Precisely Localizing Wavelength Sensitive Point-Spread Functions Engineered With a Silicon Oxide Phase Plate (original) (raw)

Point Spread Function Engineering in Fluorescence Spectroscopy

2003

fluorescence spectroscopy has rendered the microscope into a powerful tool for functional analysis of biological specimens. This thesis explores the potential of techniques, that are usually utilized for PSF-engineering, for the development of new spectroscopical applications. The derivation of a simple integral solution for the Fourier transform of the vectorial PSF lays the foundation for numerical modelling of dynamical, intensity dependent processes in the focal region. Subsequently a theory describing the combination of fluorescence correlation spectroscopy with diffraction limited, periodically modulated detection volumes is derived. This idea leads to the proposal of a 'diffusion and flow microscope' with high spatial resolution. It is readily implemented in a multifocal 4Pi microscope and its potential to extract the parameters of anisotropic diffusion as well as speed and direction of flow inside a fluid is demonstrated in simulations. Finally, experimental evidence is presented that depletion by stimulated emission can be used to identify Förster energy transfer between two molecules inside a sample.

Three-dimensional nano-localization of single fluorescent emitters

Optics Express, 2010

We present a combination of self-interference microscopy with lateral super-resolution microscopy and introduce a novel approach for localizing a single nano-emitter to within a few nanometers in all three dimensions over a large axial range. We demonstrate nanometer displacements of quantum dots placed on top of polymer bilayers that undergo swelling when changing from an air to a water environment, achieving standard deviations below 10 nm for axial and lateral localization.

Visualizing Electromagnetic Fields at the Nanoscale by Single Molecule Localization

Nano Letters, 2015

Coupling of light to the free electrons at metallic surfaces allows the confinement of electric fields to subwavelength dimensions, far below the optical diffraction limit. While this is routinely used to manipulate light at the nanoscale, 1 in electro-optic devices 2 and enhanced spectroscopic techniques, 3−6 no characterization technique for imaging the underlying nanoscopic electromagnetic fields exists, which does not perturb the field 4,7 or employ complex electron beam imaging. 8,9 Here, we demonstrate the direct visualization of electromagnetic fields on patterned metallic substrates at nanometer resolution, exploiting a strong "autonomous" fluorescence-blinking behavior of single molecules within the confined fields allowing their localization. Use of DNA-constructs for precise positioning of fluorescence dyes on the surface induces this distance-dependent autonomous blinking thus completely obviating the need for exogenous agents or switching methods. Mapping such electromagnetic field distributions at nanometer resolution aids the rational design of nanometals for diverse photonic applications.

Ultrahigh-resolution colocalization of spectrally separable point-like fluorescent probes

2001

An ultrahigh-resolution colocalization method based on the simultaneous acquisition and analysis of spectrally separated images of the excitation point-spread function of point-like fluorescent probes is reviewed. It is shown that molecular distances can be measured with accuracy better than 10 nm using conventional far-field optics. A detailed account of the methodology, theoretical considerations, signal processing, and data fitting algorithms is given.

Probing the electromagnetic field of a 15-nanometre hotspot by single molecule imaging

Nature, 2011

When light illuminates a rough metallic surface, hotspots can appear, where the light is concentrated on the nanometre scale, producing an intense electromagnetic field. This phenomenon, called the surface enhancement effect 1,2 , has a broad range of potential applications, such as the detection of weak chemical signals. Hotspots are believed to be associated with localized electromagnetic modes 3,4 , caused by the randomness of the surface texture. Probing the electromagnetic field of the hotspots would offer much insight towards uncovering the mechanism generating the enhancement; however, it requires a spatial resolution of 1-2 nm, which has been a long-standing challenge in optics. The resolution of an optical microscope is limited to about half the wavelength of the incident light, approximately 200-300 nm. Although current state-of-the-art techniques, including near-field scanning optical microscopy 5 , electron energy-loss spectroscopy 6 , cathode luminescence imaging 7 and two-photon photoemission imaging 8 have subwavelength resolution, they either introduce a non-negligible amount of perturbation, complicating interpretation of the data, or operate only in a vacuum. As a result, after more than 30 years since the discovery of the surface enhancement effect 9-11 , how the local field is distributed remains unknown.

Nanometric axial localization of single fluorescent molecules with modulated excitation

2019

Strategies have been developed in LIDAR to perform distance measurements for non-coherent emission in sparse samples based on excitation modulation. Super-resolution fluorescence microscopy is also striving to perform axial localization but through entirely different approaches. Here we revisit the amplitude modulated LIDAR approach to reach nanometric localization precision and we successfully adapt it to bring distinct advantages to super-resolution microscopy. The excitation pattern is performed by interference enabling the decoupling between spatial and time modulation. The localization of a single emitter is performed by measuring the relative phase of its linear fluorescent response to the known shifting excitation field. Taking advantage of a tilted interfering configuration, we obtain a typical axial localization precision of 7.5 nm over the entire field of view and the axial capture range, without compromising on the acquisition time, the emitter density or the lateral localization precision. The interfering pattern being robust to optical aberrations, this modulated localization (ModLoc) strategy is particularly well suited for observations deep in the samples. Images performed on various biological samples show that the localization precision remains nearly constant up to several micrometers. In the presence of coherent signals, interferometry offers unmatched sensitivity for distance measurements 1. Measuring the relative phase between the excitation in the elastically scattered or reflected signal reaches record precisions. Interferometry has thus naturally been used in some coherent microscopy configurations to obtain nanometric axial localization 2-5. However, in the case of fluorescence microscopy which is today the most widespread technique for cell imaging such an approach remains impossible because of the non- .