Interference Reflection Microscopy for Label-Free Visualization of Microtubule Dynamics
Published: July 31, 2023
Abstract
Source: Mahamdeh, M., et al. Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules. J. Vis. Exp. (2019).
In this video, we describe the interference reflection microscopy (IRM) technique to visualize microtubules growing on a coverslip surface in the presence of a suitable buffer. Upon illumination with incident light, the coverslip-buffer interface and buffer-microtubule interface reflect light, which combines to create an interference pattern, enabling the visualization of microtubules as high-contrast images against a bright background.
Protocol
1. Microscope modification and objective lens Insert a 50/50 mirror into the filter wheel of the fluorescent microscope using an appropriate filter cube (Figure 1). Handle the 50/50 mirror with care as often they have anti-reflection coating. NOTE: We used a 50/50 mirror in an empty filter cube of the microscope. The 50/50 mirror is inserted where the dichroic mirror is located. Use a high magnification/high numerical aperture (NA) oil object…
Representative Results
Figure 1. Schematic representation of IRM. (A) Epi-illumination from the light source passes through the aperture diaphragm before reaching the 50/50 mirror. The aperture diaphragm sets the beam width thus the illumination NA. The 50/50 mirror partially reflects the light up to the objective to illuminate the sample. Light reflected from the sample is collected and then projected onto the camera chip (by the tube lens) where it …
Offenlegungen
The authors have nothing to disclose.
Materials
| Microscope | Nikon | Ti-Eclipse | An inverted microscope used for perfoming the expriments |
| 50/50 beam splitter | Chroma | 21000 | When buying make sure to choose the splitter dimensions that fit the cube used in the microscope |
| NIKON PLAN FLUOR 100X/0.5-1.3 Iris objective | Nikon | MRH02902 | Imaging objective. This objective has a NA adjusting iris that was opened to NA 1.3 |
| Mucasol universal detergent | Sigma-Aldrich | Z637181-2L | Used for cleaning coverslips and slides |
| Plastic paraffin film (commerical name Parafilm M) | Sigma-Aldrich | P7793 | Used for constructing flow channels |
| Anti-TAMRA antibody | Invitrogen | A-6397 | Used to bind TAMRA-labeled molecules (e.g. microtubules) to the sample surface. RRID (AB_2536196) |
| Poloxamer 407 (commercial name Pluronic F-127) | Sigma-Aldrich | Used for blocking the channel surface to prevent nonspecific binding | |
| 40 nm gold nanoparticles | Sigma-Aldrich | 753637 | Used as a control sample |
| 20 nm gold nanoparticles | Sigma-Aldrich | 753610 | Used as a control sample |
| Zyla 4.2 Camera | Andor | Zyla 4.2 | 2048×2048 pixles (6.5µm pixel size) with quantum efficiency of 72% and 16bit dynamic range |
| Feista tracking software | https://www.bcube-dresden.de/ fiesta/wiki/FIESTA | ||
| Stabilized microtubules | Prepared in house (see references in text) |
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Diesen Artikel zitieren
Interference Reflection Microscopy for Label-Free Visualization of Microtubule Dynamics. J. Vis. Exp. (Pending Publication), e21459, doi: (2023).