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 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 …

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)