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Apr 2020
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Building a Total Internal Reflection Microscope (TIRF) with Active Stabilization (Feedback SMLM)
建立一个主动稳定的全内反射显微镜(TIRF)   

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Abstract

The data quality of high-resolution imaging can be markedly improved with active stabilization, which is based on feedback loops within the microscope that maintain the sample in the same location throughout the experiment. The purpose is to provide a highly accurate focus lock, therefore eliminating drift and improving localization precision. Here, we describe a step-by-step protocol for building a total internal reflection microscope combined with the feedback loops necessary for sample and detection stabilization, which we routinely use in single-molecule localization microscopy (SMLM). The performance of the final microscope with feedback loops, called feedback SMLM, has previously been described. We demonstrate how to build a replica of our system and include a list of the necessary optical components, tips, and an alignment strategy.

Keywords: TIRF microscopy (TIRF显微镜), Drift correction (漂移修正), Active stabilization (积极稳定), Single-molecule imaging (单分子成像), Localization microscopy (本地化显微镜), Biophysics (生物物理学)

Background

Optical microscopy is routinely used to image the spatial and temporal coordinates of individual molecules. Of the many different techniques, total internal reflection fluorescence (TIRF) microscopy is extensively used to image cells seeded onto glass coverslips. Excitation under TIRF is achieved by adjusting the laser incidence angle to a value greater than the critical angle (Axelrod, 2001; Fish, 2009). This creates an evanescent field in the specimen medium immediately adjacent to the glass-water interface, restricting the depth of the illumination to ~200 nm. As only the contact area between the glass and the cell is imaged, TIRF has an excellent signal-to-noise ratio; however, mechanical movement of the sample in 3D reduces the precision of experiments. For conditions of low-photon emission, such as single-molecule imaging, drift reduces the localization precision and therefore decreases the overall quality of the data. Here, we provide a step-by-step protocol showing how to build the feedback SMLM (Coelho et al., 2020a), thereby facilitating user development and/or integration. The protocol incorporates a TIRF microscope and active stabilization to eliminate drift (Coelho et al., 2020b).


Active stabilization is compatible with multiple types of single-molecule acquisition methods, including TIRF (Fish, 2009; Kim et al., 2020), highly inclined and laminated optical sheets (Tokunaga et al., 2008), stochastic optical reconstruction microscopy (STORM), photo-activated localization microscopy (PALM), DNA points accumulation in nanoscale topography (DNA-PAINT), in 3D (e.g., 3D-STORM (Huang et al., 2008), double-helix-PSF (Pavani et al., 2009), saddle-point PSF (Shechtman et al., 2014), 4-PI (Shtengel et al., 2009), fixed- and live-cell imaging (Shroff et al., 2008), waveguides (Diekmann et al., 2017), light-sheet approaches (Gao et al., 2014; Huang et al., 2016; Baek et al., 2017), fluorescence resonance energy transfer (Aoki et al., 2009; Poland et al., 2014 and 2015) and lifetime imaging (Krstajić et al., 2013; Suhling et al., 2015 and 2017), adaptive optics (Coelho et al., 2013 and 2020c; Burke et al., 2015), and point-detection schemes (Eggeling et al., 2009). It can be further incorporated into high-content screening (Boutros et al., 2015; Gustavsson et al., 2018), multiplexed acquisitions (Jungmann et al., 2014) and/or automatic acquisition, as well as non-fluorescence imaging methods that require focus-locking with high precision, such as atomic force microscopy (Giessibl et al., 2003; Schmidt et al., 2018).

Equipment

Illumination

  1. White LED (Mightex, catalog number: BLSLCS-4000-03-22)

  2. Infrared LED (Mightex, catalog number: BLS-LCS-4000-03-22)

  3. LED control box (Mightex, catalog number: BLS-SA02-US)

  4. Lasers (Vortran Stradus, catalog numbers: 405-100 [405 nm]; 488-150 [488 nm]); 637-180 [637 nm])


Optical components
  1. Bandpass filter (Semrock, catalog number: FF01-842/56-25)

  2. Dichroic beamsplitter (Semrock, catalog number: FF801-Di02-25x36 and Chroma, catalog number: ZT488/640rpc)

  3. Emission filter (Semrock, catalog number: Em01- R405/488/635-25)

  4. Dichroic mirrors (Chroma, catalog number: ZT442rdc and ZT594rdc)

  5. Aspheric condenser lens (Thorlabs, catalog number: ACL25416U-B)

  6. Infrared achromatic doublets lens (Thorlabs, catalog number: AC254-200-B-ML)

  7. Visible achromatic doublets lens (Thorlabs, catalog numbers: AC254-300-A-ML, AC254-200-A-ML, AC254-30-A-ML, AC254-50-A-ML)

  8. Polarization-maintaining fiber (Thorlabs, catalog number: P3-405BPM-FC-2)

  9. Elliptical mirror (Thorlabs, catalog number: BBE1-E03)

  10. Oil-immersion objective, 100× Apo SR TIRF objective, numerical aperture (NA) = 1.49, working distance (WD) = 0.12 (Nikon)


Mechanical components
  1. Fiber port (Thorlabs, catalog number: PAF2-A7A)

  2. Optical post (Thorlabs, catalog numbers: TR75/M and TR50/M)

  3. Optical post spacers (Thorlabs, catalog numbers: RS4/M, RS5/M and RS10/M)

  4. Pedestal post holder (Thorlabs, catalog number: PH100E/M and PH50E/M)

  5. Cage assembly rod (Thorlabs, catalog number: ER025 and ER4-P4)

  6. Cage XY translator (Thorlabs, catalog number: CXY1)

  7. Elliptical mirror mount (Thorlabs, catalog number: KCB1E/M)

  8. Mirror mounts (Polaris-K25S4/M)

  9. Piezoelectric mirror (Thorlabs, catalog number: Polaris-K1S3P)

  10. Threaded standard cage plates (Thorlabs, catalog number: CP33/M)

  11. Clamping forks (Thorlabs, catalog number: CF125C/M-P5)

  12. M6 cap screw and hardware kit (Thorlabs, catalog number: HW-KIT2/M) 

  13. Cage alignment plate (Thorlabs, catalog number: CPA1)

  14. Adapter C-Mount to SM1 (Thorlabs, catalog number: SM1A39 and SM1A9)

  15. Lens tubes (Thorlabs, catalog number: SM1)

  16. Actively stabilized optical table (Newport, catalog number: M-ST-46-8)

  17. Smart table controller (Newport, catalog number: ST-300)

  18. Microscope frame (Mad City Labs, catalog number: RM21-M)

  19. Cage-compatible rectangular filter holder (Thorlabs, catalog number: FFM1)

  20. Support bracket (Custom designed, CAD:  https://github.com/spcoelho/Active-Stabilization.git)

  21. Cage cube (Thorlabs, catalog number: C6W)

  22. Blank cover plate (Thorlabs, catalog number: B1C/M)

  23. Kinematic cage cube platform (Thorlabs, catalog number: B4C/M)

  24. XYZ translation stage with standard micrometers (Thorlabs, catalog number: PT3/M)

  25. Right-angle kinematic elliptical mirror mount (Thorlabs, catalog number: KCB1E/M)

  26. Camera baseplate (Manta, ¼-20 Tripod Adapter)

  27. Translation stage (Newport, catalog number: M-423-MIC)

  28. Threaded frosted glass alignment disk (Thorlabs, catalog number: DG10-1500-H1-MD)


Cameras
  1. CMOS camera (Allied Vision, Manta Camera)

  2. EMCCD (Andor, catalog number: 897)

Software

  1. Active Stabilization Software and Custom Bracket: https://github.com/spcoelho/Active-Stabilization.git

  2. NicoLase: https://github.com/PRNicovich/NicoLase


Resources
  1. How to Align a Laser: https://www.youtube.com/watch?v=qzxILY6nOmA&t=311s

  2. Coupling a Laser into a Fiber: https://www.youtube.com/watch?v=kQvhbJbDG0M

  3. Collimating a Laser Beam: https://www.youtube.com/watch?v=Z7Q17-ctQVQ

  4. TIRF Microscopy: https://www.youtube.com/watch?v=egmJIalDR48&t=1039s

Procedure

  1. TIRF Assembly

    The TIRF microscope that we describe is built on a Mad City Labs RM21 body. This microscope frame is convenient as it allows for easy access to the optical components within the frame, facilitating alignment. The RM21 frame is particularly stable, and the rectangular geometry permits simple addition of support brackets.

    1. The first step is to place the microscope body on the optical table. Figure 1 is a top-down view of a CAD design showing the location of the microscope frame in relation to the rest of the optical components. We suggest placing the body close to the center of the optical table. This then enables enclosing of the microscope in the final stages.



      Figure 1. CAD design showing a top-down view of the optical assembly. A. Without the microscope frame. B. With the microscope frame.


    2. Next, lasers are placed onto a heat sink at the appropriate height. Currently, we use Vortran lasers (detailed in the components section) that can be purchased with clean-up filters to minimize extra components. Compact and simple designs have been previously described, for example by Nicovich et al. (2017). Links in the paper provide good guidelines on assembling the lasers (including designs) and useful resources including triggering and timing. Figure 2 below shows a close-up of the design that we implemented. Three lasers (405 nm, 488 nm, and 640 nm) are placed onto a heat sink, filtered using laser filters, and combined into a single line using dichroics. In practice, more lasers can be combined if necessary (e.g., 561 nm) by expanding the design. The lasers are then aligned into a fiber. Once installed, we expect ~70% coupling efficiency.



      Figure 2. Laser assembly and fiber coupling. Three lasers (405 nm, 488 nm, and 640 nm) are placed onto a heat sink, combined using dichroics, and coupled to a laser fiber.


    3. Place the output of the optical fiber at the intended height of the expansion optics. Ensure that the laser output is straight and travels parallel to the table without deviating.

    4. Set up the laser expansion cage assembly (cage rods, ×2 right-angle mirror mounts, and ×3 cage XY translation mounts) attached to a custom RM21 bracket frame (https://github.com/spcoelho/Active-Stabilization-Design) and place at the required height. We recommend using steel pedestals to ensure greater stability. Figure 3 illustrates the assembly.



      Figure 3. Assembly of the laser expansion


    5. Align the laser so that it is centered throughout the cage assembly.

    6. Place the lenses to provide ~10-fold expansion of the laser (e.g., 30 mm and 300 mm). Ensure that these are placed at the correct distance from each other using a Shearing Interferometer.

    7. Assemble the mirror and tube lens (200 mm focal length) on top of the translation stage. The translation stage moves the lens and mirror assembly together. Ensure that the reflected beam is straight and parallel to the optical table. Place the mirror/lens centered within the frame of the microscope.

    8. Take the dichroic holders (×2) and attach to each other. Place below the position of the imaging objective at the height of the laser.

    9. Attach the dichroics, via cage rods, to the microscope frame using the RM21 bracket (https://github.com/spcoelho/Active-Stabilization-Design) (Figure 4, left hand side and Figure 5).



      Figure 4. Mirror and tube lens are on top of a translation stage located within the microscope frame shown within the final assembly



      Figure 5. Internal components without the microscope body frame


    10. Place the dichroics within their holders. One dichroic is used to reflect the laser toward the sample/objective; the second dichroic is used to separate the infrared light for active stabilization. For more details on how to assemble and configure, please refer to Coelho et al. (2020b).

    11. Align the laser so that it is centered onto the back of the imaging objective. A simple method consists of replacing the objective for a Frosted Glass Alignment Disk and observing the position of the laser.

    12. To ensure that the tube lens is at the correct distance from the back of the objective, the laser output should be as small as possible after exiting the objective. While observing the laser profile at a large distance (e.g., ceiling), adjust the position of the tube lens to ensure that a Gaussian beam with minimal diameter exits the objective. Misalignments can lead to a distorted beam profile (e.g., astigmatic).


  2. Assemble the Infrared Camera/LED Path

    1. Assemble the cage system (cage rods, right angle mirror mount, and 200 mm infrared lens) and attach it to the dichroic cube (Figure 6).

    2. Secure to the microscope frame using an RM21 bracket (https://github.com/spcoelho/Active-Stabilization-Design).

    3. Attach the infrared camera using an SM1 adapter and connect to a PC.

    4. Assemble the infrared LED, place on the XYZ translation stage, and align onto the infrared camera [for more details, see Coelho et al. (2020b)].

    5. Turn on the infrared LED and check that the illumination is centered and uniform on the infrared camera.

    6. Place polystyrene beads on the glass coverslip and record their diffraction rings.



      Figure 6. Assembly of the cage system to secure the infrared camera. The components are shown in relation to the infrared LED without the brackets/microscope frame. A. Front view. B. 45° view.


  3. Detection (Emission camera and camera stabilization path)

    This section describes how to introduce an optical feedback loop for the emission path (Figure 7). This allows to correct for movement in the detection path, facilitating high stability for prolonged acquisitions.

    1. Emission Camera

      1. To direct the fluorescence from the sample toward the camera, attach a right-angle mirror mount to tubing and connect it to the bottom of the laser dichroic holder (Figure 7).



        Figure 7. Assembly of the fluorescence emission path. A. Left: Without the white LED. Right: With the white LED. B. Left: Only the camera stabilization path. Right: Detection assembly in relation to the infrared detection.


      2. Adjust the length of the tube to match the predicted height of the fluorescent camera.

      3. Within the tube, insert the emission filter (Em01-R405/488/635-25; Semrock). This removes the laser light and/or infrared LED.

      4. Attach the tube lens (e.g., 400 mm focal length).

        For the white LED: Attach the cage cube to the free side of the right-angle mirror mount.

        Note: If extra distance between the camera and the tube lens is required, add a pair of relay lenses (e.g., 2 × 50 mm).

      5. Secure the assembly to the RM21 microscope frame using cage rods and/or SM1 tubing.

      6. Attach the camera to an optical table at the correct distance away from the lens.

      7. Place a mirror (or piezo-electric mirror) in front of the camera.

      8. Adjust the focal length of the tube lens. A simple way to get the correct distance is to flip the laser dichroic so that it reflects toward the camera. Using a very low laser power and multiple neutral density filters, focus the laser onto the camera. To get the correct distance, also remove the laser filter and last lens in the laser excitation path (TIRF lens mounted on the translation stage).

      9. Place a uniform fluorescent sample (fluorescent molecules in solution or fluorescent marker) and check the emission onto the camera.

      10. Center the emission onto the camera using the elliptical mirror underneath the objective lens. The emission should be uniform across the recorded field-of-view.

    2. Camera Stabilization

      1. Attach the white LED to the microscope frame (Figures 4 and 7B left).

      2. Assemble the cage/tube system containing a pinhole (diameter = 50 µm) and lens (e.g., f = 400 mm).

      3. Center the pinhole and adjust the position of the lens to ensure a bright Gaussian spot focused on the camera.

      4. Position the white LED spot on the edge of the imaging field-of-view (Figure 8).



        Figure 8. Camera stabilization. A. Full camera image showing LED projection toward the edge chip. B. Zoomed in region highlighted in the yellow square.

Acknowledgments

We are thankful for support from the Australia Research Council (CE140100011 to K.G., FL150100060 and CE140100036 to J.J.G.) and the National Health and Medical Research Council of Australia (APP1059278 to K.G.). This protocol is based on previous work, mainly from Coelho et al. (2020a).

Competing interests

The authors declare no competing financial interests.

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简介

[摘要]高分辨率成像的数据质量可与主动稳定可以显着提高,这是基于显微镜内反馈回路的是保持样品在整个实验过程中相同的位置。目的是提供高精度的聚焦锁定,从而消除漂移并提高定位精度。这里,我们描述一个步骤一步协议用于构建荷兰国际集团全内反射显微镜与反馈回路结合必要的样品和检测稳定,这是我们经常在单一使用-分子定位显微镜(SMLM)。与反馈回路,被称为最终显微镜的性能˚F eedback SMLM,先前被描述。我们演示了如何构建我们的系统的副本,并包括列表必要的光学组件,技巧,以及对准策略。


[背景]光学显微镜通常用于对单个分子的空间和时间坐标进行成像。在许多不同的技术中,全内反射荧光 (TIRF)显微镜被广泛用于对接种到玻璃盖玻片上的细胞进行成像。TIRF 下的激发是通过将激光入射角调整到大于临界角的值来实现的( Axelrod, 2001; Fish , 20 09 ) 。这会在紧邻玻璃-水界面的样品介质中创建一个渐逝场,将照明深度限制为 ~200 nm。由于仅对玻璃和细胞之间的接触区域进行成像,因此 TIRF 具有出色的信噪比;ħ H但是,在3D试样的机械运动减少的实验精度。对于低光子发射的条件下,如单分子成像,漂移降低了定位精度和第erefore德CREAS ES的数据的整体质量。在这里,我们提供表示如何构建步骤一步协议˚F eedback SMLM(Coelho的等人,20 20 ),吨特此便于用户开发和/或整合。该协议结合了 TIRF 显微镜和主动稳定以消除漂移( Coelho等人, 20 20 ) 。

主动稳定是与多种类型的单分子采集方法(兼容例如,TIRF (鱼,20 09 ;金。等人,20 20 ),高度倾斜并且层叠光学片小号(德永等人。,20 08 ),小号tochastic光学重构显微术(STORM),光-激活的定位显微镜(PALM),DNA分积累在纳米级形貌(DNA-PAINT)等,在三维(例如,3D-STORM (黄等人。,20 08 ),双螺旋-PSF (Pavani等人,20 09 ),鞍点PSF (Shechtman等人,20 14 ),4-PI (Shtengel等人,20 09 )等固定。-和活细胞成像( Shroff et al. , 20 08 ) 、波导( Diekmann et al. , 20 17 ) 、光片方法(Gao et al. , 20 14 ; Huang et al. , 20 16 ; Baek et al. , 20 17 ) ,荧光共振能量转移(Aoki等,20 09 ;Poland等,20 14和20 15 )和寿命成像(Krstajić等,20 13 ;Suhling等,20 15和20 17 ),自适应光学(Coelho等人,20 13和2020;B 厄克等人 ,20 15 ),和点-检测方案(Eggeling等人。,20 09 )。它可以在被进一步掺入到高含量筛选(特罗斯等人,20 15 ; Gustavsson的。等人,20 18 ),多路复用收购(容曼等人,20 14 )和/或自动采集,以及非-fluorescence成像方法其需要聚焦锁定以高精度,例如原子力显微镜(Giessibl等人,20 03 ;施密特。等人,20 18 )。

关键字:TIRF显微镜, 漂移修正, 积极稳定, 单分子成像, 本地化显微镜, 生物物理学


设备
照明

白色 LED(Mightex ,目录号:BLSLCS-4000-03-22)
红外 LED(Mightex,目录号:BLS-LCS-4000-03-22)
LED 控制箱(Mightex,目录号:BLS-SA02-US)
激光器(Vortran Stradus ,目录号小号:405-100 [ 405纳米]; 488-150 [ 488纳米] ); 637-180 [ 637 纳米])


光学元件

带通滤波器(Semrock,目录号:FF01-842/56-25)
二向色分光镜(Semrock,目录号:FF801-Di02-25x36 和 Chroma,目录号:ZT488/640rpc)
排放过滤器(Semrock ,目录号:Em01-R405/488/635-25)
二向色镜(Chroma ,目录号:ZT442rdc 和 ZT594rdc)
非球面Ç ondenser升ENS(Thorlabs公司,目录号:ACL25416U-B)
红外一个色d oublets透镜(Thorlabs公司,目录号:AC254-200-B-ML)
可见一个色d oublets透镜(Thorlabs公司,目录号小号:AC254-300-A-ML,AC254-200-A-ML,AC254-30-A-ML,AC254-50-A-ML)
偏振米aintaining ˚F IBER (Thorlabs公司,目录号:P3-405BPM-FC-2)
椭圆米irror(Thorlabs公司,目录号:BBE1-E03)
油浸物镜,100×Apo SR TIRF 物镜,数值孔径 (NA) = 1.49,工作距离 (WD) = 0.12(尼康)


机械部件

光纤端口(Thorlabs,目录号:PAF2-A7A)
光学p OST(Thorlabs公司,目录号小号:TR75 / M和TR50 / M)
光学p OST小号步行者(Thorlabs公司,产品目录号:RS4 / M,RS5 / M和RS10 / M )
基座p OST ħ以上(Thorlabs公司,目录号:PH100E / M和PH50E / M)
笼一个ssembly ř OD(Thorlabs公司,目录号:ER025和ER4-P4)
笼XY吨ranslator(Thorlabs公司,目录号:CXY1)
椭圆米irror米'mount(Thorlabs公司,目录号:KCB1E / M)
镜米ounts(北极星-K25S4 / M)
压电镜(Thorlabs ,目录号:Polaris-K1S3P)
螺纹小号TANDARD Ç年龄p酸酯(Thorlabs公司,目录号:CP33 / M)
夹紧˚F兽人(Thorlabs公司,目录号:CF125C / M-P5)
M6 Ç AP小号船员和ħ ardware ķ它(Thorlabs公司,目录号:HW-KIT2 / M)              
笼一个lignment p晚期(Thorlabs公司,目录号:CPA1)
适配器 C-Mount 至 SM1(Thorlabs,目录号:SM1A39 和 SM1A9)
透镜吨ubes(Thorlabs公司,目录NUM BER:SM1)
主动稳定光学平台(Newport,目录号:M-ST-46-8)
智能桌控制器(Newport,目录号:ST-300)
显微镜框架(Mad City Labs,目录号:RM21-M)
Cage- Ç ompatible ř ectangular ˚F ILTER ħ以上(Thorlabs公司,目录号:FFM1)
支持b球拍(定制设计,CAD:https : //github.com/spcoelho/Active-Stabilization.git)             
Cage c ube(Thorlabs,目录号:C6W)
空白Ç超过p晚期(Thorlabs公司,目录号:B1C / M)
运动学Ç年龄Ç UBE p latform(Thorlabs公司,目录号:B 4 C / M)
XYZ吨ranslation小号塔格与小号TANDARD米icrometers(Thorlabs公司,目录号:PT3 / M)
右一ngle ķ inematic Ë lliptical米irror米'mount(Thorlabs公司,目录号:KCB1E / M)
相机底板(Manta,¼-20 三脚架适配器)
翻译阶段(Newport ,目录号:M-423-MIC)
螺纹˚F rosted克小姑娘一个lignment d ISK(Thorlabs公司,目录号:DG10-1500-H1-MD)


相机

CMOS 相机(Allied Vision、Manta 相机)
EMCCD(Andor,目录号:897)



软件


1.主动稳定软件和自定义B球拍:https : //github.com/spcoelho/Active-Stabilization.git     

2. NicoLase:https : //github.com/PRNicovich/NicoLase     



资源

如何对准激光:https : //www.youtube.com/watch?v=qzxILY6nOmA&t=311s
将激光耦合到光纤中:https : //www.youtube.com/watch?v=kQvhbJbDG0M
准直激光束:https : //www.youtube.com/watch?v= Z7Q17-ctQVQ
TIRF 显微镜:https : //www.youtube.com/watch?v= egmJIalDR48 & t = 1039s


程序


TIRF Assemb LY
该TIRF显微镜是我们描述是建立在一个疯狂的城市实验室RM21体。这种显微镜框架很方便,因为它可以轻松接触框架内的光学组件,便于对齐。RM21 框架特别稳定,矩形几何形状允许简单地添加支架。

第一步是将显微镜主体放在光学平台上。图 1 是 CAD 设计的俯视图,显示了显微镜框架相对于其余光学组件的位置。我们建议将主体放置在靠近光学平台中心的位置。这然后使得包围的在最后阶段的显微镜。


图1 。CAD 设计显示了光学组件的俯视图。A.随着出显微镜帧。B.带显微镜架。


接下来,将激光器放置在适当高度的散热器上。目前,我们使用Vortran激光器(中详细描述了可以与清理过滤器购买,以尽量减少外部元件的组件部分)。紧凑和简单的设计之前已经描述过,例如 Nicovich等人。( 20 17 )。论文中的链接提供了有关组装激光器(包括设计)和有用资源(包括触发和计时)的良好指南。如下图图2密切-向上的设计是我们实施。三个激光器(405纳米,488纳米,和640纳米)被放置在一个散热片,利用激光过滤器过滤,并组合成使用分光镜单行。在实践中,如果需要,可以通过扩展设计来组合更多的激光器(例如,561 nm)。然后将激光器对准成光纤。安装后,我们预计耦合效率约为 70%。




图2 。激光组装和光纤耦合。三个激光器(405纳米,488纳米,和640纳米)被放置在一个散热片上,使用二向色组合,以及耦合到一个激光纤维。


将光纤的输出放置在扩展光学器件的预期高度。确保该激光输出是直的,并且不偏离行进平行于该表。
设置的激光膨胀笼组件(笼杆,× 2右-角镜座,和× 3笼XY TR anslation坐骑)附接至自定义RM21托架框架(https://github.com/spcoelho/Active-Stabilization-设计),并在地方的需要的高度。我们建议使用钢底座以确保更高的稳定性。图 3 说明了该组件。




图 3. 激光扩展组件的组装


对准的激光,使其整个笼中心组件。
放置的透镜亲参见〜1的激光的0倍的扩增(例如,30个和300mm)。确保该这些被放置在彼此使用剪切干涉的正确距离。
组装的反射镜和透镜管(200毫米焦距)上的顶部的平移台。平移台将镜头和反射镜组件一起移动。确保该反射光束是直的并且平行于所述光学平台上。将镜子/镜头居中放置在显微镜框架内。
取的分色持有者(× 2)和彼此附接。放置在激光高度的成像目标位置下方。
附加的分光镜,通过笼杆,以在使用该RM21支架(显微镜帧https://github.com/spcoelho/Active-Stabilization-Design)(图4,左手侧和图5)。




图4.镜和管透镜是上位于最后的组装中所示的显微镜帧内的平移台的顶




图 5. 不带显微镜机身框架的内部组件


放置在分光镜其持有之内。一个二向色镜用于将激光反射到样品/目标;吨他第二分色用于为活性STA红外光分离bilization。有关如何组装和配置的更多详细信息,请参阅Coelho等人。(2020 年)。
对准激光,使其居中在成像物镜的背面。一种简单的方法包括更换磨砂玻璃校准盘的物镜并观察激光的位置。
为了确保管透镜是在正确的距离从所述物镜的后,激光输出应该离开物镜后是尽可能小。同时观察在一个大的距离(激光轮廓例如,天花板),调整管透镜的位置,以确保该高斯光束具有最小直径离开所述目标。未对准会导致光束轮廓失真(例如,散光)。


组装的红外Ç相机/ LED路径
组装的笼SYS TEM(笼杆,直角镜支架,和200mm红外线透镜),并将它附加到所述二向色立方体(图6)。
使用n RM21 支架 ( https://github.com/spcoelho/Active-Stabilization-Design )固定到显微镜框架上。
附加的使用SM1适配器,并连接到红外摄像机一个PC。
组装的红外线LED,在地方的XYZ平移台,并对准到所述红外线摄像机[更多细节请访问科埃略等人。( 20 20 ) ] 。
打开的红外LED和检查照明居中和红外摄像机上是均匀的。
对地方聚苯乙烯珠的玻璃盖玻片,并记录他们的衍射环。




图6.大会的笼系统以固定的红外摄像机。显示的组件与没有支架/显微镜框架的红外 LED 相关。A.前视图。B. 45 °视图。


检测(发射相机和相机稳定路径)
本节介绍如何为发射路径引入光反馈回路(图 7)。这允许校正检测路径中的移动,促进长时间采集的高稳定性。

发射相机
要将样品中的荧光引导至相机,请将直角镜架连接到管道上,并将其连接到激光二向色支架的底部(图 7)。




的图7.装配的荧光发射路径。A.左:没有白色 LED。右:带有白色 LED。B.左:只有相机稳定路径。右图:与红外检测相关的检测组件。


调整的长度的管以匹配荧光照相机的预测高度。
在管内,插入的发射滤光片(EM01-R405 / 488 / 635-25; Semrock)。这将移除激光和/或红外 LED。
附加的管透镜(例如,400个毫米焦距)。
对于白色LED:附加的笼立方体直角镜支架的自由侧。

注意:如果需要在摄像机和管透镜之间额外的距离,加一对中继透镜的ES (例如,2 × 50mm)上。

使用笼杆和/或 SM1 管将组件固定到 RM21 显微镜框架上。
将相机安装到距镜头正确距离的光学平台上。
在相机前放置一面镜子(或压电镜)。             
调整 Tube Lens 的焦距。获得正确距离的一种简单方法是翻转激光二向色镜,使其反射到相机。使用一个非常低的激光功率和多个中性密度过滤器,聚焦激光到照相机上。要获得正确的距离,还需卸下激光激发路径中的激光滤光片和最后一个透镜(安装在平移台上的 TIRF 透镜)。
放置一个均匀的荧光样品(在溶液中或荧光标记物的荧光分子),并检查所述到发射的相机。
使用物镜下方的椭圆镜将发射集中到相机上。在记录的视场中,发射应该是均匀的。
相机稳定
附着的白色LED到显微镜帧(图小号4和7乙左侧)。
组装包含针孔 (直径 = 50 µm) 和透镜 (例如, f = 400 毫米)的笼子/管系统。
CENTE r是针孔和调整所述的位置的透镜,以确保一个亮ģ aussian点聚焦照相机。
将白色 LED 光斑定位在成像视野的边缘(图 8)。




图 8. 相机的稳定性。A.显示 LED 向边缘芯片投影的完整相机图像。B.放大黄色方块中突出显示的区域。


致谢


我们都感谢了FUL从澳大利亚研究理事会(CE140100011到KG,FL150100060和CE140100036到JJG)和支持的国家卫生和澳大利亚的医学研究理事会(APP1059278到KG)。个是协议我S的基于以前的工作,主要是从科埃略等人。(2020 年)。


利益争夺


作者声明没有相互竞争的经济利益。


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Coelho, S., Baek, J., Gooding, J. J. and Gaus, K. (2021). Building a Total Internal Reflection Microscope (TIRF) with Active Stabilization (Feedback SMLM). Bio-protocol 11(13): e4074. DOI: 10.21769/BioProtoc.4074.
  2. Coelho, S., Baek, J., Graus, M.S., Halstead, J.M., Nicovich, P.R., Feher, K., Gandhi, H., Gooding, J.J. and Gaus, K. (2020). Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells. Sci Adv 6(16): eaay8271.
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