Plasmonic antennas and zero mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy towards physiological concentrations

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as F\"orster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero mode waveguides and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometre scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET and FCS. Single molecule spectroscopy techniques greatly benefit from zero mode waveguides and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.Comment: WIREs Nanomed Nanobiotechnol 201

Optical assessment of gel-like mechanical and structural properties of surface layers: single particle tracking and molecular rotors

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.Thin gel-like layers form at many surfaces of natural or artificial origin. Important properties of such layers include thickness, viscosity and density. Here we discuss two optical approaches which allow assessment of these properties with high resolution. The first approach relies on centroid calculation and defocus imaging based 3D tracking of fluorescent tracer particles, which is based on standard fluorescent microscopy and allows a precision of particle detection in the range of 10nm. The size of the particle and its surface charge and polarity will determine the particle invasion into the layer. Thus simultaneous application of different colored beads with different size and properties can reveal the thickness and nature of the layer. Via tracking the thermal vibration of particles invading the layer the bulk viscosity of the layer can be calculated. The second approach uses “molecular rotor” fluorophores (MR). Due to their molecular structure, the MR’s fluorescence quantum yield increases as their internal rotation is hampered by e.g. high viscosity of the embedding medium. The MRs are several orders of magnitude smaller than the structural (macro) molecules of a gel-like layer and therefore the MRs are not necessarily directly sensitive toward the bulk viscosity of the layer. In contrast, the MRs internal rotation will be attenuated by the MRs interaction with the structural elements of the layer or the solvent included in it. Depending on their molecular structure MRs exhibit different sensitivity to the mechanical properties of the large macromolecules or the solvent in a layer. Thus, they may be used to assess the microdomain’s viscosity or density in a surface layer. Using a ratiometric imaging approach, they can be used for continuous measurements in very different experimental settings

Light Microscopy: An ongoing contemporary revolution

Optical microscopy is one of the oldest scientific instruments that is still used in forefront research. Ernst Abbe's nineteenth century formulation of the resolution limit in microscopy let generations of scientists believe that optical studies of individual molecules and resolving sub-wavelength structures were not feasible. The Nobel Prize in 2014 for super-resolution fluorescence microscopy marks a clear recognition that the old beliefs have to be revisited. In this article, we present a critical overview of various recent developments in optical microscopy. In addition to the popular super-resolution fluorescence methods, we discuss the prospects of various other techniques and imaging contrasts and consider some of the fundamental and practical challenges that lie ahead.Comment: 37 pages, 13 figure

Automated tracking of colloidal clusters with sub-pixel accuracy and precision

Quantitative tracking of features from video images is a basic technique employed in many areas of science. Here, we present a method for the tracking of features that partially overlap, in order to be able to track so-called colloidal molecules. Our approach implements two improvements into existing particle tracking algorithms. Firstly, we use the history of previously identified feature locations to successfully find their positions in consecutive frames. Secondly, we present a framework for non-linear least-squares fitting to summed radial model functions and analyze the accuracy (bias) and precision (random error) of the method on artificial data. We find that our tracking algorithm correctly identifies overlapping features with an accuracy below 0.2% of the feature radius and a precision of 0.1 to 0.01 pixels for a typical image of a colloidal cluster. Finally, we use our method to extract the three-dimensional diffusion tensor from the Brownian motion of colloidal dimers.Comment: 20 pages, 8 figures. Non-revised preprint version, please refer to http://dx.doi.org/10.1088/1361-648X/29/4/04400

Magnetic manipulation and multimodal imaging for single cell direct mechanosensing

The study of internal mechanics of single cells is paramount to understand mechanisms of mechanoregulation. External loading and cell-mediated force generation result in changes in cell shape, rheology, and the deformation of subcellular structures such as the nucleus. Moreover, alterations in the processes that regulate these responses have been further correlated to specific pathologies. Cellular deformation is often studied through application of forces in the environment of the cell, relying on strain and stress transfer through focal adhesions and the cytoskeletal system. However, the transfer of these external forces to internal mechanics can introduce uncertainties in the interpretation of subcellular responses. Our group has focused on minimally-invasive techniques for the study of internal mechanical perturbation and mechanobiology measures. We have been particularly interested in multimodal imaging methods that combine and leverage nano-scale spatial localization, visualization, biophysical and physico-chemical analysis features to reveal information that cannot be attained by any single method alone. We recently fabricated novel atomic force microscopy (AFM) cantilevers, functionalized to generate small, highly-localized magnetic fields, for the controlled force application and sensing of single cells. In combination with AFM and fluorescence microscopy detection capabilities, this technique enables the selective stimulation and monitoring of cells injected with superparamagnetic microbeads. Though the targeted magnetic force application, we are able to apply various waveforms to direct the microdisplacements of the injected beads to allow insight into the structural architecture of the cell. Coupling this with AFM techniques further yields insight into internal and external mechanics over time. This technique can be extended to include studies of intranuclear strain dynamics through fluorescent labeling of specific cellular targets and image post-processing algorithms such as hyperelastic warping. Furthermore, the ability to alter the culture environment (e.g. to manipulate osmotic pressure or enable drug delivery) allows this technique to be a powerful single cell analysis tool for a diverse set of applications. We demonstrate the feasibility of this technique through the localized application of low magnetic fields that produce bead displacements in the micrometer scale. The effects of larger induced magnetic fields in the displacement field are also presented, along with validation and viability studies, and a range of practical applications for the study of single cells

Precise Particle Tracking Against a Complicated Background: Polynomial Fitting with Gaussian Weight

We present a new particle tracking software algorithm designed to accurately track the motion of low-contrast particles against a background with large variations in light levels. The method is based on a polynomial fit of the intensity around each feature point, weighted by a Gaussian function of the distance from the centre, and is especially suitable for tracking endogeneous particles in the cell, imaged with bright field, phase contrast or fluorescence optical microscopy. Furthermore, the method can simultaneously track particles of all different sizes, and allows significant freedom in their shape. The algorithm is evaluated using the quantitative measures of accuracy and precision of previous authors, using simulated images at variable signal-to-noise ratios. To these we add a new test of the error due to a non-uniform background. Finally the tracking of particles in real cell images is demonstrated. The method is made freely available for non-commencial use as a software package with a graphical user-inferface, which can be run within the Matlab programming environment