Abbe Diffraction Limit Research Articles

Single Molecule Counting using STORM/PALM

Single-molecule counting using STORM/PALMDuring the last decade, several super-resolution microscopy techniques have been developed that enable the circumvention or even the breaking of Abbe's diffraction limit. One approach of achieving super-resolution is to control the emission state of individual fluorophores in time and the concomitant ability to localize active emitters with up to nanometer accuracy. Two of the most commonly implemented super-resolution techniques are stochastic optical reconstruction microscopy (STORM) and photo-activated localization microscopy (PALM). Each technique uses the localization of individual emitters to improve resolution as has been demonstrated with numerous intracellular structures. Beyond that, these techniques open the possibility of counting on a single molecule level. However, the ability to count is fundamentally limited by the fluorophores used for these techniques which blink many times before photobleaching. A typical STORM dye like Cy5 can cycle between tens and hundreds of times between a dark and a fluorescent state before photobleaching, thus leading to a set of localizations scattered around the true position. Whereas this property does not affect the reconstruction of images of extended objects like intracellular filaments or compartments, it severely influences the interpretation and quantification of objects for which the exact stoichiometry can be important like for membrane protein aggregates. To resolve this issue, we have implemented an algorithm that uses the spatial and temporal information of fluorophore localizations from STORM/PALM experiments to obtain a quantitative picture of the underlying molecule distribution. Our algorithm reliably operates on artificial data as well as on experimental data from biological constructs with a well-defined number of attached fluorophores.

Pump–probe spectroscopy on photoinitiators for stimulated-emission-depletion optical lithography

We report on femtosecond pump-probe experiments on two different photoinitiators in solution. These two molecules have recently appeared as attractive candidates for far-field optical lithography based on stimulated-emission-depletion (STED) inspired approaches aiming at beating Abbe's diffraction limit. For the case of 7-diethylamino-3-thenoylcoumarin (DETC), we find that stimulated emission clearly dominates over excited-state absorption, whereas the opposite holds true for the case of isopropylthioxanthone. We argue that it is desirable that stimulated emission dominates over excited-state absorption as depletion mechanism in STED photoresists. Thus, DETC is an attractive corresponding photoinitiator.

Limitations and potentials of metamaterial lenses

The seminal discovery that an ideal negative-index lens may overcome Abbe's diffraction limit has raised enormous interest in the field of metamaterials and of subwavelength focusing. This finding is based on the anomalous wave propagation in ideally isotropic and homogeneous metamaterials with negative index of refraction and low loss, provided they are available. We have designed a metamaterial lens based on one of the simplest metamaterial geometries, a cubic array of spheres, with the aim of verifying its imaging properties in a practical configuration. After a rigorous homogenization, we have shown that, for suitable designs, the effective bulk parameters may indeed provide a quasi-isotropic negative-index response, ideal for imaging applications. We have then tested the imaging properties for finite-size lenses, analyzing challenges and potentials of going beyond the diffraction limit in a practical setup. We have also explored an alternative venue to exploit the negative-index property of the designed metamaterial in a concave lens, in order to resolve subwavelength features in the far-field. Our results indicate that, although subwavelength resolution and evanescent-wave amplification are possible in metamaterial arrays, practical imaging beyond the diffraction limit is challenging and a careful design should consider the granularity, degree of isotropy, and transverse size of the metamaterial lens.

Enhanced resolution beyond the Abbe diffraction limit with wavelength-scale solid immersion lenses

The solid immersion lens (SIL) is a well-developed near-field optical device for imaging and data storage. Recent experiments have demonstrated high-quality imaging beyond the diffraction limit by nanoscale lenses in an SIL-type implementation [Nature 460, 498 (2009)]; we call these nSIL. A question arises as to what resolution is obtainable with an nSIL. From full three-dimensional, finite-difference time-domain calculations, we demonstrate that the FWHM of the focal spot of an objective-lens-nSIL system can be reduced by greater than 25% compared to a regular macroscopic SIL.

ChemInform Abstract: Scanning Near-Field Optical Microscopy and Spectroscopy as a Tool for Chemical Analysis

Research into the nanometer-scale region is currently of relevance in many branches of modern science and engineering, such as in microelectronics, supramolecular chemistry, and in a biological context. A great deal of attention is given to the design of devices, usually towards understanding the function of existing molecular machines. Central to this task are powerful diagnostic tools capable of recording chemical information with spatial resolution in the nanometer range. While elemental analysis of surfaces with a lateral resolution of a few dozen nanometers is almost routine, analysis of species with a resolution of <1 µm is very difficult. Scanning-tunneling and atomic-force microscopies usually do not give any chemical information. By combining scanning near-field microscopy (SNOM)-the optical member of the family of scanning-probe microscopies-with spectroscopy, it is possible to obtain information from sample areas as little as 50 nm in diameter. In SNOM, a light source is scanned above the object of interest at a distance of a few nanometers. In the near field, the illuminated area is not subject to the Abbe diffraction limit, but merely by the size of the illuminating source. High quality SNOM probes can be reproducibly prepared by a chemical-etching method. These etched probes have an transmission up to 1000-fold higher than commercial (pulled) SNOM tips and can withstand higher laser power. This last advantage allows not only high resolution imaging, but also localized spectroscopic investigations of surfaces and even nanosampling by pulsed-laser ablation. The ablated material can be transported over a considerable distance, which opens the possibility for its subsequent analysis with a complementary, highly sensitive analytical method, such as mass spectrometry.

Studying Surface Chemistry beyond the Diffraction Limit: 10 Years of TERS

The use of an illuminated scanning probe tip to greatly enhance Raman scattering from the sample underneath the tip is one of the most intriguing developments in optical spectroscopy, and the steeply increasing number of publications per year shows that chemists, physicists and biologists alike recognize the importance and great potential of this technique. With tip-enhanced Raman spectroscopy (TERS), one of the main goals in surface science has been achieved, namely the combination of scanning probe microscopy and optical spectroscopy such as Raman spectroscopy. Important here is the use of the tip as an optical antenna to substantially increase the emitted radiation and to simultaneously improve the optical resolution much beyond the Abbe diffraction limit. This permits the correlation of topographic and chemical information of the same surface region. The synergy of detailed insight in morphology and the chemical nature of the target species facilitates data interpretation significantly and enables characterization of interfaces at the nanometer scale. A wide variety of substrates and sample molecules have been studied with TERS since the first publication of tip-enhanced Raman spectra, and the technique has reached a first level of maturity on its 10th birthday, with TERS applications extending into various research fields from surface chemistry over biology to nanoscale physics.

Optical Saturation as a Versatile Tool to Enhance Resolution in Confocal Microscopy

One of the most actively developing areas in fluorescence microscopy is the achievement of spatial resolution below Abbe's diffraction limit, which restricts the resolution to several hundreds of nanometers. Most of the approaches in use at this time require a complex optical setup, a difficult mathematical treatment, or usage of dyes with special photophysical properties. In this work, we present a new, to our knowledge, approach in confocal microscopy that enhances the resolution moderately but is both technically and computationally simple. As it is based on the saturation of the transition from the ground state to the first excited state, it is universally applicable with respect to the dye used. The idea of the method presented is based on a principle similar to that underlying saturation excitation microscopy, but instead of applying harmonically modulated excitation light, the fluorophores are excited by picosecond laser pulses at different intensities, resulting in different levels of saturation. We show that the method can be easily combined with the concept of triplet relaxation, which by tuning the dark periods between pulses helps to suppress the formation of a photolabile triplet state and effectively reduces photobleaching. We demonstrate our approach imaging GFP-labeled protein patches within the plasma membrane of yeast cells.

Optical Superresolution: Imaging Beyond Abbe's Diffraction Limit

Optical Superresolution: Imaging Beyond Abbe's Diffraction Limit

Near-field focusing and magnification through self-assembled nanoscale spherical lenses

The performance of a light microscope is intrinsically constrained by the Abbe diffraction limit. No matter how close to optical perfection it is, an imaging system cannot resolve two objects that are beyond this natural limit, which is dependent on the wavelength of the observed light and its angular distribution. Several methods have been devised to beat the diffraction limit, but these have generally required esoteric excitation schemes, so remain impractical. Lee et al. are working on a new way of beating the limit, using nanoscale spherical lenses that self-assemble by bottom-up integration of cup-shaped organic molecules called calixarenes. Lenses produced in this way have very short focal lengths that can generate near-field magnification beyond the diffraction limit, enabling the resolution of features of the order of 200 nm. The lenses can be placed at will on a surface and, among other things, can be used to reduce the size of deep-ultraviolet lithography features. Cup-shaped molecules of calix[4]hydroquinone self-assemble on a surface into a lens shape; these lenses are shown to generate near-field magnification beyond the diffraction limit, enabling the resolution of features of the order of 200 nanometres. Such spherical nanolenses provide new pathways for lens-based near-field focusing and high-resolution optical imaging at very low intensities, which are useful for, among other things, bio-imaging and near-field lithography. It is well known that a lens-based far-field optical microscope cannot resolve two objects beyond Abbe’s diffraction limit. Recently, it has been demonstrated that this limit can be overcome by lensing effects driven by surface-plasmon excitation1,2,3, and by fluorescence microscopy driven by molecular excitation4. However, the resolution obtained using geometrical lens-based optics without such excitation schemes remains limited by Abbe’s law even when using the immersion technique5, which enhances the resolution by increasing the refractive indices of immersion liquids. As for submicrometre-scale or nanoscale objects, standard geometrical optics fails for visible light because the interactions of such objects with light waves are described inevitably by near-field optics6. Here we report near-field high resolution by nanoscale spherical lenses that are self-assembled by bottom-up integration7 of organic molecules. These nanolenses, in contrast to geometrical optics lenses, exhibit curvilinear trajectories of light, resulting in remarkably short near-field focal lengths. This in turn results in near-field magnification that is able to resolve features beyond the diffraction limit. Such spherical nanolenses provide new pathways for lens-based near-field focusing and high-resolution optical imaging at very low intensities, which are useful for bio-imaging, near-field lithography, optical memory storage, light harvesting, spectral signal enhancing, and optical nano-sensing.

Imaging with subwavelength resolution by a generalized superlens at infrared wavelengths

We demonstrate experimentally negative refraction by a photonic crystal prism and imaging of a point source by a photonic crystal slab at 1.5 microm wavelength. The photonic crystal structures were nanofabricated in a InGaAsP/InP heterostructure platform, and optical characterization was performed using a near-field scanning optical microscope. By designing a suitable lens surface termination, an image spot size of 0.12lambda2 was achieved, demonstrating superlens imaging with subwavelength resolution well below Abbe's diffraction limit (0.5lambda2).

Superresolution digital holographic microscopy for three-dimensional samples

An approach that allows superresolution imaging of three-dimensional (3-D) samples by numerical refocusing is presented in the field of digital holographic microscopy. Based on the object's spectrum shift produced by tilted illumination, we present a time multiplexing superresolved approach to overcome the Abbe's diffraction limit. The proposed approach uses a microscope in a Mach-Zehnder interferometric architecture with the particularity that the output plane does not coincide with the image plane. Thus, a set of off-axis non-image plane holograms are sequentially recorded for every tilted beam used in the illumination stage. After that and by using simple digital post-processing and numerical reconstruction, a 3-D superresolved sample volume is reconstructed slice-by- slice in terms of the definition of a synthetic aperture (SA) that expands the cutoff frequency of the microscope lens. Experimental results showing the capabilities of the proposed approach are presented.

Axial superresolution by synthetic aperture generation

The use of tilted illumination onto the input object in combination with time multiplexing is a useful technique to overcome the Abbe diffraction limit in imaging systems. It is based on the generation of an expanded synthetic aperture that improves the cutoff frequency (and thus the resolution limit) of the imaging system. In this paper we present an experimental validation of the fact that the generation of a synthetic aperture improves not only the lateral resolution but also the axial one. Thus, it is possible to achieve higher optical sectioning of three-dimensional (3D) objects than that defined by the theoretical resolution limit imposed by diffraction. Experimental results are provided for two different cases: a synthetic object (micrometer slide) imaged by a 0.14 numerical aperture (NA) microscope lens, and a biosample (swine sperm cells) imaged by a 0.42 NA objective.

Superresolved Holographic Microscopy

Superresolution methods in digital holographic microscopy provide a useful tool to overcome the Abbe's diffraction limit when using modest microscope lenses. The process improves the cutoff frequency of the microscope lens by means of the generation of a synthetic aperture based on time multiplexing and using 3 main stages: optical coding, optical decoding, and digital postprocessing. After the whole process, a superresolved image is obtained by Fourier transformation of the synthetic aperture.

Nanoprocessing with nanojoule near-infrared femtosecond laser pulses

Abstract Near-infrared (NIR) femtosecond laser pulses are novel tools for ultraprecise intracellular and intratissue surgery, cell isolation and nanostructering of biomaterials as well as for high-resolution multiphoton tomography. The required high transient laser intensity of TW/cm2 for multiphoton ablation can be achieved with low nanojoule energy laser pulses when using focusing optics of high numerical aperture. Most of the experiments have been performed with a compact turn-key femtosecond laser scanning system (FemtOcut, JenLab). The tight focusing results in a sub-femtoliter multiphoton interaction volume which enables nanoprocessing in the sub-200 nm range without any destructive photodisruptive collateral effects. FWHM cut sizes and nanostructures in silicon as low as 70 nm as well as knocking out of specific sub-70 nm genomic regions have been realized at 800 nm laser wavelength. Working with a precision of one order better than “Abbe's diffraction limit”, we demonstrate nano- and microdissection for applications in genomics and proteomics, sub-100 nm nanomachining for cell adhesion studies, destruction of intracelluar organelles and intratissue cells, targeted transfection as well as ocular refractive nanosurgery. NIR femtosecond lasers will become important tools in laser nanomedicine.

Phase contrast and DIC illumination for AFM hybrids

High-resolution optical microscopy is an essential pre-requisite for life science force microscopy, particularly for applications in cell biology and medicine. Identification and validation of cells is typically established with techniques like phase contrast microscopy or differential interference contrast microscopy. The option to select or monitor individual cells online with such light microscopy techniques while performing atomic force microscopy (AFM) measurements is therefore extremely beneficial. Here, we report two conceptually different strategies to implement these light microscopy techniques in a fully functional AFM head at the ultimate resolution of the Abbe diffraction limit.

Near-field microscopy by elastic light scattering from a tip.

We describe ultraresolution microscopy far beyond the classical Abbe diffraction limit of one half wavelength (lambda/2), and also beyond the practical limit (ca. lambda/10) of aperture-based scanning near-field optical microscopy (SNOM). The 'apertureless' SNOM discussed here uses light scattering from a sharp tip (hence scattering-type or s-SNOM) and has no lambda-related resolution limit. Rather, its resolution is approximately equal to the radius a of the probing tip (for commercial tips, a < 20 nm) so that 10 nm is obtained in the visible (lambda/60). A resolution of lambda/500 has been obtained in the mid-infrared at lambda = 10 microm. The advantage of infrared, terahertz and even microwave illumination is that specific excitations can be exploited to yield specific contrast, e.g. the molecular vibration offering a spectroscopic fingerprint to identify chemical composition. S-SNOM can routinely acquire simultaneous amplitude and phase images to obtain information on refractive and absorptive properties. Plasmon- or phonon-resonant materials can be highlighted by their particularly high near-field signal level. Furthermore, s-SNOM can map the characteristic optical eigenfields of small, optically resonant particles. Lastly, we describe theoretical modelling that explains and predicts s-SNOM contrast on the basis of the local dielectric function.

Saturated patterned excitation microscopy with two-dimensional excitation patterns

The techniques of patterned excitation microscopy (PEM, also referred to in the literature as structured illumination, harmonic excitation light microscopy, or laterally modulated excitation microscopy), has recently been extended to the non-linear regime, permitting a further increase in resolution breaking the Abbe diffraction limit (saturated PEM, saturated patterned excitation microscopy (SPEM)). Fluorescence saturation was suggested as the non-linear effect employed to achieve this aim. Here a two-dimensional extension of the linear and the non-linear patterned excitation technique is introduced and simulations of the expected resolution improvement are presented. The simulations account for photon statistics, a sub-optimal degree of modulation and a high amount of background fluorescence in the sample. The resulting point-spread-functions achieve a full width at half maximum of 215 nm (widefield), 118 nm (linear PEM), and 57 nm (saturated PEM, 9×9 orders). For higher resolution, an increased number of detected photons and of raw data images are required. A potential method for substantially decreasing the required number of raw images in PEM and SPEM is discussed.

Status of THz-to-Visible Nanospectroscopy Development.

Quite unexpectedly, THz and infraredspectroscopy has now a real chance to solveproblems in the nanosciences. This rests ona new microscope technique that overcomesthe Abbe diffraction limit, by using thenear field of a metal antenna in closeproximity to a scanned sample surface. HereI briefly summarize present activities inthe microwave, mid-infrared and visiblespectral ranges. It seems straightforwardand highly desirable to fill the existinggap between about 20 GHz and 20 THz, andattain spatial resolution of 10 nm andbelow also in this important part of theelectromagnetic spectrum.

FRET: A Spectral Ruler for Interacting Molecules Involved in Apoptosis

Abstract Fluorescence Resonance Energy Transfer (FRET) is a process by which a fluorophore (the donor) in the excited state transfers its energy to a neighboring fluorophore (the acceptor) non-radiatively through dipole-dipole interactions. Since the efficiency of energy transfer varies as the inverse of the sixth power of the distance separating the donor and acceptor chromophores, for FRET to occur the distance between the two molecules cannot exceed 10 to 100 angstroms (1 to l0nm). The combination of FRET and optical microscopy allows examination and quantitation of dynamic molecular interactions between cellular constituents at resolutions beyond the Abbe diffraction limit of light microscopy. Through the microscope one may detect FRET by an overall decrease in fluorescence emission of the donor with a concomitant increase in fluorescence emission of the acceptor.

High-resolution video-enhanced light microscopy

Video-enhanced differential interference contrast (VE-DIC) methods can produce high contrast images of isolated macromolecular complexes and cellular fine structures invisible by eye or photographic recording methods through the light microscope. Objects 10 nm - 20 nm wide, much smaller than the wavelength of green light, can be seen at high contrast, although lateral resolution is limited by the Abbe diffraction limit (about 200 nm for 550 green light through objective NA = 1.4). In addition, VE-DIC microscopy can produce thin optical sections through transparent thick specimens with z-axis resolution less than 200 nm. We have been developing this technology to provide video assays (30 frames per sec) of the dynamics of individual molecular complexes. Applications include measurement of the polymerization kinetics of individual 25 nm diameter microtubules in living cells and in reconstituted preparations, the assembly of individual polymers of sickle cell hemoglobin in vitro, the translation of mechanochemical enzymes like dynein and kinesin along individual microtubules in vitro and the movements of individual sister kinetochores during mitosis. Lateral movements of diffraction limited images of spherical particles can be tracked with 1 nm precision. We have used a semi-automatic version of the Gelles tracking algorithm to measure the movements of individual kinetochores and centrosomes in living cells at 1 sec intervals with 100-200 nm precision.