Patterning photoresist with extreme control over dose and placement is the first crucial step in semiconductor manufacturing. However, how can the activation of modern complex resist components be accurately measured at sufficient spatial resolution? No exposed nanometre-scale resist pattern is sufficiently sturdy to unalteredly withstand inspection by intense photon or electron beams, not even after processing and development.This paper presents experimental proof that infrared atomic force microscopy (IR-AFM) is sufficiently sensitive and gentle to chemically record vulnerable yet valuable lithographic patterns in a chemically amplified resist after exposure prior to development. Accordingly, IR-AFM metrology provides long-sought insights into changes in the chemical and spatial distribution per component in a latent resist image, both directly after exposure and during processing. With these to-be-gained understandings, a disruptive acceleration of resist design and processing is expected.

Patterning photoresist with extreme control over dose and placement is the first crucial step in semiconductor manufacturing. However, how can the activation of modern complex resist components be accurately measured at sufficient spatial resolution? No exposed nanometre-scale resist pattern is sufficiently sturdy to unaltered withstand inspection by intense photon or electron beams, not even after processing and development. This paper presents experimental proof that infrared atomic force microscopy (IR-AFM) is sufficiently sensitive and gentle to chemically record vulnerable yet valuable lithographic patterns in a chemically amplified resist after exposure prior to development. Accordingly, IR-AFM metrology provides long-sought insights into changes in the chemical and spatial distribution per component in a latent resist image, both directly after exposure and during processing. With these to-be-gained understandings, a disruptive acceleration of resist design and processing is expected.

In order to extract ever more performance from semiconductor devices on the same device area, the semiconductor industry is moving towards device structures with increasingly complex material combinations and 3D geometries. To ensure cost effective fabrication of next generation devices, metrology solutions are needed that tackle the specific challenges that come from these developments such as 3 dimensional imaging of structures and imaging of deeply buried structures under arbitrary, complex layers. Compared to existing metrology solutions for high end manufacturing, ultrasonic inspection techniques have advantages: they are unaffected by optically opaque layers, the acoustic wavelength (60nm @ 100GHz in SiO2) can be smaller than optical wavelengths and the measurement depth can be larger. However, traditional acoustic microscopy tops out at a few GHz due to manufacturing tolerances and the required liquid couplant. We propose to combine very high frequency ultrasound with scanning probe microscopy. By locating the transducer above the cantilever tip, it guides sound into the sample with a dry tip-sample contact. This allows for very high acoustic frequencies and a resolution of O(wavelength).

While Atomic Force Microscopy is mostly used to investigate surface properties, people have almost since its invention sought to apply its high resolution capability to image also structures buried within samples. One of the earliest techniques for this was based on using ultrasound excitations to visualize local differences in effective tip-sample stiffness caused by the presence of buried structures with different visco-elasticity from their surroundings. While the use of ultrasound has often triggered discussions on the contribution of diffraction or scattering of acoustic waves in visualizing buried structures, no conclusive papers on this topic have been published. Here we demonstrate and discuss how such acoustical effects can be unambiguously recognized and can be used with Atomic Force Microscopy to visualize deeply buried structures.

In this paper, we present an AFM based subsurface measurement technique that can be used for overlay and critical dimensions (CD) measurements through optically opaque layers. The proposed method uses the surface elasticity map to resolve the presence and geometry of subsurface structures. To improve the imaging performance of the AFM based subsurface measurements, we made use of photothermal excitation of the AFM cantilever together with a frequency modulation scheme. The experimental results show a significant improvement in the quality of the image, which leads to a more accurate and reliable CD and overlay measurement.

Innate immunity is critical in the early containment of influenza A virus (IAV) infection and surfactant protein D (SP-D) plays a crucial role in innate defense against IAV in the lungs. Multivalent lectin-mediated interactions of SP-D with IAVs result in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phagocytic cells. Previous studies showed that porcine SP-D (pSP-D) exhibits distinct antiviral activity against IAV as compared to human SP-D (hSP-D), mainly due to key residues in the lectin domain of pSP-D that contribute to its profound neutralizing activity. These observations provided the basis for the design of a full-length recombinant mutant form of hSP-D, designated as "improved SP-D" (iSP-D). Inspired by pSP-D, the lectin domain of iSP-D has 5 amino acids replaced (Asp324Asn, Asp330Asn, Val251Glu, Lys287Gln, Glu289Lys) and 3 amino acids inserted (326Gly-Ser-Ser). Characterization of iSP-D revealed no major differences in protein assembly and saccharide binding selectivity as compared to hSP-D. However, hemagglutination inhibition measurements showed that iSP-D expressed strongly enhanced activity compared to hSP-D against 31 different IAV strains tested, including (pandemic) IAVs that were resistant for neutralization by hSP-D. Furthermore, iSP-D showed increased viral aggregation and enhanced protection of MDCK cells against infection by IAV. Importantly, prophylactic or therapeutic application of iSP-D decreased weight loss and reduced viral lung titers in a murine model of IAV infection using a clinical isolate of H1N1pdm09 virus. These studies demonstrate the potential of iSP-D as a novel human-based antiviral inhalation drug that may provide immediate protection against or recovery from respiratory (pandemic) IAV infections in humans.

Extracting quantitative information about dimensions and material properties of buried structures is continuing to be an important but difficult task in metrology. Examples of questions asking for this capability include critical dimension metrology of fins such as the profile (bottom width, top width, height) or the presence and extent of voids. In recent years TNO has demonstrated the concept of using Atomic Force Microscopy (AFM) in combination with ultrasound to image buried structures based on their (visco-)elasticity in a technique called Subsurface Scanning Ultrasound Resonance Force Microscopy (SSURFM). We have successfully imaged structures less than 10nm wide, as well as structures buried up to a micrometer deep. However, extracting quantitative information from this data is not trivial, as the induced stress field in the sample depends on many parameters in a non-linear way: experimental parameters such as applied force, tip size and tip shape, and geometry and material properties of the buried structures themselves. Therefore, measurements based on this technique have a point spread function which varies in a complicated way with the sample properties that need to be measured. However, a solid understanding of the physics and mechanics involved, and the modeling of the expected structures and their response to externally applied stress, enable quantitative measurements. We specifically show our progress on characterizing a sample comprising fins from a 7nm node manufacturing test run.

As the pitch approaches the 10nm node, in order to meet current and future patterning challenges, high resolution techniques are required, complementary to extreme ultraviolet lithography (EUVL) for high volume manufacturing of nanodevices. These complementary techniques should have the following specifications: 1) High patterning resolution, below 10 nm; 2) capability of patterning in 3D; 3) sufficient wafer-scale throughput; 4) the capability of closed loop metrology and 5) the capability of measuring nondestructively through layers, for alignment and overlay applications. Scanning probe microscopy (SPM) has shown a great degree of nano-scale control, and a great potential to address the challenges found in metrology. There has been a broad development of SPM-based methods for patterning and metrology purposes although its exploitation for technological applications is limited due to the modest throughput of scanning probe based techniques. In this article we present experimental results that include the proof-of-principle of using SSURFM to locate existing buried nanopatterns (lines of 50 nm) and subsequently using our patterning technology to manufacture nanocontact holes aligned to the existing buried lines. In combination with the high throughput parallel scanning probe, this example demonstrates the great potential and the suitability of the group of technologies developed at TNO (consisting of the patterning and the subsurface nanoimaging) for alignment and overlay, especially through opaque layers.

Nondestructive subsurface nanoimaging through optically opaque media is considered to be extremely challenging and is essential for several semiconductor metrology applications including overlay and alignment and buried void and defect characterization. The current key challenge in overlay and alignment is the measurement of targets that are covered by optically opaque layers. Moreover, with the device dimensions moving to the smaller nodes and the issue of the so-called loading effect causing offsets between between targets and product features, it is increasingly desirable to perform alignment and overlay on product features or so-called on-cell overlay, which requires higher lateral resolution than optical methods can provide. Our recently developed technique known as SubSurface Ultrasonic Resonance Force Microscopy (SSURFM) has shown the capability for high-resolution imaging of structures below a surface based on (visco-)elasticity of the constituent materials and as such is a promising technique to perform overlay and alignment with high resolution in upcoming production nodes. In this paper, we describe the developed SSURFM technique and the experimental results on imaging buried features through various layers and the ability to detect objects with resolution below 10 nm. In summary, the experimental results show that the SSURFM is a potential solution for on-cell overlay and alignment as well as detecting buried defects or voids and generally metrology through optically opaque layers.

Imaging of nanoscale structures buried in a covering material is an extremely challenging task, but is also considered extremely important in a wide variety of fields. From fundamental research into the way living cells are built up to process control in semiconductor manufacturing would all benefit from the capability to image nanoscale structures through arbitrary covering layers. Combining Atomic Force Microscopy (AFM) with ultrasound has been shown a promising technology to enable such imaging in various configurations. Here we report the development of an alternative method of combining AFM with ultrasound which we call SubSurface Ultrasonic Resonance Force Microscopy (SSURFM) and which is based on a combination of the two most common variants described in literature, which each have their specific strong points: Ultrasonic Force Microscopy (UFM) and Contact Resonance AFM (CR-AFM). We show the excellent performance of this combination on a number of samples designed specifically to mimic relevant conditions for the application as a metrology technique in the semiconductor manufacturing process. We also discuss the physics of the image contrast mechanism which is based on sensing local changes in visco-elastic properties of the sample bygenerating large indentations in the surface.

Inspection of EUV mask substrates and blanks is demanding. We envision this is a good target application for massively parallel Atomic Force Microscopy (AFM). We envision to do a full surface characterization of EUV masks with AFM enabling 1nm true 3D resolution over the entire surface. The limiting factor to do this is in the sensor itself: throughput is limited by the time that a cantilever needs to adjust its oscillation amplitude to the surface topography while scanning. We propose to use heavily damped cantilevers to maximize the measurement bandwidth. We show that using up to 20.000 cantilevers in parallel we can then reach a throughput of one 152×152mm2 substrate per 2 days with 1nm resolution.

In this paper we present a new AFM based nano-patterning technique that can be used for fast defect repairing of high resolution photomasks and possibly other high-speed nano-patterning applications. The proposed method works based on hammering the sample with tapping mode AFM followed by wet cleaning of the residuals. On the area where a specific pattern should be written, the tip-sample interaction force is tuned in a controlled manner by changing the excitation frequency of the cantilever without interrupting the imaging process. Using this method several patterns where transferred to different samples with imaging speed. While the pattern was transferred to the sample in each tracing scan line, the patterned sample was imaged in retracing scan line, thus the outcome was immediately visible during the experiment.

We have developed a high speed, miniature scanning probe microscope (MSPM) integrated with a Positioning Unit (PU) for accurately positioning the MSPM on a large substrate. This combination enables simultaneous, parallel operation of many units on a large sample for high throughput measurements. The size of the MSPM is 19 × 45 × 70 mm(3). It contains a one-dimensional flexure stage with counter-balanced actuation for vertical scanning with a bandwidth of 50 kHz and a z-travel range of more than 2 μm. This stage is mechanically decoupled from the rest of the MSPM by suspending it on specific dynamically determined points. The motion of the probe, which is mounted on top of the flexure stage is measured by a very compact optical beam deflection (OBD). Thermal noise spectrum measurements of short cantilevers show a bandwidth of 2 MHz and a noise of less than 15 fm/Hz(1/2). A fast approach and engagement of the probe to the substrate surface have been achieved by integrating a small stepper actuator and direct monitoring of the cantilever response to the approaching surface. The PU has the same width as the MSPM, 45 mm and can position the MSPM to a pre-chosen position within an area of 275×30 mm(2) to within 100 nm accuracy within a few seconds. During scanning, the MSPM is detached from the PU which is essential to eliminate mechanical vibration and drift from the relatively low-resonance frequency and low-stiffness structure of the PU. Although the specific implementation of the MSPM we describe here has been developed as an atomic force microscope, the general architecture is applicable to any form of SPM. This high speed MSPM is now being used in a parallel SPM architecture for inspection and metrology of large samples such as semiconductor wafers and masks.

The development of nerve guidance conduits is constantly evolving as the need arises for therapies for spinal cord injury. In addition to providing a path for regrowing axons to reconnect with their appropriate targets, the structural and biochemical cues provided by these conduits should be permissive for directional neurite outgrowth and be protective against inhibition in the vicinity of the injury site. Here, we adapted the use of iso-electric focusing to drive the alignment of supramolecular fibrils into self-assembled collagen hydrogels (∼300 µm diameter), and tested those hydrogels for the ability to direct and enhance the migration of neurites. Structural characterization revealed anisotropic alignment of nanofibrillar aggregates (∼20 nm diameter), arranged in micron-scale bundles (∼1 to 2 µm diameter) similar to the hierarchical size scales observed in native tissues. Neurite outgrowth extended bidirectionally along the axes of aligned hydrogels. Furthermore, it was shown that, as opposed to poly-D-lysine, neurite outgrowth on aligned hydrogels is not inhibited in the presence of myelin-associated glycoprotein (p > 0.05). These results highlight for the first time a structural and biochemical role for iso-electrically aligned collagen hydrogels in controlling neuronal growth, and indicate that the short-term signaling associated with these hydrogels can be used in adjunct therapy following injury to the spinal cord.

The mechanical properties of cells are reported to be regulated by a range of factors including interactions with the extracellular environment and other cells, differentiation status, the onset of pathological states, as well as the intracellular factors, for example, the cytoskeleton. The cell cycle is considered to be a well-ordered sequence of biochemical events. A number of processes reported to occur during its progression are inherently mechanical and, as such, require mechanical regulation. In spite of this, few attempts have been made to investigate the putative regulatory role of the cell cycle in mechanobiology. In the present study, Atomic Force Microscopy (AFM) was employed to investigate the elastic modulus of synchronised osteoblasts. The data obtained confirm that osteoblast elasticity is regulated by cell cycle phase; specifically, cells in S phase were found to have a modulus approximately 1.7 times that of G1 phase cells. Confocal microscopy studies revealed that aspects of osteoblast morphology, namely F-actin expression, were also modulated by the cell cycle, and tended to increase with phase progression from G0 onwards. The data obtained in this study are likely to have implications for the fields of tissue- and bio-engineering, where prior knowledge of cell mechanobiology is essential for the effective replacement and repair of tissue. Furthermore, studies focused on biomechanics and the biophysical properties of cells are important in the understanding of the onset and progression of disease states, for example cancer at the cellular level. Our study demonstrates the importance of the combined use of traditional and relatively novel microscopy techniques in understanding mechanical regulation by crucial cellular processes, such as the cell cycle.

Mechanical forces play a critical role in the regulation of cell spreading and cytoskeletal organization. It has been demonstrated that the force required to compress a spread cell is significantly higher than that required to compress a cell with a rounded morphology [1]. This increase in force can not be attributed to morphological changes alone [2], highlighting the importance of cytoskeletal remodeling during cell spreading in the resistance of cells to compressive forces.

Muscle contractions begin in early embryonic life, generating forces that regulate the correct formation of the skeleton. In this paper we test the hypothesis that the biophysical stimulation generated by muscle forces may be a causative factor for the changes in shape of the knee joint as it grows. We do this by predicting the spatial and temporal patterns of biophysical stimuli, where cell proliferation and rudiment shape changes occur within the emerging tissues of the joint over time. We used optical projection tomography (OPT) to create anatomically accurate finite element models of the embryonic knee at three time points (stages) of development. OPT was also used to locate muscle attachment sites and AFM was used to determine material properties. An association was found between the emergence of joint shape, cell proliferation and the pattern of biophysical stimuli generated by embryonic muscle contractions. Elevated rates of growth and cell proliferation in the medial condyle were found to co-localise with elevated patterns of biophysical stimuli including maximum principal stresses and fluid flow, throughout the time period studied, indicating that cartilage growth and chondrocyte proliferation in the epiphysis is potentially related to local patterns of biophysical stimuli. The development of the patella and articular cartilages, which is known to be affected by in ovo immobilisation, could be contributed to by specific patterns of fluid flow, pore pressure and stress in the joint interzone. This suggests that both cartilage growth and tissue differentiation in the embryonic joint is regulated by specific patterns of biophysical stimuli and that these stimuli are needed for the correct development of the joint.

A new method for extracting quantitative data from amplitude modulation dynamic force-distance measurements is developed. The method is based on the harmonic oscillator model of vibrating atomic force microscope cantilevers, and is capable of extracting both the conservative and dissipative parts of the tip-sample interaction from a measurement of oscillation amplitude and phase as a function of distance. Numerical simulations are used to demonstrate the validity of the method. Further proof of the accuracy of this method is provided by a measurement of electrostatic forces between an AFM tip and a graphite sample.

Fluorescent correlation spectroscopy (FCS) was used to measure binding affinities of ligands to ligates that are expressed by phage-display technology. Using this method we have quantified the binding of the 14-3-3 signaling protein to artificial peptide ligand. As a ligand we used the R18 artificial peptide expressed as a fusion in the cpIII coat protein that is present in 3 to 5 copies in an M13 phage. Comparisons of binding affinities were made with free R18 ligands using FCS. The result showed a relatively high binding affinity for the phage-displayed R18 peptide compared with binding to free fluorescently labeled R18. Quantification was supported by titration of the phage numbers using atomic force microscopy (AFM). AFM was shown to accurately determine phage numbers in solution as a good alternative for electron microscopy. It was shown to give reliable data that correlated perfectly with those of the viable phage numbers determined by classical bacterial infection studies. In conclusion, a very fast and sensitive method for the selection of new peptide ligands or ligates based on a quantitative assay in solution has been developed.