Submicron Imaging Research Articles

Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High-Resolution Nuclear Magnetic Resonance Analysis of Soybean Seeds, Embryos and Single Cells

AbstractChemical analysis of soybean seeds, somatic embryos and single cells were carried out by Fourier Transform Infrared (FT-IR), Fourier Transform Near Infrared (FT-NIR) Microspectroscopy, Fluorescence and High-Resolution NMR (HR-NMR). The first FT-NIR chemical images of biological systems approaching 1 micron (1μ) resolution are presented here. Chemical images obtained by FT-NIR and FT-IR Microspectroscopy are presented for oil in soybean seeds and somatic embryos under physiological conditions. FT-NIR spectra of oil and proteins were obtained for volumes as small as 2μ3. Related, HR-NMR analyses of oil contents in somatic embryos are also presented here with nanoliter precision. Such 400 MHz 1H NMR analyses allowed the selection of mutagenized embryos with higher oil content (e.g. ~20%) compared to non-mutagenized control embryos. Moreover, developmental changes in single soybean seeds and/or somatic embryos may be monitored by FT-NIR with a precision approaching the picogram level. Indeed, detailed chemical analyses of oils and phytochemicals are now becoming possible by FT-NIR Chemical Imaging/ Microspectroscopy of single cells. The cost, speed and analytical requirements of plant breeding and genetic selection programs are fully satisfied by FT-NIR spectroscopy and Microspectroscopy for soybeans and soybean embryos. FT-NIR Microspectroscopy and Chemical Imaging are also shown to be potentially important in functional Genomics and Proteomics research through the rapid and accurate detection of high-content microarrays (HCMA). Multi-photon (MP), pulsed femtosecond laser NIR Fluorescence Excitation techniques were shown to be capable of Single Molecule Detection (SMD). Therefore, such powerful techniques allow for the most sensitive and reliable quantitative analyses to be carried out both in vitro and in vivo. Thus, MP NIR excitation for Fluorescence Correlation Spectroscopy (FCS) allows not only single molecule detection, but also molecular dynamics and high resolution, submicron imaging of femtoliter volumes inside living cells and tissues. These novel, ultra-sensitive and rapid NIR/FCS analyses have numerous applications in important research areas, such as: agricultural biotechnology, food safety, pharmacology, medical research and clinical diagnosis of viral diseases and cancers.

Biomedical research at LIPSION – Present state and future developments

Since its commissioning in 1998 the high energy ion nanoprobe LIPSION has been developed into a versatile tool for a variety of applications in biomedical research. It includes quantitative trace element analysis with sub-micron spatial resolution and 3D-element imaging, as well as 2D- and 3D-microscopy of density distributions. The analytical methods base on particle induced X-ray emission spectrometry (PIXE) and PIXE-tomography, Rutherford backscattering spectrometry (RBS), as well as scanning transmission ion microscopy (STIM) and STIM-tomography.The continuous developments led to improved capabilities for trace element analysis. For sub-micron analysis the spatial resolution could be improved to 0.3μm for high resolution work and to 0.5μm for routine analysis. On the other hand, LIPSION was optimized for high sample throughput in quantitative element imaging maintaining a lateral resolution of 1–2μm. Recently the methods of PIXE- and STIM-tomography of biological specimens have been extended to limited angle tomography to avoid the complicated preparation of free-standing samples.Besides its analytical applications, the nanoprobe is also used for targeted irradiation of living cells with counted single ions for radiobiological research. It is capable of irradiating up to 20,000 cells per hour. Furthermore, we have developed a technique to achieve confined cell growth based on the proton beam writing technique (PBW) and agar. The paper presents an overview of the current biomedical research fields at LIPSION and gives an outlook on prospective developments.

Sub-micron imaging on high-topography wafers using spray coating and projection lithography

We propose a method to image inside deep trenches (50 μm) using spray-coated resist and the ASML PAS 5500/100 system with the new functionality multi-step imaging. Multi-step imaging allows extending the focus offset range of the PAS 5500/100 system from ±30 μm to ±200 μm. Isolated trenches and contact holes were both imaged inside the deep trenches and on the surface of the wafer to study the versatility of the new functionality. A resolution of 700 nm in 3 μm thick photoresist, at the bottom of 50 μm deep, 200 μm wide trench, was obtained with this process. Finally, multi-focus exposure that consists in exposing the same image several times at various focus offsets was performed in order to image thick photoresist on high topographic substrates.

Prospects for sub-micron solid state nuclear magnetic resonance imaging with low-temperature dynamic nuclear polarization.

We evaluate the feasibility of (1)H nuclear magnetic resonance (NMR) imaging with sub-micron voxel dimensions using a combination of low temperatures and dynamic nuclear polarization (DNP). Experiments are performed on nitroxide-doped glycerol-water at 9.4 T and temperatures below 40 K, using a 30 mW tunable microwave source for DNP. With DNP at 7 K, a 0.5 microL sample yields a (1)H NMR signal-to-noise ratio of 770 in two scans with pulsed spin-lock detection and after 80 db signal attenuation. With reasonable extrapolations, we infer that (1)H NMR signals from 1 microm(3) voxel volumes should be readily detectable, and voxels as small as 0.03 microm(3) may eventually be detectable. Through homonuclear decoupling with a frequency-switched Lee-Goldburg spin echo technique, we obtain 830 Hz (1)H NMR linewidths at low temperatures, implying that pulsed field gradients equal to 0.4 G/d or less would be required during spatial encoding dimensions of an imaging sequence, where d is the resolution in each dimension.

A new SIMS paradigm for 2D and 3D molecular imaging of bio-systems

With the implementation of focused primary ion beams, secondary ion mass spectrometry (SIMS) has become a significant technique in the rapidly emerging field of mass spectral imaging in the biological sciences. Liquid metal ion guns (LMIG) offered the prospect of sub-100 nm spatial resolution, however this aspiration has yet to be reached for molecular imaging. This brief review shows that using LMIG the limitations of the static limit and low ionization probability will restrict useful imaging to around 2 mum spatial resolution with high-yield molecules. The only prospect of going beyond this in the absence of factors of 100 increase in ionization probability is to use polyatomic ion beams such as C (60) (+) , for which bombardment induced damage is low. In these cases sub-micron imaging becomes possible, using voxels together with molecular depth profiling and 3D imaging. The discussion shows that conventional ToF-SIMS instrumentation then becomes a limitation in that the pulsed ion beam has a very low duty cycle which results in inordinately long analysis times, and pulsing the beam means that high-mass resolution and high spatial resolution are mutually incompatible. New instrumental configurations are described that allow the use of a dc ion beam and separate the mass spectrometry for the ion formation process. Early results from these instruments suggest that sub-micron analysis and imaging with high mass resolution and good ion yields are now realizable, although the low ion yield issue still needs to be solved.

Structures in Multicomponent Polymer Films: Their Formation, Observation and Applications in Electronics and Biotechnology

Several strategies to form multicomponent films of functional polymers, with micron, submicron and nanometer structures, intended for plastic electronics and biotechnology are presented. These approaches are based on film deposition from polymer solution onto a rotating substrate (spin-casting), a method implemented already on manufacturing lines. Film structures are determined with compositional (nanometer) depth profiling and (submicron) imaging modes of dynamic secondary ion mass spectrometry, near-field scanning optical microscopy (with submicron resolution) and scanning probe microscopy (revealing nanometer features). Self-organization of spin-cast polymer mixtures is discussed in detail, since it o®ers a one-step process to deposit and align simultaneously domains, rich in di®erent polymers, forming various device elements: (i) Surface segregation drives self-stratification of nanometer lamellae for solar cells and anisotropic conductors. (ii) Cohesion energy density controls morphological transition from lamellar (optimal for encapsulated transistors) to lateral structures (suggested for light emitting diodes with variable color). (iii) Selective adhesion to substrate microtemplates, patterned chemically, orders lateral structures for plastic circuitries. (iv) Submicron imprints of water droplets (breath figures) decorate selectively micron-sized domains, and can be used in devices with hierarchic structure. In addition, selective protein adsorption to regular polymer micropatterns, formed with soft lithography after spin-casting, suggests applications in protein chip technology. An approach to reduce lateral blend film structures to submicron scale is also presented, based on (annealed) films of multicomponent nanoparticles

Use of NanoSIMS in the search for early life on Earth: ambient inclusion trails in a c . 3400 Ma sandstone

Ambient inclusion trails (AIT) are enigmatic microtubular structures created by the migration of mineral crystals through a lithified substrate. The decomposition of organic material has been suggested as the driving force for the crystal migration, but has yet to be rigorously tested. AIT may hold potential as a biosignature for investigating early life on Earth if the associated organic material can be shown to be biological. This paper attempts to test the formation mechanism and biogenicity of AIT from the c . 3400 Ma Strelley Pool sandstone of Western Australia using NanoSIMS technology. In doing so, we demonstrate the unique ability of the NanoSIMS to combine sub-micron scale imaging with in situ chemical and isotopic data, thereby enhancing our ability to evaluate the biogenicity criteria for Archaean microstructures. Enrichments of a suite of major elements (C, N, P, S) and trace elements (Co, Fe, Ni, Zn), often associated with biological processes, are found within several AIT in this sandstone. C and N enrichments are most common along AIT margins, and correlate with depletions of Si, O, Ca and Mg, indicating that this material is indeed organic in nature. δ 13 C values of this carbonaceous material average −26‰. Petrographic observations show that some of the AIT occur in the centre of detrital sandstone grains where they were sealed from later fluid flow and therefore preserve primary Archaean (bio)geochemistry. In contrast, AIT found around the outer edges of sandstone grains may contain more recent organic material introduced by later fluid migration and are an unreliable biosignature. Using the petrographic and geochemical data a multi-stage model for AIT formation and subsequent diagenetic modification is proposed. The possible sources of the primary organic material are discussed and we conclude that the data are consistent with a biological origin for these AIT. An abiogenic origin is more difficult to sustain but cannot yet be completely excluded for AIT in general.

Focused ion beam manipulation and ultramicroscopy of unprepared cells

The focused ion beam (FIB) technique of nanomachining combined with simultaneous scanning electron microscopy (SEM) was used for submicron manipulation and imaging of unprepared (fresh) cells to demonstrate the potentiality of the FIB/SEM technique for ultramicroscopic studies. Sectioning at the nanoscale level was successfully performed by means of ion beam-driven milling operations that reveal the ultrastructure of fresh yeast cells. The FIB/SEM has many advantages over other ultramicroscopy techniques already applied for unprepared/fresh biological samples.

Soft x-ray submicron imaging detector based on point defects in LiF

The use of lithium fluoride (LiF) crystals and films as imaging detectors for EUV and soft-x-ray radiation is discussed. The EUV or soft-x-ray radiation can generate stable color centers, emitting in the visible spectral range an intense fluorescence from the exposed areas. The high dynamic response of the material to the received dose and the atomic scale of the color centers make this detector extremely interesting for imaging at a spatial resolution which can be much smaller than the light wavelength. Experimental results of contact microscopy imaging of test meshes demonstrate a resolution of the order of 400nm. This high spatial resolution has been obtained in a wide field of view, up to several mm2. Images obtained on different biological samples, as well as an investigation of a soft x-ray laser beam are presented. The behavior of the generated color centers density as a function of the deposited x-ray dose and the advantages of this new diagnostic technique for both coherent and noncoherent EUV sources, compared with CCDs detectors, photographic films, and photoresists are discussed.

Assessment of the Analytical Capabilities of Scanning Capacitance and Scanning Spreading Resistance Microscopy Applied to Semiconductor Devices

In this work we compare the performance of Scanning Capacitance Microscopy and Scanning Spreading Resistance Microscopy for the characterization of doping profiles in semiconductor devices. Particular attention is devoted to parameters of paramount importance for the failure analysis and the characterization of silicon devices, like the quantitative one-dimensional profiling accuracy, the electrical junction delineation and the two-dimensional sub-micron imaging capability.

Effects of objective numerical apertures on achievable imaging depths in multiphoton microscopy

Multiphoton microscopy is a powerful technique for achieving three-dimensional submicron imaging in biological specimens. However, specimen optical parameters such as refractive indices and scattering coefficients can result in the loss of image resolution and decreased signal in depth. These factors are coupled to the focusing objective's numerical aperture (NA) in limiting the achievable imaging depths. In this work, we performed multiphoton imaging on aqueous fluorescent solution, human skin, and rat tail tendon to show that, under the same immersion condition, lower NA objectives can examine more deeply into biological specimens and should be used when optimal imaging depths is desired.

Submicron imaging with a planar silver lens

Optical imaging through a thin planar silver layer has been achieved by utilizing near-field lithography techniques. A 120 nm thick silver lens that was placed 60 nm below a patterned mask, imaged the mask’s features onto a photosensitive material located 60 nm below the silver. The entire structure was exposed from above with a mercury lamp. Features sizes as small as 350 nm (at a 700 nm period) were imaged onto the photosensitive material, demonstrating the lensing ability of the planar silver slab.

Submicron imaging in the EUV spectral range using high-harmonic radiation

Nowadays, high-harmonic generation is a state-of-the art technique for laboratory extreme ultra-violet (EUV) sources providing electromagnetic radiation with photon energies up to some hundreds of eV. In contrast to other laser-based EUV-sources, the emitted pulses have pulse durations that are shorter or in the order of the driving laser pulse duration, i.e. fs or less. We report on first experiments using a high-harmonic source for microscopy imaging with radiation of 96 eV close to the silicon-L-edge at 100 eV. A simple setup with a zone plate as objective and a mica test sample yields a spatial resolution of 0.8 μm limited by the pixel size of the back-illuminated ccd used as detector. The advantage of the presented microscopy technique is less found in the achievable spatial resolution but in the combination of moderate spatial and high temporal resolution.

Submicron imaging of buried integrated circuit structures using scanning confocal electron microscopy

Two-dimensional images of model integrated circuit components were collected using the technique of scanning confocal electron microscopy. For structures embedded about 5 μm below the surface of a silicon oxide dielectric, a lateral resolution of 76±9 nm was measured. Elemental mapping via x-ray emission spectrometry is demonstrated. A parallax analysis of images taken for various tilt angles to the electron beam allowed determination of the spacing between two wiring planes. The results show that scanning confocal electron microscopy is capable of probing buried structures at resolutions that will be necessary for the inspection of next-generation integrated circuit technology.

A variable cryogenic temperature near-field scanning optical microscope

We describe a novel variable (cryogenic) temperature near-field scanning optical microscope (VT-NSOM) designed specifically for submicron imaging of materials and devices over a temperature range of 12–300 K. In high vacuum, we cool only the sample stage of the compact NSOM, thereby maintaining a large scan area (35 μm×35 μm) at low temperatures and enabling rapid (∼30 min) temperature changes. With incorporation into an external conventional optical microscope, the VT-NSOM is capable of imaging a single submicron feature over the entire temperature range. We demonstrate the performance of the instrument by examining the photoresponse of threading dislocation defects in relaxed GeSi films.

SYNCHROTRON RADIATION SCANNING PHOTOEMISSION MICROSCOPY: INSTRUMENTATION AND APPLICATION IN SURFACE SCIENCE

X-ray photoelectron spectroscopy has become a true microscopic technique at third generation soft X-ray synchrotron sources, finding applications in many domains of academic and applied research. This paper describes the present status of scanning photoemission microscopy, where by using photon optics the photon beam can be focused to micron or submicron dimensions and imaging or spectroscopy can be performed. It discusses different photon focusing optical elements and describes the major components of the constructed scanning microscopes. The applications of imaging and spectroscopy with high lateral resolution in surface science are illustrated, and some recent results obtained in different laboratories are briefly reviewed.

E‐Beam SNMS: A complementary surface analysis technique

AbstractSputtered neutral mass spectrometry (SNMS) with ionization by low‐energy electron beam offers several analytical advantages that complement the capabilities of the major surface analysis techniques: XPS, AES and SIMS. These include easy operation on insulator specimens, reduced influence of topography or sample tilt, low spread of elemental relative sensitivity factors (RSFs) across the periodic table, near‐constant RSFs in different matrices and hence quantification from 100% down to trace concentrations, ppm detection limits, microprobe operation for depth profiling and submicron imaging and depth resolutions as for SIMS (≥1 nm). In addition to these analytical performances, the equipment shares much in common with quadrupole SIMS facilities and, as with that technique, is easily integrated into multitechnique instruments.This paper compares performances of SNMS and SIMS on the same samples and presents some imaging and useful yield data from a close‐coupled ionizer, which is a new development on the 410 SIMSLAB. Comparisons are also made between, SIMS, SNMS and AES.

Soft X-ray submicron imaging experiments with nanosecond exposure

A high throughput soft X-ray optical system was designed, constructed and tested for obtaining magnified images of nonradiating objects at wavelengths λ=175–200 Å. The optical system consists of normal incidence multilayer mirrors including a condenser and Schwarzschild objective with 10× magnification and it was applied together with laser produced plasma as an X-ray source. The magnified images with 0.7 μm resolution were produced in one shot exposure at 0.8-1 J of incident laser energy. These results show the potential for development of table-top soft X-ray microscopes with 1 nanosecond exposure and submicron spatial resolution.

High performance acrylic polymers for chemically amplified photoresist applications

Chemically amplified resists have recently achieved importance as materials useful for high density microlithographic applications requiring submicron imaging. In an effort to extend the chemical amplification concept to applications requiring thick film resists, we have developed a family of acrylic polymers which provide high performance, aqueous developing positive resists when formulated with the appropriate photo-acid generators. The polymers were prepared from methyl methacrylate (MMA), t-butyl methacrylate (TBMA) and methacrylic acid (MAA), each of which serves a separate function in the formed terpolymer. An unusual degree of control over the aqueous development kinetics can be achieved by manipulation of the terpolymer composition. The lithographic properties of resist formulations based on these polymers have been examined employing optical exposure at wavelengths from 248 to 514 nm, and e-beam exposure.

Submicron imaging at 248.3nm: A lithographic performance review and initial process performance screening of Megaposit SNR 248-1.0 photo resist.

This paper reviews the initial lithographic performance of the Megaposit® SNR™ 248-1.0 Photo Resist during the imaging of 0.35, 0.40, 0.45, 0.50 and 0.55μm line/space pairs on a 0.42NA/0.5 coherence/ KrF stepper. The test results show that process window overlap can be maintained. to a normalized geometry size of 0.59 k1 for line/space pairs. This degree of overlap is shown to be comparable to a normalized value of 0.76 k1 during the simultaneous imaging of line/space pairs, isolated lines and isolated spaces. This result appears to be consistent with the g-line results published previously1. It is also shown that at the exposure dose required to size the target geometry, the resist induced bias, due to lateral development is negligible and provides the possibility to extend the working resolution to smaller geometries if the intensity minimum of the aerial image can be suppressed. Finally, an additional screening experiment in which soft bake temperat re, post exposure bake temperature, resist thickness and develop time were varied, This latter study suggests that detailed optimization studies should examine lower PEB temperatures, 140C and less, and shorter develop times, 90s and less, at both EMIN and EMAX thicknesses.