Polarization Contrast Microscopy Research Articles

Excitation polarization angle-resolved single-laser dual-polarization energy transfer on the cell surface

A quick and robust approach for the simultaneous detection of donor and acceptor anisotropies and FRET efficiency is presented based on changing the angle of polarization of the exciting light in a single-laser dual channel ratiometric detection scheme of FRET in a flow cytometer. The convenience of the method lies in that for the extra assessment of anisotropies it does not necessitates alteration of the detection side of the optics conventionally used in measuring FRET in the steady state. The detection of anisotropies is pushed to the side of excitation where anisotropies are enabled by just a few sequential measurements at different polarization angles of the exciting light. The power of the method is illustrated by comparing results on the homo-associations and inter-subunit FRET on the MHCI receptor on the surface of Kit-225K6 T cells obtained with the angle-resolved approach to those obtained by the dual-T format approach as a validation. In flow conditions, but not in imaging microscopy, a minor drawback of this method is that due to the sequential nature of anisotropy detection, the mutual cell-by-cell correlations of anisotropies with each other and with FRET are lost. The merit of the approach is that besides the degree of molecular associations probed by the FRET efficiency, information can also be gained on the rotational dynamics and extent of homo-FRET prevailing in the the donor and acceptor dye systems during the hetero-FRET process, and as a consequence, orientation factor for FRET can also be estimated. FRET efficiencies at the different polarization angles are shifted depending on the donor and acceptor anisotropy, enabling quantitation of anisotropy changes by FRET efficiency. Attention is called for polarization biases when using even depolarized excitation and detecting not concerning with fluorescence polarization. When established in polarization contrast microscopy, the approach might be applied for also controlling FRET.A new formula for the determination of the sensitivity calibration factor for FRET α (or Get) has also been deduced based on the donor and acceptor anisotropies.

Polarization contrast scattering spectroscopy of individual metal nanoantennas

Metal nanoantennas give rise to strongly localized and enhanced electric fields. The enhancement is largest at the plasmon resonance, which, therefore, needs to be characterized. Here, we use polarization contrast microscopy for imaging and spectroscopy of single metal nanoantennas. The method relies on the strong suppression of incident linearly polarized light with a cross-polarized analyzer in the detection path and exploits the distinct polarization dependence of the plasmonic response of the antennas. The technique enables an easy-to-use background-free measurement of plasmon resonances of single metal nanoantennas in the visible and infrared spectral range.

Automated quantification of renal fibrosis with Sirius Red and polarization contrast microscopy.

Interstitial fibrosis is commonly measured by histology. The Masson trichrome stain is widely used, with semiquantitative scores subjectively assigned by trained operators. We have developed an objective technique combining Sirius Red staining, polarization contrast microscopy, and automated analysis. Repeated analysis of the same sections by the same operator (r = 0.99) or by different operators (r = 0.98) was highly consistent for Sirius Red, while Masson trichrome performed less consistently (r = 0.61 and 0.72, respectively). These techniques performed equally well when comparing sections from the left and right kidneys of mice. Poor correlation between Sirius Red and Masson trichrome may reflect different specificities, as enhanced birefringence with Sirius Red staining is specific for collagen type I and III fibrils. Combining whole‐section imaging and automated image analysis with Sirius Red/polarization contrast is a rapid, reproducible, and precise technique that is complementary to Masson trichrome. It also prevents biased selection of fields as fibrosis is measured on the entire kidney section. This new tool shall enhance our search for novel therapeutics and noninvasive biomarkers for fibrosis.To listen to podcast click here

Slow Single-Molecule Diffusion in Liquid Crystals

We report on measurements of single-molecule Brownian motion in liquid crystals, unravelling the anisotropic mobility of individual dye molecules. This anisotropic Brownian motion is directly correlated with the structural properties in a smectic A (8CB) and a nematic (5CB) liquid crystal sample cell on the micrometer scale using polarization contrast microscopy. A considerably slower mobility of dye molecules is found as compared to self-diffusion measurements by NMR, while anisotropy values compare well to recent literature data. This is suggested to be related to local distortions of the director structure around the dye molecules.

Rotational Dynamics of Laterally Frozen Nanoparticles Specifically Attached to Biomembranes

The strongly polarized light scattering of gold nanorods at their longitudinal plasmon frequency allows for the tracking of single gold nanorod lateral positions and orientations via optical dark-field microscopy. We monitor both lateral and rotational diffusion of polymer-coated gold nanorods attached to artificial biomembranes on solid supports. The attachment is mediated by the biotin-streptavidin receptor−ligand system, but weak interaction is also observed in the absence of streptavidin. In the latter case, we observe a two-dimensional lateral diffusion of the nanorods with a diffusion coefficient of 0.5 μm2 s−1. This lateral motion is strongly reduced with the addition of streptavidin. However, the particles are still able to rotate, and we study their rotational motion using polarization contrast microscopy. The rotational diffusion time in the range of 100 ms depends on the biotin concentration in the membrane, hence the number of anchor points, and on the temperature. Cooling the membrane beyond ...