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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)

SC Simao Coelho *
JB Jongho Baek *
JG J. Justin Gooding
KG Katharina Gaus

 (*contributed equally to this work) 发布: 2021年07月05日第11卷第13期 DOI: 10.21769/BioProtoc.4074 浏览次数: 7850

评审: Sijie WeiVikash VermaAnonymous reviewer(s)

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

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

English
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文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

    3. ris ris Download Citation in RIS Format

分类

生物工程 > 生物医学工程

生物物理学 > 显微技术

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