherche（普罗旺斯 – 阿尔卑斯 – 科特迪瓦-d'Azur补助的ANR-08-PCVI 0034-02，国家情报局2010汪曾祺1214 01）基金会争取，LA RECHERCHEMédicale（队报labéliséeFRM-2009）。 VR是支持从法甲国立CONTRE LE癌症的奖学金。
Materials
| Reagent | Company | Catalogue number | Quantity |
| Cos-7 cell line | ATCC | CRL-1651 | 5,000 cells/well |
| HBSS without Ca2+ | GIBCO | 14175 | 1 ml |
| 0.05% Trypsin EDTA | GIBCO | 25300 | 1 ml |
| 8-well Lab-tek | NUNC | 155441 | 1 |
| QDot-605 streptavidin | Invitrogen | Q10101MP | 20 mM |
| Biotinylated Fab (for Fab synthesis, see reference 21) | |||
| Fab from mAb 108 | ATCC | HB-9764 | 200 μg |
| NHS-Biotin | Thermo Scientific | 21435 | 18.5 μg |
| Complete medium | |||
| DMEM | GIBCO | 41965 | 500 ml |
| Fetal Bovine Serum | SIGMA | F7524 | 50 ml |
| L-Glutamine | GIBCO | 25030 | 5 ml |
| HEPES | GIBCO | 15630 | 5 ml |
| Sodium Pyruvate | GIBCO | 11360 | 5 ml |
| Imaging medium | |||
| HBSS with Ca2+ | GIBCO | 14025 | 25 ml |
| HEPES | GIBCO | 15630 | 250 μl |
| Equipment | Company | Reference |
| Inverted microscope | Nikon | Eclipse TE2000U |
| Fluorescent lamp | Nikon | Intensilight C-HGFIE |
| 1.3 NA 100x objective | Nikon | Plan Fluor 1.30 |
| 1.49 NA 100x objective | Nikon | APO TIRF 1.49 |
| Camera | Roper Scientific | Cascade 512 B |
| Thermostated box | Life Imaging Services | The Box |
Appendix: example Script of MTT supplementary analysis
function MTT_example(file_name)
%%% Basic examples showing how to recover MTT output results
%%% to plot each trace and to build the histogram
%%% of fluorescence intensities
if nargin<1 % no file_name provided?
files = dir(‘*.stk’);
if isempty(files), disp(‘no data in current dir’), return, end
file_name = files(1).name; % default: first stk file
disp([‘using’ file_name ‘by default’])
end
file_param = [file_name ‘_tab_param.dat’]; % output file
%% Load data
cd(‘output23′) % or (‘output22’), according to version used
% Disclaimer: version 2.2 only generates 7 parameters,
% an extra parameter, noise, was added in version 2.3
% To read all parameters at once, in a single table
% tab_param = fread_all_param(file_param);
% tab_i = tab_param(2:8:end, :); tab_j = …
% To read all parameters (except frame_number) in separate tables
% [tab_i,tab_j,tab_alpha,tab_radius,tab_offset,tab_blk,tab_noise] = fread_all_data_spt(file_param);
tab_i = fread_data_spt(file_param, 3); % index is 3 because trace number & frame number, non informative, are discarded!
tab_j= fread_data_spt(file_param, 4);
tab_alpha = fread_data_spt(file_param, 5);
tab_blk = fread_data_spt(file_param, 8);
%% Loop over traces
N_traces = size(tab_i,1);
% Tables are N_traces lines by N_frames colums
for itrc = 1:N_traces
No_blk_index = tab_blk(itrc, :)>0; % non blinking steps only
plot(tab_i(itrc, No_blk_index), tab_j(itrc, No_blk_index))
xlabel(‘i (pixel)’), ylabel(‘j (pixel)’)
title([‘trace # ‘ num2str(itrc)])
disp(‘Please strike any key for next trace’), pause
end
%% Fluo histogram
N_datapoints = sum(tab_blk(:)>0); % non blinking steps only
hist(tab_alpha(tab_blk>0),2*sqrt(N_datapoints)) % using 2sqrt(N) bins
xlabel(‘intensity (a.u.)’), ylabel(‘occurrence’)
title(‘histogram of particles fluorescence intensity’)
References
- Serge, A., Bertaux, N., Rigneault, H., Marguet, D. Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat. Methods. 5, 687-694 (2008).
- Schmidt, T., Schutz, G. J., Baumgartner, W., Gruber, H. J., Schindler, H. Imaging of single molecule diffusion. Proc. Natl. Acad. Sci. U S A. 93, 2926-2929 (1996).
- Lommerse, P. H. Single-molecule imaging of the H-ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane. Biophys. J. 86, 609-616 (2004).
- Marguet, D., Lenne, P. F., Rigneault, H., He, H. T. Dynamics in the plasma membrane: how to combine fluidity and order. EMBO. J. 25, 3446-3457 (2006).
- Saxton, M. J., Jacobson, K. Single-particle tracking: applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26, 373-399 (1997).
- Dahan, M. Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science. 302, 442-445 (2003).
- Harms, G. S. Single-molecule imaging of l-type Ca(2+) channels in live cells. Biophys. J. 81, 2639-2646 (2001).
- Iino, R., Koyama, I., Kusumi, A. Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface. Biophys. J. 80, 2667-2677 (2001).
- Sako, Y., Minoghchi, S., Yanagida, T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat. Cell Biol. 2, 168-172 (2000).
- Schutz, G. J., Kada, G., Pastushenko, V. P., Schindler, H. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. Embo. J. 19, 892-901 (2000).
- Seisenberger, G. Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science. 294, 1929-1932 (2001).
- Jacobson, K., Sheets, E. D., Simson, R. Revisiting the fluid mosaic model of membranes. Science. 268, 1441-1442 (1995).
- Saffman, P. G., Delbruck, M. Brownian motion in biological membranes. Proc. Natl. Acad. Sci. U S A. 72, 3111-3113 (1975).
- Singer, S. J., Nicolson, G. L. The fluid mosaic model of the structure of cell membranes. Science. 175, 720-731 (1972).
- Papoulis, A. . Probability, Random Variables and Stochastic Process 277. , (2001).
- Van Trees, H. L. . Detection, Estimation, and Modulation Theory, Wiley Inter-Science. , (1968).
- Moerner, W. E. Single-molecule mountains yield nanoscale cell images. Nat. Methods. 3, 781-782 (2006).
- Rust, M. J., Bates, M., Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods. 3, 793-795 (2006).
- Betzig, E. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 313, 1642-1645 (2006).
- Manley, S. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods. 5, 155-157 (2008).
- Andrew, S. M. Enzymatic digestion of monoclonal antibodies. Methods Mol. Med. 40, 325-331 (2000).
- Hell, S. W., Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780-782 (1994).
- Klar, T. A., Hell, S. W. Subdiffraction resolution in far-field fluorescence microscopy. Opt. Lett. 24, 954-956 (1999).
- Meilhac, N., Guyader, L. L. e., Salome, L., Destainville, N. Detection of confinement and jumps in single-molecule membrane trajectories. Phys. Rev. E. Stat. Nonlin. Soft. Matter Phys. 73, 011915 (2006).
- Saxton, M. J. Single-particle tracking: effects of corrals. Biophys. J. 69, 389-398 (1995).
- Serge, A., Fourgeaud, L., Hemar, A., Choquet, D. Receptor activation and homer differentially control the lateral mobility of metabotropic glutamate receptor 5 in the neuronal membrane. J. Neurosci. 22, 3910-3920 (2002).
- Simson, R., Sheets, E. D., Jacobson, K. Detection of temporary lateral confinement of membrane proteins using single-particle tracking analysis. Biophys. J. 69, 989-993 (1995).
- Jacobson, K., Dietrich, C. Looking at lipid rafts. Trends Cell Biol. 9, 87-91 (1999).
- Kusumi, A., Sako, Y., Yamamoto, M. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys. J. 65, 2021-2040 (1993).
- Livneh, E. Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility. J. Cell Biol. 103, 327-331 (1986).
- Medintz, I. L., Uyeda, H. T., Goldman, E. R., Mattoussi, H. Quantum dot bioconjugates for imaging, labelling and. 4, 435-446 (2005).
- Wu, X., Bruchez, M. P. Labeling cellular targets with semiconductor quantum dot conjugates. Methods Cell Biol. 75, 171-183 (2004).
- Mohammadi, M. Aggregation-induced activation of the epidermal growth factor receptor protein tyrosine kinase. Biochimie. 32, 8742-8748 (1993).
- Howarth, M. Monovalent, reduced-size quantum dots for imaging receptors on living cells. Nat. Methods. 5, 397-399 (2008).
- Bertaux, N., Marguet, D., Rigneault, H., Sergé, A. Multiple-target tracing (MTT) algorithm probes molecular dynamics at cell surface. Protocol Exchange. , (1038).
- Groc, L. Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies. The Journal of neuroscience : the official journal of the Society for Neuroscience. 27, 12433-12437 (2007).
- Cui, B. One at a time, live tracking of NGF axonal transport using quantum dots. Proceedings of the National Academy of Sciences of the United States of America. 104, 13666-13671 (2007).
- He, H. T., Marguet, D. Detecting nanodomains in living cell membrane by fluorescence correlation spectroscopy. Annu. Rev. Phys. Chem. 62, 417-436 (2011).
- Cebecauer, M., Spitaler, M., Serge, A., Magee, A. I. Signalling complexes and clusters: functional advantages and methodological hurdles. J. Cell. Sci. 123, 309-320 (2010).
- Kao, H. P., Verkman, A. S. Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophys. J. 67, 1291-1300 (1994).
