Diffraction-limited Illumination Research Articles

Microsphere assistance in interference microscopy with high numerical aperture objective lenses

Various attempts have been discussed to overcome the lateral resolution limit and thus to enlarge the fields of application of optical interference microscopy. Microsphere-assisted microscopy and interferometry have proven that the imaging of structures well below Abbe’s resolution limit through near-field assistance is possible if microspheres are placed on the measured surface and utilized as near-field assisting imaging elements. The enhancement of the numerical aperture (NA) by the microspheres as well as photonic nanojets was identified to explain the resolution enhancement, but also whispering gallery modes and evanescent waves are assumed to have an influence. Up to now, to the best of our knowledge, there is no complete understanding of the underlying mechanisms and no model enabling to examine ideal imaging parameters. This contribution is intended to clarify how much the lateral resolution of an already highly resolving Linnik interferometer equipped with 100 × NA 0.9 objective lenses can be further improved by microspheres. Our simulation model developed so far is based on rigorous near-field calculations combined with the diffraction-limited illumination and imaging process in an interference microscope. Here, we extend the model with respect to microsphere-assisted interference microscopy providing a rigorous simulation of the scattered electric field directly above the sphere. Simulation and experimental results will be compared in the three-dimensional spatial frequency domain and discussed in context with ray-tracing computations to achieve an in-depth understanding of the underlying mechanism of resolution enhancement by the microsphere.

High-fidelity nano-FTIR spectroscopy by on-pixel normalization of signal harmonics

Abstract Scattering-type scanning near-field optical microscopy (s-SNOM) and Fourier transform infrared nanospectroscopy (nano-FTIR) are emerging tools for physical and chemical nanocharacterization of organic and inorganic composite materials. Being based on (i) diffraction-limited illumination of a scanning probe tip for nanofocusing of light and (ii) recording of the tip-scattered radiation, the efficient suppression of background scattering has been critical for their success. Here, we show that indirect tip illumination via far-field reflection and scattering at the sample can produce s-SNOM and nano-FTIR signals of materials that are not present at the tip position – despite full background suppression. Although these artefacts occur primarily on or near large sample structures, their understanding and recognition are of utmost importance to ensure correct interpretation of images and spectra. Detailed experimental and theoretical results show how such artefacts can be identified and eliminated by a simple signal normalization step, thus critically strengthening the analytical capabilities of s-SNOM and nano-FTIR spectroscopy.

Wide-vergence, multi-spectral adaptive optics scanning laser ophthalmoscope with diffraction-limited illumination and collection.

Visualizing and assessing the function of microscopic retinal structures in the human eye is a challenging task that has been greatly facilitated by ophthalmic adaptive optics (AO). Yet, as AO imaging systems advance in functionality by employing multiple spectral channels and larger vergence ranges, achieving optimal resolution and signal-to-noise ratios (SNR) becomes difficult and is often compromised. While current-generation AO retinal imaging systems have demonstrated excellent, near diffraction-limited imaging performance over wide vergence and spectral ranges, a full theoretical and experimental analysis of an AOSLO that includes both the light delivery and collection optics has not been done, and neither has the effects of extending wavefront correction from one wavelength to imaging performance in different spectral channels. Here, we report a methodology and system design for simultaneously achieving diffraction-limited performance in both the illumination and collection paths for a wide-vergence, multi-spectral AO scanning laser ophthalmoscope (SLO) over a 1.2 diopter vergence range while correcting the wavefront in a separate wavelength. To validate the design, an AOSLO was constructed to have three imaging channels spanning different wavelength ranges (543 ± 11 nm, 680 ± 11 nm, and 840 ± 6 nm, respectively) and one near-infrared wavefront sensing channel (940 ± 5 nm). The AOSLO optics and their alignment were determined via simulations in optical and optomechanical design software and then experimentally verified by measuring the AOSLO's illumination and collection point spread functions (PSF) for each channel using a phase retrieval technique. The collection efficiency was then measured for each channel as a function of confocal pinhole size when imaging a model eye achieving near-theoretical performance. Imaging results from healthy human adult volunteers demonstrate the system's ability to resolve the foveal cone mosaic in all three imaging channels despite a wide spectral separation between the wavefront sensing and imaging channels.

A resistor network simulation model for laser-scanning photo-current microscopy to quantify low conductance regions in organic thin films

Organic field effect transistors (OFETs) show considerable variation from device to device and batch to batch. For the basic understanding of the microscopic origins of these variations, it is crucial to develop both experimental and theoretical methods that allow to localize ineffective regions in a device. Raster techniques, which manipulate conductivity locally, combined with global current readout may provide this information, e.g. it has been suggested that diffraction limited illumination of OFETs by laser scanning photo-current microscopy provides trap density distribution maps. However, the question arises whether local variation of conductivity is indeed suited for localization of such defects given that the detected photo-current passes through the whole network of conductance in the thin film device. In this study, we present a simulation model based on resistor networks to investigate the effect of defective regions in organic thin films. We show that varying conductances locally allows indeed to reconstruct the spatial distributions of ineffective areas. We also demonstrate how such simulations can be applied to interpret photo-current microscopy maps in terms of trap densities.

Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility.

Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity.

Arrays of Nanoapertures for Examining Membrane Organization and Dynamics

Nanoscale domains in the plasma membrane may be responsible for a variety of cellular signaling processes via membrane receptor clustering and lipid phase partitioning. However, current experimental approaches are limited in spatial and/or temporal resolution to address many membrane domain hypotheses. We have developed a new approach utilizing an array of nanoapertures to examine membrane organization and dynamics with near-field optical fluorescence microscopy without incorporating a scanning probe or disturbing the membrane. These nanoapertures are glass-filled, cylindrical pores (>50 nm diameter) in a thin aluminum film on a fused silica support, and they provide a planar surface for unperturbed cell adherence and growth. A nanoaperture confines the transmitted excitation light to a sub-diffraction limited spot directly above aperture, providing a 40-fold decrease in the illuminated area versus diffraction-limited illumination of the plasma membrane. Otherwise conventional microscopy excitation sources and fluorescent probes are used to enable fluorescence correlation spectroscopy (FCS) with 50 nanometer and 1 microsecond resolution. Further, these apertures provide two key benefits for FCS in addition to improved resolution: assured alignment of numerous illumination spots for cross-correlations and assured focusing of illumination on the cellular membrane as opposed to focusing within the cytoplasm. Chromatic aberrations and slight laser misalignment that may complicate far-field, two-color FCS are not of concern here because the illumination profile is determined by the aperture directly. This technique has been applied to both model and living cell membranes to examine the diffusion of lipids and proteins in nanoscale dimensions. In particular, results will be presented demonstrating the effectiveness of this technique to observe diffusion of membrane proteins and lipids in varying phases and cross-correlation that occurs after cross-linking a selected component.

Quasi-confocal fluorescence sectioning with dynamic speckle illumination

We present a simple modification to a conventional wide-field fluorescence microscope that provides depth discrimination in thick tissues. The technique consists of illuminating a sample with a sequence of independent speckle patterns and displaying the rms of the resultant sequence of fluorescence images. The advantage of speckle illumination is that it provides diffraction-limited illumination granularity that is highly contrasted even in scattering media. We demonstrate quasi-confocal imaging in a mouse olfactory bulb labeled with green fluorescent protein.

Nano-Sizing of Specific Gene Domains in Intact Human Cell Nuclei by Spatially Modulated Illumination Light Microscopy

Although light microscopy and three-dimensional image analysis have made considerable progress during the last decade, it is still challenging to analyze the genome nano-architecture of specific gene domains in three-dimensional cell nuclei by fluorescence microscopy. Here, we present for the first time chromatin compaction measurements in human lymphocyte cell nuclei for three different, specific gene domains using a novel light microscopic approach called Spatially Modulated Illumination microscopy. Gene domains for p53, p58, and c-myc were labeled by fluorescence in situ hybridization and the sizes of the fluorescence in situ hybridization "spots" were measured. The mean diameters of the gene domains were determined to 103 nm (c-myc), 119 nm (p53), and 123 nm (p58) and did not correlate to the genomic, labeled sequence length. Assuming a spherical domain shape, these values would correspond to volumes of 5.7 x 10(-4) microm(3) (c-myc), 8.9 x 10(-4) microm(3) (p53), and 9.7 x 10(-4) microm(3) (p58). These volumes are approximately 2 orders of magnitude smaller than the diffraction limited illumination or observation volume, respectively, in a confocal laser scanning microscope using a high numerical aperture objective lens. By comparison of the labeled sequence length to the domain size, compaction ratios were estimated to 1:129 (p53), 1:235 (p58), and 1:396 (c-myc). The measurements demonstrate the advantage of the SMI technique for the analysis of gene domain nano-architecture in cell nuclei. The data indicate that chromatin compaction is subjected to a large variability which may be due to different states of genetic activity or reflect the cell cycle state.

Intensity spatial filtering applied to defect detection in integrated circuit photomasks

An intensity spatial filter has been devised and used for the semiautomatic photoelectric detection of 2.5-µ defects in 5-cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> photolithography masks for silicon integrated circuits. The filter is based on a simple geometric approximation to the form factor or envelope function for the intensity in the Fourier-transform plane, and permits small-area diffraction-limited illumination. This approach complements the Watkins' method.