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Jul 2019

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Assembly and Imaging Set up of PIE-Scope
PIE-Scope的组装和成像设置   

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Abstract

Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. The PIE-scope can be retrofitted on existing microscopes, although the drawings we provide are meant to work on ThermoFisher DualBeams with small mechanical modifications those can be adapted on other brands.

Keywords: PIE-scope (光子-离子-电子显微镜), Cryo-lamella (低温板层), Cryo-CLEM (Cryo-CLEM), Correlative microscopy (相关显微镜), Cryo-light microscopy (低温光学显微镜)

Background

Cryo-electron tomography (cryo-ET) is currently the principal method for investigating the structure of proteins and protein complexes directly in their native environment (Beck and Baumeister, 2016). Cells are generally too thick to be imaged using cryo-ET and, to overcome the issue of cell thickness, the most common and successful approach is to use a cryo-focused ion beam microscope (FIB) to thin the sample and produce flat electron-transparent lamellae of approximately 300 nm thick (Marko et al., 2006 and 2007). It is important to note that cells feature an extremely crowded and complex environment; therefore, the lamellae must be prepared targeting regions containing the event or structure under study. The approach that is generally taken is based on correlative microscopy (CLEM), where fluorescence microscopy is used to identify the location of a region of interest and then, through image correlation the same region is found using the FIB (Sartori et al., 2007; Arnold et al., 2016). The complete cryo-CLEM workflow links light microscopy (live cell imaging and cryo-LM), cryo-FIB and cryo-ET. The workflow generally starts with imaging living cells or tissue in order to obtain information about the process dynamics. When appropriate, the sample is vitrified through plunge or high-pressure freezing. From this moment onwards, every time the sample is transferred inside or outside a microscope the chances for contamination and damage increase.

All the risks associated with cryo-transfers described above can be mitigated using an integrated approach, where a light microscope is integrated into the vacuum chamber of the electron microscope. This approach has been successfully implemented for CLEM where the introduction of a light microscope objective in a TEM column permitted switching between TEM and LM imaging, known as the iLEM (Agronskaia et al., 2008). On the cryo-FIBs, the first example of an integrated correlative system is the PIE-scope (Gorelick et al., 2019). The PIE-scope (which stands for Photon-Ion-Electron microscope) is a peripheral add on that can be retrofitted on existing ThermoFisher DualBeam microscopes. The hardware setup assembled is shown in Figure 1 and the assembly is shown in Video 1.


Figure 1. Overview of the PIE-scope hardware installed on a ThermoFisher Helios G4. A) is a view of the outside component of the PIE-scope optical path. The position of the major parts is marked on the figure. B) is an internal view of the chamber, showing how the assembly can fit inside an existing FIB/SEM microscope. C) View of the computer control, we have a dedicated PIE-scope PC next to the PC controlling the FIB/SEM and an additional support PC for developmental purposes and to allow developers to work while the microscope is being used for data collection. This is not required in a user-based facility since the support PC provided with the instrument is sufficient to control the PIE-scope. All major parts (of the PIE-scope and the FIB/SEM microscope) are marked in the figures.


Video 1. S3 exploded PIE


The goal of this protocol is to help labs to replicate the assembly, install the required hardware and software. This will be particularly useful in the context of automating complex sample preparation workflows where automation will be critical (Buckley et al., 2020).

Materials and Reagents

  1. Vitrified (cryo-preserved) cells or tissue
  2. Cryo-EM grids (gold for cell culture, copper for yeast or bacterial suspension)
  3. Autogrids (Thermo Fisher, catalog number: 1205101 )

Equipment

  1. DualBeam microscope (Thermo Fisher Helios G4 or Aquilos)
  2. Custom components (the published drawings and assembly can be found on Zenodo at https://zenodo.org/record/3260173#.XsShV2gzb-g while updated drawings can be obtained from https://www.demarco-lab.com/resources)
  3. Piezo Z-drive (Smaract, model: SLC-2445-D-S-HV )
  4. Dichroic switch (Smaract, model: SLC-2445-D-S )
  5. Motor Driver (Smaract, model: MCS-6CC-USB-TAB )
  6. Vacuum feedthrough for z-drive (Smaract, model: LEMO1B-FGJ-SJG-FGG-14 )
  7. Motor driver sensor module (Smaract, model: MCS-3S-EP-SDS15-TAB )
  8. Camera, Basler, acA1920-155 μm USB 3.0
  9. Quad-line dichroic (Chroma, model: 89402bs )
  10. Quad-channel emission filter (Chroma, model: 89402m )
  11. Tube lens (200 mm) (Thorlabs, model: AC254-200-A-ML )
  12. Excitation tube lens (400 mm) (Thorlabs, model: AC254-400-A-ML)
  13. Emission mirror (Thorlabs, model: BBE1-E02 )
  14. Objective long mirror (Edmund Optics, 32366 )
  15. Flange mirror (Edmund Optics, 83536 )
  16. Laser combiner ( 405-488-561-640 ), Toptica, iCHROME CLE
  17. Flange mirror mount (Thorlabs, model: KMSS/M )
  18. Excitation mirror mount (Thorlabs, model: KMSS/M )
  19. Pinhole (Thorlabs, model: SM1D12D )
  20. Tube-lens mount (Thorlabs, model: SMR1/M )
  21. C-mount to SM1 adapter (Thorlabs, model: SM1A10TS )
  22. Objective (20x/0.5) (Olympus, MPLFNL 20X/0.45 )
  23. Vacuum flange (Thorlabs, model: VPCH42-A )
  24. FC/APC fibre to SM1 adapter (Thorlabs, model: SM1FCA )
  25. Adjustable tube mount (Thorlabs, model: SM1V10 )
  26. Tube mount (Thorlabs, model: SM1L10 )
  27. O-rings and gaskets (Lesker, various to fit the flanges)
  28. 10/90 R/T BeamSplitter (Thorlabs, model: BS025 )

Software

Currently, there are two options available to control the PIE-scope.


Software requirements for option 1:

  1. Python 3.5 or higher
  2. ThermoFisher Autoscript 4.1 or higher
  3. PyPylon 1.0 or higher
  4. Python libraries: Numpy
  5. National Instruments Labview 2015 or higher

Software requirements for option 2:

  1. Python 3.5 or higher
  2. Python libraries: Numpy, PyQt, Scitek-image
  3. PyPylon 1.0 or higher
  4. ThermoFisher Autoscript 4.1 or higher

All python code and packages, with the exception for the commercial software, can be downloaded from our lab resource page or the Github page (https://github.com/demarcolab). The general software architecture is described in Figure 2.


Figure 2. Diagram showing the relationship between the hardware and software components. The white boxes identify the APIs used to control the various components.

Procedure

  1. PIE-scope assembly
    This section is of interest for laboratories that intend to assemble a copy of the current PIE-scope design. The design we propose is to be fitted on the front GIS (gas injection system) flanges of the vacuum chamber of the microscope. The flange used is GIS2, for which the vacuum feedthrough and the mirror support have been designed. Accordingly, if there is a GIS on any of the front ports it should be moved on GIS ports 4 or 5 if possible. If those ports are already in use and one GIS must stay at the front we suggest to use GIS port 3 and leave port 1 free. The presented design requires custom-built components, and all drawings can be downloaded from https://www.demarco-lab.com/resources. In case the proposed design does not fit a specific microscope configuration (e.g., there are collisions with detectors or manipulators) it is possible to change the position or orientation of the PIE-scope inside the chamber by modifying the design of the custom components.
       Further, not all components must be replicated using custom parts: the illumination and excitation arms of the PIE scope can be assembled using 30 mm Thorlabs cages, Thorlabs CERNA components or similar. The early prototypes of the PIE-scope have been realized using Thorlabs cage assemblies, but to increase the alignment stability, and to improve the compactness of the system we decided to opt for a monolithic aluminium body.

    The assembly consists of 2 major components:
    • The focus drive assembly
    • The atmospheric setup
    The focus drive assembly consists of a monolithic brace that is fixed on 2 M6 bolts located on the vacuum chamber wall between the front GIS ports and the FIB column. The brace holds a long mirror (Edmund Optics) using spring-loaded pins. The piezo linear positioner used for focusing (Smaract) is bolted directly on the front of the brace. The objective is mounted on the positioner using a custom adapter, the drawings provided will work with any Olympus objective, and the positioner will be able to operate with any objective weighting less than 150 g.
        The electrical feedthrough for the focus drive (LEMO) can be placed on any available flange, we used GIS port 3.
       The atmospheric setup starts at the vacuum flange. The vacuum feedthrough of the light path is achieved through a quartz window. For this purpose, we use a high vacuum compatible window flange (Thorlabs). The flange is mounted on an adapter that converts the GIS flange into a CF-40. The adapter also allows mounting the microscope body to the vacuum chamber.

    The microscope body consists of 2 main components:
    • A sturdy bracket to steer the beam path such that the sum of the reflection angles from the back focal plane of the objective to the light source and detectors is a multiple of 90 deg.
    • The main body, which consists of an excitation arm and an emission arm. This component also hosts the dichroic mirror, the fibre adapter, the tube lens and the detector.
    The mirror located on the bracket is mounted on an adjustable mirror mount to steer across 2 angles and therefore compensate for the mechanical tolerances (which the assembly of this component are close to 1 mm). A dichroic mirror (Chroma) and a 90/10 Beam splitter (Thorlabs) are mounted, through a custom adapter, on a motorized linear positioner (Smaract). Switching between the beamsplitter and the dichroic provides the choice between reflected light and fluorescence imaging.
        The tube lens is directly mounted on the main body and its position cannot be adjusted. At the extremities of the two arms of the microscope body, there are the detector and the optical fibre. The decision to design the emission arm with a 90 deg kink was made to make the overall assembly more compact and accordingly less prone to vibrations. In principle, this arm can be made straight, especially if the components used for the body are based on Thorlabs Cerna or similar, but one must be aware that the arm will be protruding beyond the current footprint of the instrument at the front, and might result on the way when loading samples through the main door of the DualBeam.
       The laser source is a Toptica iCLE-50, which ensures 50mW of laser power across four channels (405, 488, 561, 640 nm). In our experience, a lower power will be suitable, and we calculated that 10mW is sufficient for this microscope. The detector currently is a Basler acA1920-155 um USB 3.0. This is a cost-effective solution that has also the advantage to be extremely light and compact if higher sensitivity is required we sustain suggest to use a Hamamatsu flash 4 V2 or V3 camera. The current design of the PIE-scope body has been done taking into account the weight and size of this camera.
       All positioners are from Smaract, accordingly, it is possible to control them from a single motor drive. There are multiple options, some come with USB interface and some with Ethernet, depending on the choice of the drive one will have to choose the PIE-scope GUI. The USB drive is only compatible with the Labview interface, while the Ethernet version allows its use with both. Accordingly, we suggest purchasing the ethernet version.
       The PIE-scope is controlled through a computer connected to the same local network of the microscope PC of the FIB/SEM. The support PC that is always sold with the FIB/SEM is already suitable for the task, but from experience, it is easier to have a dedicated computer so that extra ethernet and USB 3.0 ports can be added. We currently use an HPz4 workstation with an extra Ethernet card to ensure enough ports are available. The PC has 1 connection to the general network to transfer the data and 1 connected to the local microscope network to communicate with the microscope PC. A third connection is required to connect the Smaract MCS motor drive. Lasers and camera are connected via USB 3.0. The Basler camera is a heated CMOS camera which requires 2A of current from the USB port, it must be noted that not all ports fulfil the specification and we suggest purchasing a dedicated USB 3.0 expansion card. Further, to enable live processing and image segmentation, we suggest at least 32 Gb of memory and a dedicated GPU.
       To control the PIE-scope, we provide 2 options: one completely python-based, where all functions are integrated; and one which is Labview based where live camera view is not integrated due to restrictions of the Basler SDK. Both versions of the software contain all required controls to enable imaging with the FIB, the SEM and the light microscope. All images are stored in a single directory and the naming is unequivocally unique (using timestamps) and always contain the imaging modality. This makes it easy to reconstruct the sequence of events during future image analyses. Image correlation is currently only 2D, but it is possible (in the python UI) to define the position of milling patterns directly from the correlated image, therefore it is possible to use the fluorescence signal to directly define the location of the lamellae.
    An example of the workflow is shown in Figure 3 and Video 2.


    Figure 3. Suggested sample workflow when using the PIE-scope

    Video 2. S1 PIEscope CorrVideo

  2. Using the PIE-scope
    The first step, after the LM has been mounted and aligned, is to measure the exact position of the optical axis relative to the coincidence point of the FIB/SEM. This step requires a fiducial that can be recognized in all modalities. The calibration consists in estimating the shift between the coincident point and the expected position and adjusted by centring the fiducial. Write down the absolute coordinates the shift values are then inserted in the control software to automatically perform direct movements between imaging modalities.
       In the software, we assumed 2 positions (see Figure 1 and Video 1), which can be customized: (i) LM imaging, where the sample orientation is normal to the optical axis of the LM; (ii) FIB imaging, where the sample orientation is normal to the optical axis of the FIB. Once the imaging positions have been defined it is possible to perform pre-computed relative movements (shifts, tilt, and compucentric rotations) to image the same ROI under different modalities. The control software interface allows direct control of the FIB/SEM, a visual comparison between the FIB/SEM and LM images and saving the data in organized sub-directories.
        In PIE-scope we implemented a basic correlation procedure (Figures 4-5), which leads the user to identify the location of the region of interest in the FIB or SEM image. Although using the proposed procedure is optional and specific use-cases might benefit from custom-designed image processing, we find that the availability of a general method already present and embedded in the software greatly enhances the usability. The PIE-scope correlation is performed through custom made python scripts that allow selecting multiple points on the LM and FIB/SEM images to calculate the appropriate transformation. 2D correlation is performed simply by applying an affine transformation that includes anisotropic scaling to match the pixel spacing resulting from imaging a sample from different tilts. This procedure is best suited for 2D correlation and, according to previous reports (Kukulski et al., 2011; Schorb and Briggs, 2014), it can lead to correlation precisions which are better than 100 nm.
       Once completed, the correlated image can be used to directly select the locations of the milling patterns for the FIB (Figure 6).


    Figure 4. Overview of the GUI. In blu the control panel which includes the focusing options, the correlation commands and the setup for multi-channel and volume imaging using the fluorescence microscope. The red box identifies the light microscopy panel, while the green box shows the FIB/SEM imaging panel


    Figure 5. The correlation panel allows for manually selecting matching features in light and focused ion beam microscopy. This part of the suite is available offline to allow the preparation of correlated images.


    Figure 6. The milling parameter selection. Once the correlated image is generated it is possible to define milling boxes or patterns directly from the overlay. This increases the efficiency in the selection of the locations to mill.

Acknowledgments

This work was funded through the ARC centre of excellence in Advanced Molecular Imaging and by the ARC Laureate fellowship program. The original article describing the PIE-scope is Gorelick et al. (2019).

Competing interests

The authors declare no competing interests.

References

  1. Agronskaia, A. V., Valentijn, J. A., van Driel, L. F., Schneijdenberg, C. T., Humbel, B. M., van Bergen en Henegouwen, P. M., Verkleij, A. J., Koster, A. J. and Gerritsen, H. C. (2008). Integrated fluorescence and transmission electron microscopy. J Struct Biol 164(2): 183-189.
  2. Arnold, J., Mahamid, J., Lucic, V., de Marco, A., Fernandez, J. J., Laugks, T., Mayer, T., Hyman, A. A., Baumeister, W. and Plitzko, J. M. (2016). Site-specific cryo-focused ion beam sample preparation guided by 3D correlative microscopy. Biophys J 110(4): 860-869.
  3. Beck, M. and Baumeister, W. (2016). Cryo-electron tomography: can it reveal the molecular sociology of cells in atomic detail? Trends Cell Biol 26(11): 825-837.
  4. Gorelick, S., Buckley, G., Gervinskas, G., Johnson, T. K., Handley, A., Caggiano, M. P., Whisstock, J. C., Pocock, R., de Marco A. (2019). PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy. eLife 8: e45919.
  5. Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M. and Briggs, J. A. (2011). Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J Cell Biol 192(1): 111-119. 
  6. Marko, M., Hsieh, C., Moberlychan, W., Mannella, C. A. and Frank, J. (2006). Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy of frozen-hydrated biological samples. J Microsc 222(Pt 1): 42-47. 
  7. Marko, M., Hsieh, C., Schalek, R., Frank, J. and Mannella, C. (2007). Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nat Methods 4(3): 215-217. 
  8. Sartori, A., Gatz, R., Beck, F., Rigort, A., Baumeister, W. and Plitzko, J. M. (2007). Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. J Struct Biol 160(2): 135-145. 
  9. Schorb, M. and Briggs, J. A. (2014). Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity. Ultramicroscopy 143: 24-32.
  10. Buckley, G., Gervinskas, G., Taveneau, C., Venugopal, H., Whisstock, J. C., de Marco, A. (2020). Automated cryo-lamella preparation for high-throughput in-situ structural biology. J Struct Biol 210(2): 107488.

简介

[摘要] 低温电子断层扫描(cryo-ET)是一种能够直接在细胞环境中解析大分子复合物结构的方法。但是,原位Cryo-ET的样品制备需要大量人工,并且可能需要通过低温聚焦离子束(FIB)研磨和相关光学显微镜来制备低温薄片,以确保感兴趣的事件存在于薄片中。在这里,我们介绍了一个集成的低温FIB和光学显微镜设置,称为E 上的P hoton I 电子显微镜(PIE-scope),可以直接和快速分离包含目标蛋白质复合物的细胞区域。尽管我们提供的图纸旨在在ThermoFisher DualBeams上进行细微的机械改造,但可以在其他品牌上使用,但PIE显微镜可以在现有显微镜上进行改装。

[背景 ] 低温电子断层扫描(低温ET)是目前用于直接在其天然环境中研究的蛋白和蛋白复合物的结构的主要方法(Beck和鲍迈斯特,2016) 。细胞通常太厚而无法使用cryo-ET成像,因此,要克服细胞厚度的问题,最常见且成功的方法是使用低温聚焦离子束显微镜(FIB)来稀释样品并产生平面电子。大约300纳米厚的透明薄片(Marko 等,2006和2007)。重要的是要注意细胞具有极为拥挤和复杂的环境; 因此,必须针对包含研究中的事件或结构的区域制备薄片。通常采用的方法是基于相关显微镜(CLEM),其中荧光显微镜用于识别感兴趣区域的位置,然后通过图像相关使用FIB找到相同区域(Sartori 等,2007)。; Arnold 等,2016 )。完整的cryo-CLEM工作流程将光学显微镜(活细胞成像和cryo-LM),cryo-FIB和cryo-ET连接在一起。工作流程通常从对活细胞或组织成像开始,以获得有关过程动态的信息。在适当的情况下,通过插入或高压冷冻将样品玻璃化。从这一刻起,每次将样品转移到显微镜内部或外部时,污染和损坏的机会就会增加。

可以使用集成方法来减轻与上述低温转移相关的所有风险,在集成方法中,将光学显微镜集成到电子显微镜的真空室中。这种方法已经成功地用于CLEM,其中在TEM柱中引入光学显微镜物镜可以在TEM和LM成像之间切换,这被称为iLEM (Agronskaia 等,2008)。在低温FIB上,集成相关系统的第一个示例是PIE范围(Gorelick 等,2019)。PIE显微镜(代表光子离子电子显微镜)是外围设备,可以在现有的ThermoFisher DualBeam 显微镜上进行改装。组装的硬件设置如图1所示,组装过程如视频1所示。


图1.安装在ThermoFisher Helios G4上的PIE-scope硬件概述。A)是PIE-scope光路外部组件的视图。主要部件的位置在图中标出。B)是腔室的内部视图,显示了该组件如何安装在现有的FIB / SEM显微镜内。C)从计算机控制的角度来看,我们在控制FIB / SEM的PC旁边有一台专用的PIE-scope PC,另外还有一台用于开发目的的支持PC,它允许开发人员在使用显微镜进行数据收集时进行工作。这不是在用户需要的- 因为仪器提供支持PC是足以控制PIE-范围基础设施。图中标出了所有主要部件(PIE显微镜和FIB / SEM显微镜)。

视频1. S3爆炸了PIE

该协议的目的是帮助实验室复制程序集,安装所需的硬件和软件。在自动化至关重要的复杂样品制备工作流程自动化的情况下,这将特别有用(Buckley 等,2020)。

关键字:光子-离子-电子显微镜, 低温板层, Cryo-CLEM, 相关显微镜, 低温光学显微镜




材料和试剂



玻璃化(冷冻保存)的细胞或组织

Cryo-EM网格(用于细胞培养的金,用于酵母或细菌悬浮液的铜)

自动格栅(Thermo Fisher,货号:1205101)


设备



双束显微镜(赛默飞世太阳神G4或Aquilos )

定制组件(已发布的图纸和装配体可以在Zenodo 上找到,网址为https://zenodo.org/record/3260173#.XsShV2gzb-g,而更新的图纸可以从https://www.demarco-lab.com/resources获得)

压电Z驱动器(Smaract ,型号:SLC-2445-DS-HV)

二向色开关(Smaract ,型号:SLC-2445-DS)

马达驱动器(Smaract ,型号:MCS-6CC-USB-TAB)

Z驱动器的真空馈通(Smaract ,型号:LEMO1B-FGJ-SJG-FGG-14)

电机驱动器传感器模块(Smaract ,型号:MCS-3S-EP-SDS15-TAB)

相机,Basler,acA1920-155 μ米USB 3.0

四线二向色性(色度,型号:89402bs)

四通道发射滤光片(色度,型号:89402m)

套筒镜(200毫米)(Thorlabs,型号:AC254-200-A-ML)

激发管透镜(400毫米)(Thorlabs,型号:AC254-400-A-ML)

发射镜(Thorlabs,型号:BBE1-E02)

物镜长镜(Edmund Optics,32366)

法兰镜(Edmund Optics,83536)

激光合成器(405-488-561-640),Toptica ,i CHROME CLE

法兰镜安装架(Thorlab s,型号:KMSS / M)

激励镜支架(Thorlabs,型号:KMSS / M)

针孔(Thorlabs,型号:SM1D12D)

套筒镜安装架(Thorlabs,型号:SMR1 / M)

C型安装到SM1适配器(Thorlabs,型号:SM1A10TS)

物镜(20x / 0.5)(奥林巴斯,MPLFNL 20X / 0.45)

真空法兰(Thorlabs,型号:VPCH42-A)

FC / APC 光纤转SM1适配器(Thorlabs,型号:SM1FCA)

可调式管架(Thorlabs,型号:SM1V10)

管安装座(Thorlabs,型号:SM1L10)

O形圈和垫圈(Lesker ,各种以适合法兰)

10/90 R / T BeamSplitter (Thorlabs,型号:BS025)


软件



当前,有两个选项可用于控制PIE范围。



选项1的软件要求


Python 3.5或更高版本

ThermoFisher Autoscript 4.1或更高版本

PyPylon 1.0或更高

Python库:Numpy

National Instruments Labview 2015或更高版本


选项2的软件要求:


Python 3.5或更高版本

Python库:Numpy,PyQt,Scitek图像

PyPylon 1.0或更高

ThermoFisher Autoscript 4.1或更高版本

除了商业软件外,所有python代码和软件包都可以从我们的实验室资源页面或Github 页面( https://github.com/demarcolab)下载。通用软件架构如图2所示。



D:\ Reformatting \ 2020-8-3 \ 1902883--1511 Alex Marco 810684 \ Figs jpg \图2.jpg


图2.图显示了硬件和软件组件之间的关系。白框标识用于控制各种组件的API。



程序



PIE-scope组装

对于打算组装当前PIE范围设计副本的实验室,本部分非常有用。我们建议的设计应安装在显微镜真空室的前GIS(注气系统)法兰上。所使用的法兰是GIS2,已为其设计了真空馈通和反光镜支架。因此,如果在任何前端口上都有GIS,则应尽可能在GIS端口4或5上移动它。如果这些端口已在使用中,并且一个GIS必须留在前面,我们建议使用GIS端口3,而保留端口1空闲。呈现的设计需要定制的组件,所有图纸都可以从https://www.demarco-lab.com/resources下载。如果建议的设计不适合特定的显微镜配置(例如,与检测器或操纵器发生碰撞),则可以通过修改自定义组件的设计来更改腔室内PIE 显微镜的位置或方向。


  此外,并非所有组件都必须使用定制零件来复制:PIE示波器的照明和激发臂可以使用30 mm Thorlabs笼,Thorlabs CERNA组件或类似组件组装。PIE-scope的早期原型是使用Thorlabs笼式组件实现的,但是为了增加对准稳定性并提高系统的紧凑性,我们决定选择整体式铝制机身。



该组件包括2个主要组件:


聚焦驱动器组件

大气设置

聚焦驱动器组件由整体式支架组成,该支架固定在位于前GIS端口和FIB柱之间的真空室壁上的2个M6螺栓上。支架使用弹簧销固定一个长镜(埃德蒙光学)。用于聚焦的压电线性定位器(Smaract )直接用螺栓固定在支架的正面。使用自定义适配器将物镜安装在定位器上,提供的图纸可用于任何Olympus物镜,并且定位器将能够在重量小于150 g的任何物镜下运行。


  聚焦驱动器(LEMO)的电气馈通可以放置在任何可用的法兰上,我们使用GIS端口3。


  大气设置从真空法兰开始。光路的真空馈通是通过石英窗实现的。为此,我们使用了高真空兼容的窗户法兰(Thorlabs)。法兰安装在将GIS法兰转换为CF-40的适配器上。该适配器还允许将显微镜主体安装到真空室。



显微镜主体由2个主要组件组成:


坚固的托架,用于控制光束路径,以使从物镜的后焦平面到光源和检测器的反射角之和为90度的倍数。

主体由激励臂和发射臂组成。该组件还承载二向色镜,光纤适配器,套管透镜和检测器。

位于支架上的反光镜安装在可调节的反光镜安装座上,可在2个角度上转向,因此可补偿机械公差(该组件的装配接近1毫米)。通过定制适配器将二向色镜(Chroma)和90/10分束器(Thorlabs)安装在电动线性定位器(Smaract )上。在分光镜和二向色镜之间切换可在反射光和荧光成像之间进行选择。


  套筒透镜直接安装在主体上,其位置无法调节。在显微镜主体的两个臂的末端,有检测器和光纤。做出设计90度扭结的发射臂的决定是为了使整个组件更紧凑,因此更不易发生振动。原则上,该臂可以伸直,特别是如果用于身体的部件是基于Thorlabs Cerna 或类似的东西时,但必须意识到该臂会伸出到仪器前方的当前占地面积之外,并且可能通过DualBeam的大门装载样品时的结果。


  激光源是Toptica iCLE-50,可确保在四个通道(405、488、561、640 nm)上产生50mW的激光功率。根据我们的经验,较低的功率将是合适的,并且我们计算出10mW足够用于此显微镜。该检测器当前是Basler acA1920-155 um USB 3.0。这是一种经济高效的解决方案,如果需要更高的感光度,则还具有非常轻便和紧凑的优势,我们建议使用Hamamatsu flash 4 V2或V3相机。PIE镜主体的当前设计已考虑到此相机的重量和尺寸。


  所有定位器均来自Smaract ,因此,可以通过单个电动机驱动器对其进行控制。有多种选择,有些带有USB接口,有些带有以太网,根据驱动器的选择,必须选择PIE-scope GUI。USB驱动器仅与Labview 接口兼容,而以太网版本则允许两者同时使用。因此,我们建议购买以太网版本。


  通过连接到FIB / SEM显微镜PC的同一本地网络的计算机控制PIE-scope。始终随FIB / SEM一起出售的支持PC已经适合该任务,但是从经验来看,拥有一台专用计算机会更容易,因此可以添加额外的以太网和USB 3.0端口。当前,我们将HPz4 工作站与额外的以太网卡一起使用,以确保有足够的端口可用。PC与普通网络有1个连接以传输数据,而与本地显微镜网络有1个连接以与显微镜PC通信。需要第三个连接来连接Smaract MCS电机驱动器。激光和相机通过USB 3.0 连接。Basler相机是加热的CMOS相机,需要USB端口提供2A的电流,必须注意,并非所有端口都符合规格,因此我们建议购买专用的USB 3.0扩展卡。此外,为了实现实时处理和图像分割,我们建议至少使用32 Gb的内存和专用GPU。             


  为了控制PIE范围,我们提供了2种选择:一种完全基于python,其中集成了所有功能;另一种是基于python的。一种是基于Labview 的软件,由于Basler SDK的限制,无法集成实时摄像机视图。这两个版本的软件均包含所有必需的控件,以实现使用FIB,SEM和光学显微镜的成像。所有图像都存储在一个目录中,并且命名是唯一的(使用时间戳),并且始终包含成像模式。这使得在将来的图像分析过程中重建事件序列变得容易。图像相关性目前仅为2D,但可以(在python UI中)直接从相关图像中定义铣削图案的位置,因此可以使用荧光信号直接定义薄片的位置。


工作流的一个示例如图3和视频2所示。



D:\ Reformatting \ 2020-8-3 \ 1902883--1511 Alex Marco 810684 \ Figs jpg \ fig3--word.jpg


图3.使用PIE范围时建议的示例工作流程






视频2. S1 PIEscope CorrVideo



使用PIE范围

LM已安装并对准后,第一步是测量光轴相对于FIB / SEM的重合点的准确位置。此步骤需要一个可以在所有方式中识别的基准。校准包括估计重合点和预期位置之间的偏移,并通过将基准点居中进行调整。写下绝对坐标,然后将偏移值插入控制软件,以自动执行成像模态之间的直接运动。


  在软件中,我们假定2个位置(参照˚F igure 1和V IDEO 1),该可定制:(我)LM成像,其中所述样品取向垂直于LM的光轴; (ii)FIB成像,其中样品方向垂直于FIB的光轴。一旦定义了成像位置,便可以执行预先计算的相对运动(移位,倾斜和复合中心旋转),以在不同模式下成像相同的ROI。控制软件界面允许直接控制FIB / SEM,在FIB / SEM和LM图像之间进行可视比较,并将数据保存在有组织的子目录中。


  在PIE-scope中,我们实施了一个基本的关联过程(图4-5),该过程使用户可以识别FIB或SEM图像中感兴趣区域的位置。尽管使用建议的过程是可选的,并且特定的用例可能会受益于定制设计的图像处理,但是我们发现,已经存在并嵌入在软件中的通用方法的可用性极大地增强了可用性。通过定制的python脚本执行PIE范围关联,该脚本允许在LM和FIB / SEM图像上选择多个点以计算适当的变换。只需通过应用仿射变换即可执行2D相关,该仿射变换包括各向异性缩放以匹配从不同角度对样本进行成像得到的像素间距。此过程最适合二维关联,并且根据以前的报告(Kukulski 等,2011 ;Schorb和Briggs,2014),它可以产生优于100 nm的关联精度。


  完成后,相关图像可以用于直接选择FIB的铣削图案的位置(图6)。



D:\ Reformatting \ 2020-8-3 \ 1902883--1511 Alex Marco 810684 \ Figs jpg \图4.jpg


图4. GUI概述。在蓝色中,控制面板包括聚焦选项,相关命令以及使用荧光显微镜进行多通道和体积成像的设置。红色框表示光学显微镜面板,绿色框表示FIB / SEM成像面板



D:\ Reformatting \ 2020-8-3 \ 1902883--1511 Alex Marco 810684 \ Figs jpg \图5.jpg


图5.相关面板允许在光学和聚焦离子束显微镜中手动选择匹配特征。该套件的这一部分可以脱机使用,以准备相关图像。



D:\ Reformatting \ 2020-8-3 \ 1902883--1511 Alex Marco 810684 \ Figs jpg \图6.jpg


图6.铣削参数选择。一旦生成了相关图像,就可以直接从覆盖图中定义铣削框或图案。这提高了铣削位置选择的效率。



致谢



这项工作是由ARC 高级分子成像卓越中心和ARC获奖者研究金计划资助的。描述PIE范围的原始文章是Gorelick 等。(2019 )。



利益争夺



作者声明没有利益冲突



参考文献



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Copyright Gorelick et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gorelick, S., Dierickx, D. A., Buckley, G., Whisstock, J. C. and Marco, A. d. (2020). Assembly and Imaging Set up of PIE-Scope. Bio-protocol 10(19): e3768. DOI: 10.21769/BioProtoc.3768.
  2. Gorelick, S., Buckley, G., Gervinskas, G., Johnson, T. K., Handley, A., Caggiano, M. P., Whisstock, J. C., Pocock, R., de Marco A. (2019). PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy. ELife 8: e45919.
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