Interference Reflection Microscopy for Label-Free Visualization of Microtubule Dynamics

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

1. 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.
2. Use a high magnification/high numerical aperture (NA) oil objective.
   NOTE: In this protocol, we used a 100x/1.3 NA objective.

2. Chamber preparation ....

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 obje.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	2048x2048 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)