Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy

Abstract

SummaryWhen studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labeling or there is concern that labelling will disrupt its function. Commonly used techniques for observing unlabeled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments, and interferometric scattering has a limited field of view. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the image quality of interference reflection microscopy is similar to or exceeds that of all other techniques and, with minimal microscope modification, can be used to study the dynamics of unlabeled microtubules.

The dynamic and structural properties of axonemal tubulins support the high length stability of cilia

“…We characterized the dynamic properties of the various axonemal tubulins using interference reflection microscopy (IRM) to perform label-free dynamic MT assays 32 . Purified tubulin was assembled onto GMP-CPP stabilized MT seeds polymerized from bovine brain tubulin and attached to the coverslip (Fig.…”

“…For dynamic MT assays, axonemal tubulins extensions were grown from TAMRA-labeled GMPCPP-stabilized bovine brain MT seeds and imaged by IRM 32 . Coverslips (Marienfeld, Germany) were cleaned using piranha solution and rendered hydrophobic by silinization 71 .…”

High-Throughput, High-Resolution Interferometric Light Microscopy of Biological Nanoparticles

“…24 The illumination NA effect on the image contrast in Köhler geometry is also experimentally studied in reflected interferometric microscopy using a glass substrate. 39 The low-NA illumination is obtained by underfilling the objective pupil using a symmetric circular source function. We refer to the SPIR microscopy with a low-NA implementation as conventional SPIR in which the emphasis has been on achieving high-contrast nanosized object detection.…”

Pathogenic Mutations in the Kinesin-3 Motor KIF1A Diminish Force Generation and Movement Through Allosteric Mechanisms

“…Finally, the protein-bead mixture was diluted in 40 μL assay buffer (60 mM HEPES, 50 mM KoAc, 2 mM MgCl2, 1 mM EGTA, 1 mM DTT, 10 μM taxol, 2 mM ATP, 50 mM glucose, gloxy, 1.25 mg/ml α-casein, 10% glycerol) and flowed into the slide chamber. All optical trapping experiments were performed with a LUMICKS C-Trap ® , which combines optical tweezers with 3-color TIRF microscopy and interference reflection microscopy (IRM) to visualize unlabeled MTs(88).Inducible peroxisome dispersion assayPlasmids for expression of WT or mutant rat KIF1A(339)-LZ motors tagged with monomeric NeonGreen and an FRB domain were cotransfected into COS-7 cells with a plasmid for expression of PEX3-mRFP-FKBP at a ratio of 6:1 with TransITLT1 transfection reagent (Mirus). Eight hours after transfection, rapamycin (Calbiochem, Millipore Sigma) was added to final concentration of 44 nM to promote FRB and FKBP heterodimerization and recruitment of motors to peroxisomes.…”