Total internal reflection fluorescence (TIRF) microscopy is widely used to examine the localization and dynamics of molecular events near the coverslips1. TIRF relies on the use of an evanescent wave generated at the interface between two media of different refractive indices (RI) to selectively illuminate fluorophores at this interface.
Since its axial-resolution and background suppression is superior to most other optical microscopy methods, TIRF has often been used to study dynamic events with single molecule sensitivity. However, precise measurement of such small point sources requires uncompromising positional accuracy in all three dimensions. Unfortunately, most commercial systems are at best capable of actively correcting for Z-axis instability. Developed by Drs. Robert Tjian and Steven Chu of the Howard Hughes Medical Institute, the AIC single molecule TIRF (smTIRF) system is part of our Transcription Imaging Consortium. The key feature that differentiates the AIC smTIRF system from other commercial products is how the stage is stabilized for long-term data acquisition in all three dimensions. The system is equipped with a NA 1.49 60X objective, a three-axis nano-positioning stage, 405nm, 488nm, 532 nm and 640 nm lasers, and two separate 512 × 512-pixel EMCCD cameras (195 nm per pixel) for simultaneous dual channel image acquisition.
The unique capabilities of smTIRF at the AIC
The x-y-z thermal stage drift can generate deleterious effect on TIRF microscopy, especially when precise spatiotemporal measurements are needed for single molecule imaging. To compensate for this stage drift, we use an 850 nm LED to capture reference images of beads on the sample coverslip with a separate CCD camera (IRCCD). The 3D position of each bead is determined in real time at 30 Hz, and averaged over 30 frames. Compensatory Δx, Δy, and Δz corrections are then conveyed to the nano-positioning stage at 1 Hz for feedback controlled position stabilization2. A schematic of the system and bead positioning system is shown in Figure 1.
With its enhanced signal-to-noise ratio, and thin axial resolution, TIRF is generally well suited for the examination of molecular dynamics at the specimen-coverslip interface, such as the studies on focal adhesion, cell membrane receptors, endocytosis and extracellular matrix. The smTIRF at the AIC is further tailor-designed for prolonged (hours), multicolor imaging at the single molecule level, or in single cell applications wherein nanoscale x-y-z spatial information/stability is crucial. At Janelia, we have successfully used the smTIRF system to study the dynamic behavior of transcription factors both in vitro and in live cells3 (See Movie 1 and Figure 2]. Because the smTIRF combines high temporal resolution (ca. 25ms) with extended imaging times (>1hr, subject to sample/flurophore stability) due to its excellent positional stability, unique experiments can be conducted. For instance, the fast dynamic behavior of cell surface receptors can be longitudinally monitored within the framework of slower cell-cycle dependent mechanisms over multiple cell divisions.
Movie 1: Sox2 transcription factor binding to DNA targets with varying affinity. See Chen, et al., Cell 156(6), 1274–1285 (2014)
Strengths
• Drift correction in all three dimensions
• Single molecule sensitivity
• Wide-field detection and strong background suppression
• Simultaneous dual-color detection. Two-camera set-up maximizes available FOV.
• Suitable for studying fast dynamic biological activities over prolonged periods
• No restriction on the fluorophores
• Lateral resolution: 200 – 250 nm
• Axial resolution: 100 nm (typical), 30-80 nm (best)
• Temporal resolution: ca. 25ms.
Limitations
• Best suited for biological activities on membrane surfaces or those that can be covalently bound or functionalized to the surface of a coverslip.
Further Reading
1. Axelrod, D. Chapter 7: Total internal reflection fluorescence microscopy. Methods Cell Biol. 89, 169–221 (2008).
2. Revyakin, A. et al. Transcription initiation by human RNA polymerase II visualized at single-molecule resolution. Genes Dev 26, 1691–1702 (2012).
3. Chen, J. et al. Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell 156, 1274–1285 (2014).