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Updated: Jan 20, 2026
Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules
Published on: August 8, 2019
Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules
Mohammed Mahamdeh1, Jonathon Howard2
1Department of Molecular Biophysics and Biochemistry, Yale University; Harvard Medical School, Harvard University.
Abstract:
There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and wide-field illumination microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.
Insights
Interference reflection microscopy (IRM) offers a cost-effective alternative to fluorescent labeling for visualizing biomolecules near surfaces. This method provides high-quality imaging of microtubule dynamics, comparable to TIRF microscopy, without interfering with molecular activity.
Area of Science:
- Biophysics
- Microscopy techniques
- Cell biology
Background:
- Total-internal reflection fluorescence (TIRF) microscopy is widely used for visualizing biomolecules near surfaces.
- TIRF microscopy requires fluorescent labeling, which can alter molecular activity and lead to photobleaching or photodamage.
- Alternative methods are needed for label-free visualization of biomolecular dynamics.
Purpose of the Study:
- To demonstrate that interference reflection microscopy (IRM) can achieve imaging quality comparable to TIRF microscopy for visualizing biomolecules near surfaces.
- To present a simple method for modifying a standard fluorescence microscope to perform IRM.
- To highlight IRM as a potentially general and cost-effective technique for in vitro studies of large biomolecules and oligomers.
Main Methods:
- Modification of a standard fluorescence microscope to enable interference reflection microscopy (IRM).
- Application of IRM for imaging microtubule dynamics in vitro.
- Utilizing image analysis software for high-precision measurements.
Main Results:
- IRM provides imaging quality similar to TIRF microscopy for microtubules.
- IRM is a simpler and more economical alternative to differential interference contrast microscopy and interferometric scattering microscopy.
- IRM is less sensitive to surface imperfections and impurities compared to darkfield microscopy.
- High-precision measurements (up to 20 nm) of microtubule length with a 10 Hz bandwidth are achievable with IRM and sCMOS cameras.
Conclusions:
- Interference reflection microscopy (IRM) is a viable and advantageous technique for visualizing large biomolecules and their dynamics near surfaces.
- IRM offers a cost-effective, label-free alternative to TIRF microscopy, preserving molecular function.
- The described IRM setup allows for precise, high-speed measurements, expanding possibilities for in vitro biophysical studies.
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