LED-based interference-reflection microscopy combined with optical tweezers for quantitative three-dimensional microtubule imaging

Abstract

Optical tweezers combined with various microscopy techniques are a versatile tool for single-molecule force spectroscopy. However, some combinations may compromise measurements. Here, we combined optical tweezers with total-internal-reflection-fluorescence (TIRF) and interference-reflection microscopy (IRM). Using a light-emitting diode (LED) for IRM illumination, we show that single microtubules can be imaged with high contrast. Furthermore, we converted the IRM interference pattern of an upward bent microtubule to its three-dimensional (3D) profile calibrated against the optical tweezers and evanescent TIRF field. In general, LED-based IRM is a powerful method for high-contrast 3D microscopy.

Polycationic gold nanorods as multipurposein vitromicrotubule markers

“…4B) showed a mostly homogeneous dark contrast for the microtubules as expected for objects in surface proximity. 23 Qualitatively, microtubules appeared darker on APTES surfaces compared to PEGylated antibody surfaces indicating a smaller microtubule-surfaces distance for the APTES surface. Based on the TIRF and overlay images ( Fig.…”

Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

“…To avoid drift and diffusive light from any light source other than the illumination source, all adjustments during an experiment are controlled via motorized components from outside the dark inner microscope room. In addition, to visualize the sample, a bright field and interference reflection microscope (IRM, [23,24]) are included ( Fig. 1).…”

Interferometric Scattering (iSCAT) Microscopy and Related Techniques

“…In the decade that followed right up to the present day we see a broad array of research efforts, drawn from numerous communities, exploring interferometric detection of single nano-objects such as viruses, DNA, microtubules, exosomes, and proteins [75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91]. Interestingly, interferometric microscopies are also flourishing in the general context of label-free imaging of cells and membranes even if nanoparticles are not at the center of attention [75,76,90,[92][93][94][95][96][97][98][99][100][101][102][103][104][105][106]. The underlying physics of these methods remains the same although a plethora of acronyms such as interference reflectance imaging sensing (IRIS) [77], rotating coherent scattering (ROCS) [98], interference plasmonic imaging (iPM) [88], coherent bright-field imaging (COBRI) [107], stroboscopic interference scattering imaging (stroboSCAT) [108], interferometric scattering mass spectrometry (iSCAMS) [109] are on the rise.…”

“…What is important to realize, however, is that while most iSCAT measurements are performed with a laser, this is not a necessity if the distance between the nanoparticle and the place where the reference is picked up is not larger than the coherence length of the source. In other words, measurements involving nanoparticles very close to a cover glass can be just as well done using incoherent sources such as LEDs [75,83,85,145].…”

“…As pointed out in the introductory section, interferometric microscopy has a long and rich history. In the past decade, however, iSCAT and its related techniques have ushered in a revival of interferometric imaging for cell biology applications, such as imaging microtubules [75,76,91,187], actin [121], the acto-myosin network [188], as well as organelles and micron or smaller-sized vesicle containers which assist in the transport of material throughout the cell [165]. Echoing previous efforts from the holographic community [189,190], interferometric efforts are now also focusing on identifying the small changes in the cell membrane, where previously the whole cell was profiled.…”