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An astigmatic optical profilometer provides numerous advantages such as compact size, low cost, and high resolution. In 2018, a z -axis modulation mode of the astigmatic optical profilometer was developed to realize quantitative height measurement for a surface comprising complex materials. However, the current imaging rate of the z -axis modulation mode is time-consuming. In this article, a resonant scanner was proposed to execute a high-speed z -axis scanning motion. By inducing the resonant mode, the resonant scanner enabled both a large travel range of over 87 μ m and high oscillation frequency of 1.576 kHz. Additionally, a data analysis process was proposed to calibrate the nonlinear movement of the resonant scanner. Experimental results demonstrated that the developed optical profilometer successfully captured the quantitative height and reflectivity of the complex material comprising chrome patterns on a glass substrate. Furthermore, an imaging rate of 256 s/frame was achieved, which was approximately 50 times faster than that of a previous system employing a commercial flexure-guided scanner.

Utilizing the photopolymer based volume-holography phase grating, a cubic-phase optical element is fabricated for the generation of 1D and 2D Airy beams. The characteristic properties of the generated Airy beams are experimentally verified and discussed.

Trillions of USD have been dedicated to the development of consumer electronic technologies, to produce drones, wireless chargers, spy cameras, and DVD/Blu-ray optical data storage systems. Consequently, these have been perfected into compact, reliable, high-performance, and low-cost devices. This work reviews the complete repurposing (hacking) of consumer electronics for biomedical imaging and sensing applications.

Conventional microscale 3D printing techniques mostly rely on the raster scanning method, which needs constant changing of printer head/light beam/substrate directions to print a solid structure. Therefore, throughput is a longstanding bottleneck and it is more challenging to print microfeatures in large areas. This study demonstrates the possibility of 3D printing microfeatures on a fast-spinning disc. A Blu-ray drives based high-throughput 3D printer (BRIGHT3D) is developed to demonstrate the spin printing on disc concept and evaluate the highest linear printing speed. The BRIGHT3D integrates two Blu-ray drives that are synchronized by a customized controller. The printing substrate is a standard Blu-ray RW disc spun by a spindle motor. Both drives utilize the same optical pick-up unit (OPU), which equips a voice coil motor (VCM) for the disc wobbling compensation. The bottom OPU detects the disc wobbling and feeds the VCM control signal back to top OPU for maintaining laser (405 nm, 658 microwatts) focused on the spinning substrate disc. The BRIGHT3D can directly spin-coat (up to 6,440 rpm) commercial photopolymers with a controllable thickness on top of the substrate disc. The top OPU laser was switched with a frequency of 1~500 kHz (duty cycle: 80 %) for the preliminary spinning 3D printing evaluation. Microfeatures can be cured by the BRIGHT3D while the disc is spinning at a speed of 265 rpm, which has a linear speed from inner diameter, 20 mm, to the outer diameter, 58.5 mm, of 0.55~1.63 meters per second. After removing the photopolymer residues by 75% ethanol, various microscale features on the disc can be seen and measured by scanning electron microscopy. Microscale lines (height/width: 1.43/8.25 microns) and dots (length: 5.97 microns) were successfully printed on the disc. The BRIGHT3D is aiming for multiple layer printing on the disc to realize sophisticated features of high-throughput 3D printing in the near future.

The initial formation process of nanobubbles at solid–water interfaces remains unclear because of the limitations of current imaging techniques. To directly observe the formation process, an astigmatic high-speed atomic force microscope (AFM) was modified to enable imaging in the liquid environment. By using a customized cantilever holder, the resonance of small cantilevers was effectively enhanced in water. The proposed high-speed imaging technique yielded highly dynamic quasi-two-dimensional (2D) gas structures (thickness: 20–30 nm) initially at the graphite–water interface. The 2D structures were laterally mobile mainly within certain areas, but occasionally a gas structure might extensively migrate and settle in a new area. The 2D structures were often confined by substrate step edges in one lateral dimension. Eventually, all quasi-2D gas structures were transformed into cap-shaped nanobubbles of higher heights and reduced lateral dimensions. These nanobubbles were immobile and remained stable under continuous AFM imaging. This study demonstrated that nanobubbles could be stably imaged at a scan rate of 100 lines per second (640 μm/s).

Hsp70s use ATP hydrolysis to disrupt protein-protein associations and to move macromolecules. One example is the Hsc70- mediated disassembly of the clathrin coats that form on vesicles during endocytosis. Here, we exploited the exceptional features of these coats to test three models-Brownian ratchet, power-stroke and entropic pulling-proposed to explain how Hsp70s transform their substrates. Our data rule out the ratchet and power-stroke models and instead support a collision-pressure mechanism whereby collisions between clathrin-coat walls and Hsc70s drive coats apart. Collision pressure is the complement to the pulling force described in the entropic pulling model. We also found that self-association augments collision pressure, thereby allowing disassembly of clathrin lattices that have been predicted to be resistant to disassembly. These results illuminate how Hsp70s generate the forces that transform their substrates.

Force-distance (FD) curve-based atomic force microscopy (AFM) has been widely applied for characterizing the surface morphology and mechanical properties of soft materials at nanometer resolution. In this paper, we demonstrate FD curve-based AFM using a newly developed algorithm to detect contact points (CPs) from each FD curve and to determine properties such as stiffness, adhesion, and deformation. The performance of our method is compared with that of the tapping and peak force tapping (PFT) modes by imaging the same area of a heterogeneous blend of soft and hard polymers by using the same tip. The morphology obtained with our CP detection-based method is comparable to that obtained using the tapping mode, but PFT shows strong deviation in height images obtained on soft polymer regions. We also acquire FD curves at a much higher sampling rate than PFT. A site-dependent decaying oscillation following snap-in is detected, and the frequency of the decaying oscillation appears to be related to the stiffness of the region, pointing to a new direction for characterizing viscoelastic properties of sample surfaces with a high spatial resolution.

Scanning tunneling microscope (STM) is a powerful tool for studying the structural and electronic properties of materials at the atomic scale. The combination of low temperature and high magnetic field for STM and related spectroscopy techniques allows us to investigate the novel physical properties of materials at these extreme conditions with high energy resolution. Here, we present the construction and the performance of an ultrahigh vacuum

Scanning tunneling microscope (STM) is a powerful tool for studying the structural and electronic properties of materials at the atomic scale. The combination of low temperature and high magnetic field for STM and related spectroscopy techniques allows us to investigate the novel physical properties of materials at these extreme conditions with high energy resolution. Here, we present the construction and the performance of an ultrahigh vacuum 3He fridge-based STM system with a 7 Tesla superconducting magnet. It features a double deck sample stage on the STM head so we can clean the tip by field emission or prepare a spin-polarized tip in situ without removing the sample from the STM. It is also capable of in situ sample and tip exchange and preparation. The energy resolution of scanning tunneling spectroscopy at T = 310 mK is determined to be 400 mK by measuring the superconducting gap with a niobium tip on a gold surface. We demonstrate the performance of this STM system by imaging the bicollinear magnetic order of Fe1+xTe at T = 5 K

Atomic force microscopy (AFM) has been used to measure cellular stiffness at different osmolarities to investigate the effect of osmotic pressure on cells. However, substantial direct evidence is essential to clarify the phenomena derived from the experimental results. This study used both the single-point and force mapping methods to measure the effective Young's modulus of the cell by using temporal and spatial information. The single-point force measurements confirmed the positive correlation between cellular stiffness and osmolarity. The force mapping measurements provided local stiffness on the cellular surface and identified the cytoskeleton distribution underneath the plasma membrane. At hyper-osmolarity, the cytoskeleton was observed to cover most of the area underneath the plasma membrane, and the effective Young's modulus on the area with cytoskeleton support was determined to be higher than that at iso-osmolarity. The overall increase in cellular Young's modulus confirmed the occurrence of cytoskeleton compression at hyper-osmolarity. On the other hand, although the average Young's modulus at hypo-osmolarity was lower than that at iso-osmolarity, we observed that the local Young's modulus measured on the areas with cytoskeleton support remained similar from iso-osmolarity to hypo-osmolarity. The reduction of the average Young's modulus at hypo-osmolarity was attributed to reduced cytoskeleton coverage underneath the plasma membrane.

Astigmatic detection systems have been used in hybrid systems to produce both contact and noncontact profilometers, which provide advantageous features such as low cost, small laser spot, high bandwidth, and compact size. However, current astigmatic optical profilometers cannot provide quantitative height measurement on a surface consisting of complex materials. In this paper, a novel method called z-axis modulation is proposed to overcome this limitation. A homemade astigmatic optical profilometer was developed, and an analytical process for height calculation was also developed. As demonstrated by the experimental results, z-axis modulation can provide accurate height measurement. Furthermore, optical properties such as reflectivity can also be measured.

Recent advances in neural stem cell (NSC) transplantation have led to an inspiring progress in alleviating central nervous system (CNS) damages and restoring brain functions from diseases or injuries. One challenge of NSC transplantation is directed differentiation of transplanted NSCs into desired neuronal subtypes, such as neurons, to compensate the adverse impact of brain injury; another challenge lies in the lack of tools to noninvasively monitor the dynamics of NSC differentiation after transplantation in vivo. In this study, we developed a polymer nanovehicle for morphogen sustained release to overcome the drawbacks of conventional methods to realize the long-term directed NSC differentiation in vivo. Moreover, we constructed a bicistronic vector with a unique neuron specific gene tubb3 promoter to drive reporter gene expression for real-time imaging of NSC differentiation and migration. The developed uniform nanovehicle showed efficient NSC uptake and achieved a controlled release of morphogen in cytosol to consistently stimulate NSC differentiation into neurons at a sustainably effective concentration. The spatiotemporal imaging results showed a multiplexed migration, proliferation, differentiation, and apoptosis orchestra of transplanted NSCs regulated by nanovehicles in TBI mice. The imaging results also uncovered the peak time of NSC differentiation in vivo. Although we observed only a handful of NSCs ultimately migrated to the TBI area and differentiated into neurons, those neurons were functional, ameliorating the detrimental impact of TBI. The imaging findings enabled by the nanovehicle and the neuron specific bicistronic vector provide additional understanding of the in vivo behaviors of transplanted NSCs in neuronal regenerative medicine.

We have developed a high-sensitivity atomic force microscopy (AFM) mode operated in aqueous environment based on the torsional resonance of the cantilever. It is found that the torsional mode can achieve a good spatial resolution even with a relatively large tip. We have used this mode to image different soft materials in water, including DNA molecules and purple membrane. High-resolution images of purple membrane can be obtained at a relatively low ion concentration under a long-range electrostatic force. Thus the torsional mode allows investigators to probe surface structures and their properties under a wide range of solution conditions.

Toxocarosis is a zoonosis caused by the transmission of the Toxocara canis (T. canis) larvae to humans. Its infectious third-stage larvae can invade the brains of paratenic hosts. The resultant brain damage can result in cerebral toxocarosis (CT). Astrocytes have important neurotrophic and neuroprotective functions in the brain. Substantial studies have shown that astrocyte apoptosis may contribute to the pathogenesis of many acute and chronic neurodegenerative disorders. We propose an alternation detection method, a combination of the astigmatic detection microscopy (ADM) and atomic force microscopy (AFM) techniques, to investigate the apoptosis of astrocytes triggered with T. canis larval excretory/secretory (Tc E/S) antigen. The variation in the pathology of a cell's morphological changes was investigated with ADM and AFM analyses and then confirmed by western blotting. The results showed that the round cells increased as the concentration of Tc E/S antigen and incubated time increased. In addition, the mean height of apoptotic cells was approximately twice that of untreated normal cells, which meant there was correlation between the Tc E/S antigen treatment and cell height. For each cleaved caspase-3 in the cells cocultured with Tc E/S antigen and incubated for 9 h, the corresponding intensities increased about 34-fold (34.4 ± 1.8) compared with those of the control cells. This method can provide researchers with a perspective for understanding the limited information on the mechanism of astroglial injury and death during a T. canis larval invasion in a brain infection.

Eukaryotic flagella are responsible for the motile organelles that cause the migration of mammalian sperms. The lashing force and torque of the sperm flagellum contain critical information regarding the sperm health, as important evaluation factors for sperm screening. The objective of the study was to investigate the lashing force and torque of a sperm under physiological conditions using atomic force microscopy (AFM). At a distance of about 18.5 μm from its head-tail junction, a lashing force of 0.96 ± 0.20 nN was measured. Its corresponding lashing torque was 1.77(± 0.37)× 10(-14) N·m. The torque increases in proportion to the square of the head-tail junction distance. Our results reasonably conclude that the axonemal motility is linear dependent on the flagellum length of the sperm. Our developed measurement system can consistently determine the lashing force and torque of a sperm, which can contribute to further studies concerning the mechanism of sperm transport and fertilization.

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30–300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging., As vesicles fuse to the plasma membrane, they form intermediate Ω-shaped structures followed by either closure of the pore or full merging with the plasma membrane. Here Wen et al. show that dynamic actin assembly provides membrane tension to promote Ω merging in neuroendocrine cells and synapses.

We report the investigation of the self-assembly mechanism of nanofibers, using a small peptide amphiphile (NapFFKYp) as a model. Combining experimental and simulation methods, we identify the self-assembly pathways in the solution and on the substrates, respectively. In the solution, peptide amphiphiles undergo the nucleation process to grow into nanofibers. The nanofibers can further twist into high-ordered nanofibers with aging. On the substrates, peptide amphiphiles form nanofibers and nanosheet structures simultaneously. This surface-induced nanosheet consists of rod-like structures, and its thickness is substrate-dependent. Most intriguingly, water can transform the nanosheet into the nanofiber. Molecular dynamic simulation suggests that hydrophobic and ion-ion interactions are dominant forces during the self-assembly process.

The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We report that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed spontaneously, without the addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C(+) cells. Adding to the recent paradigm of signal transduction through higher order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling by a receptor at the cell surface.

Cancer evolves to evade or compromise the surveillance of the immune system, and cancer immunotherapy aims to harness the immune system in order to inhibit cancer development. Unmethylated CpG dinucleotide-containing oligonucleotides (CpG), a class of potent adjuvants that activate the toll-like receptor 9 (TLR9) located in the endolysosome of many antigen-presenting cells (APCs), are promising for cancer immunotherapy. However, clinical application of synthetic CpG confronts many challenges such as suboptimal delivery into APCs, unfavorable pharmacokinetics caused by limited biostability and short in vivo half-life, and side effects associated with leaking of CpG into the systemic circulation. Here we present DNA-inorganic hybrid nanovaccines (hNVs) for efficient uptake into APCs, prolonged tumor retention, and potent immunostimulation and cancer immunotherapy. hNVs were self-assembled from concatemer CpG analogs and magnesium pyrophosphate (Mg2PPi). Mg2PPi renders hNVs resistant to nuclease degradation and thermal denaturation, both of which are demanding characteristics for effective vaccination and the storage and transportation of vaccines. Fluorophore-labeled hNVs were tracked to be efficiently internalized into the endolysosomes of APCs, where Mg2PPi was dissolved in an acidic environment and thus CpG analogs were exposed to hNVs. Internalized hNVs in APCs led to (1) elevated secretion of proinflammatory factors, and (2) elevated expression of co-stimulatory factors. Compared with molecular CpG, hNVs dramatically prolonged the tissue retention of CpG analogs and reduced splenomegaly, a common side effect of CpG. In a melanoma mouse model, two injections of hNVs significantly inhibited the tumor growth and outperformed the molecular CpG. These results suggest hNVs are promising for cancer immunotherapy.

We have developed a tumor-specific phosphatase-triggered co-assembly strategy of indocyanine green (ICG)-doped nanofibers for cancer theranostics (1). This strategy relies on targeted peptide self-assemblies to improve the critical tumor uptake of ICG and its photoacoustic and photothermal properties. We demonstrated complete tumor elimination by photo-thermal therapy with high therapeutic accuracy within 24-48 hours post-injection. We have conducted extensive AFM investigations in vitro to delineate the fundamental understanding of the phosphatase-instructed formation of nanofibers. This process is crucial to its theranostic capability, and can be generalized to the self-assembly pathways of other triggered peptide hydrogelators. Our AFM results reveal a coexistence of multiple conformational structures such as the nanosheets and nanofibers with distinct nanomechnical properties. Additionally, AFM results also reveal that water can induce conformational change from nanosheets to nanofibers, which shows the dominant hydrophobic interactions during the self-assembly process. Peptide hydrogels are widely used in various biological applications, such as drug delivery and tissue engineering owing to their fibrous structure that provides the advantages of high water content and biocompatibility. This work illustrates that AFM is a powerful biophysical tool in nanomedicine for probing the structure and mechanical properties of complex biopolymer systems. Ref(1) “Tumor-Specific Formation of Enzyme-Instructed Supramolecular Self-Assemblies as Cancer Theranostics.” Huang P, Gao Y, Lin J, Hu H, Liao HS, Yan X, Tang Y, Jin A, Song J, Niu G, Zhang G, Horkay F, Chen X. ACS Nano. 2015, DOI: 10.1021/acsnano.5b03874.

Alzheimer's disease (AD) is characterized by progressive dementia and eventual loss of bodily functions that, historically, is associated with the formation of plaques composed of amyloid beta (Aβ), a 36-43 amino acid peptide. A much more complex picture is now emerging that involves many different species of Aβ oliogomers along the assembly pathway. We have used atomic force microscopy (AFM) to investigate Aβ assembly dynamics in order to observe fibril morphology and to characterize fibril elongation and nanomechanics in physiologically relevant buffers on mica as well as on supported lipid bilayers. We have especially focused on resolving small oligomers that is currently thought to initiate deterioration of brain function in AD and its drug intervention and on defining early stage oligomer to fibril growth by integrations between optical spectroscopy determination of protein conformation and AFM observations. We aim to apply quantitative kinetic schemes to identify the number of steps and kinetic constants using time-resolved AFM, thioflavin T fluorescence and circular dichroism data to define the transition from lag to log phase and the broader assembly mechanism.

Biological atomic force microscopy (Bio-AFM) can quantify changes in cellular and macromolecular morphology and elasticity at the nanoscale and over a broad time scale, from fast dynamics to long-term cellular evolutions. Herein, we report quantitative Bio-AFM characterization of mechanobiological features of lung cancer and other cells in response to chemotherapeutics and multifunctional theranostic nanoparticles. Multidrug resistance (MDR) presents a daunting challenge for successful cancer management. Comprehensive investigation of mechanobiological characters of cancer cells with MDR provides insights in directing anti-cancer therapy. In our study, we have embarked on using multimodal and multiphasic Bio-AFM to identify cellular biomechanical markers and to investigate interactions among cells and theranostic nanoparticles as an aid to developing more efficient therapeutics. We have found a spectrum of distinct biomechanical signatures, such as stiffness moduli and adhesions that differ under physiological conditions between drug sensitive and resistant cancer cells. Multifunctional theranostic nanoparticles, such as polymeric nanovehicles (1), single-walled carbon nanotubes (SWCNTs), and carbon dots (Cdots) with various surface coatings are observed to have differential influences on cellular responses. As part of work to broaden the applicability of Bio-AFM for cellular imaging, we are investigating the development of docetaxel-sensitivity and resistance in a non-small-cell lung cancer cell line. Ref (1) “Polymeric Nanovehicle Regulated Spatiotemporal Real-Time Imaging of the Differentiation Dynamics of Transplanted Neural Stem Cells after Traumatic Brain Injury.” Wang Z, Wang Y, Wang Z, Zhao J, Gutkind JS, Srivatsan A, Zhang G, Liao HS, Fu X, Jin A, Tong X, Niu G, Chen X. ACS Nano. 2015. 9:6683-95. doi:10.1021/acsnano.5b00690

We demonstrate the use of an astigmatic detection system (ADS) for resonance frequency identification of polymer microcantilever sensors. The ADS technology is based on a DVD optical head combined with an optical microscope (OM). The optical head has a signal bandwidth of 80 MHz, allowing thermal fluctuation measurements on cantilever beams with a subnanometer resolution. Furthermore, an external excitation can intensify the resonance amplitude, enhancing the signal- to-noise ratio. The full width at half maximum (FWHM) of the laser spot is 568 nm, which facilitates read-out on potentially submicrometer-sized cantilevers. The resonant frequency of SU-8 microcantilevers is measured by both thermal fluctuation and excited vibration measurement modes of the ADS.

Despite the effort of developing various nanodelivery systems, most of them suffer from undesired high uptakes by the reticuloendothelial system, such as liver and spleen. Herein we develop an endogenous phosphatase-triggered coassembly strategy to form tumor-specific indocyanine green (ICG)-doped nanofibers (5) for cancer theranostics. Based on coordinated intermolecular interactions, 5 significantly altered near-infrared absorbance of ICG, which improves the critical photoacoustic and photothermal properties. The phosphatase-instructed coassembly process, as well as its theranostic capability, was successfully conducted at different levels ranging from in vitro, living cell, tissue mimic, to in vivo. Specifically, the tumor uptake of ICG was markedly increased to 15.05 ± 3.78%ID/g, which was 25-fold higher than that of free ICG (0.59 ± 0.24%ID/g) at 4 h after intravenous injection. The resulting ultrahigh T/N ratios (15) clearly differentiated tumors from the surrounding normal tissue. Complete tumor elimination with high therapeutic accuracy has been successfully achieved upon laser irradiation (0.8 W/cm(2), 5 min) within 24-48 h postinjection. As the first example, in vivo formation of tumor-specific ICG-doped nanofiber for PTT theranostics owns the immense potential for clinical translation of personalized nanomedicine with targeted drug delivery as well as for cancer theranostics.

An important challenge in targeted drug delivery and vaccine development is to create the most effective interface between the carrier and target cells. Parasitic diseases, for which no approved vaccines are available, remain a world health problem with over one million severe cases per year attributed to malaria alone. In parallel to developing potential malaria antigen constructs (1), we have also bioengineered and characterized nanoscale virus-like particles (VLPs) with tools such as atomic force microscopy (AFM) and quantitative nanomechanical mapping (QNM) in hopes that these might serve as possible carriers for drugs or vaccines because of how they uniquely interact with human cells. Through using AFM and QNM techniques we have revealed some morphological features of our own bioengineered VLP, a protein capsid of bacteriophage Qβ (d = 25 nm), and analyzed deformation of the capsid under compressive force to quantify its elastic properties. We have found that these particles exhibit a linear relationship between compressive force and deformation as would a Hookean spring. Significant deformation (> 5 nm) was observed at forces of 0.1 - 0.5 nN. Our calculated spring constant for our Qβ capsid, 0.059 N/m, indicates that our VLP is more pliable than others of similar protein structure, which is possibly a favorable characteristic for the capsid to bind with the cell membrane in an in vivo carrier scenario. We are also using this analytical technique for the characterization of chemically conjugated (malaria) vaccines. Ref (1) “Development of a Pfs25-EPA malaria transmission blocking vaccine as a chemically conjugated nanoparticle.” Shimp RL Jr, Rowe C, Reiter K, Chen B, Nguyen V, Aebig J, Rausch KM, Kumar K, Wu Y, Jin AJ, Jones DS, Narum DL. Vaccine. 2013. 31:2954-62. doi: 10.1016/j.vaccine.2013.04.034.

Atomic force microscopy (AFM) is an advanced tool that enables the visualization of dynamic behaviors and the quantification of mechanical properties under physiological conditions of biological samples from macromolecules to cells and beyond. We have constructed a high-speed AFM toward a temporal resolution of 1s or shorter using a digital versatile disc (DVD) pickup head to detect deflections from a small cantilever. In addition, a flexure-guided scanner and a sinusoidal scan method were implemented to reach 100 line/s scan rate and successful images of clathrin cages and amyloid-β fibrils in buffers. To better define nanomechanical properties of biomedical samples ranging from nanomedicine constructs to live cells and EMC-like matrices (1), we have relied on optimizing commercial AFM systems and on using force-volume and quantitative nanomechanical mapping methods. This work showcases the value of high-speed and quantitative AFM in visualizing dynamic biological behavior at nanometer scale and in defining nanomechanical properties for understanding fundamental biomedical questions. Ref (1) “Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions,” Doyle AD, Carvajal N, Jin A, Matsumoto K, and Yamada KM, Nature Commun., in press (2015).

For precisely measuring the mass variation, different modes of cantilever-based mass sensor were developed. The dynamic mode applied for mass sensor has advantages of less thermal drift and high mass sensitivity. However, mass sensor is usually operated in liquid environment for bio-chemical examination, but the liquid viscosity significantly dampens the performance of the dynamic mode. In this paper, the high order resonances are proposed for increasing the mass sensitivity in liquid. For comprehending the influence on the mass sensitivity, we analytically and experimentally studied the performances of the unmodified commercial cantilever the modified cantilever, which is made of commercial cantilever and processed to derive different shapes with mass variation. And in their thermal fluctuation spectra, the first five order resonances are detected for comparison. The mass sensitivity −5.397 Hz/pg of the fifth order resonance is 94 times that of the first order resonance of −0.057 Hz/pg.

This paper proposes a new developed AFM pickup head by using the holographic optical element (HOE) for the atomic force microscope. The holographic optical element is very compact and has been applied in the DVD-pickup head with a stable and precise sensing performance. By comparison with the optical lever method of the ordinary AFM, the holographic optical element makes AFM an easy optical adjustment and a compact configuration. Through this miniaturizing and easy adjusting progress, AFM can also open wider application field. In this paper, we present the development and its performance of the new developed HOE-AFM, which is experimentally verified that it can realize nanometer resolution through measuring the calibration grating and the graphite specimen.

High-speed atomic force microscopy (HS-AFM) enables visualizing dynamic behaviors of biological molecules under physiological conditions at a temporal resolution of 1s or shorter. A small cantilever with a high resonance frequency is crucial in increasing the scan speed. However, detecting mechanical resonances of small cantilevers is technically challenging. In this study, we constructed an atomic force microscope using a digital versatile disc (DVD) pickup head to detect cantilever deflections. In addition, a flexure-guided scanner and a sinusoidal scan method were implemented. In this work, we imaged a grating sample in air by using a regular cantilever and a small cantilever with a resonance frequency of 5.5 MHz. Poor tracking was seen at the scan rate of 50 line/s when a cantilever for regular AFM imaging was used. Using a small cantilever at the scan rate of 100 line/s revealed no significant degradation in the topographic images. The results indicate that a smaller cantilever can achieve a higher scan rate and superior force sensitivity. This work shows the potential for using a DVD pickup head in future HS-AFM technology.

The astigmatic detection system (ADS) based on commercial optical pickup head was demonstrated to achieve a sub-nanometer sensitivity in detecting the vertical movement of an object surface in air. The detection laser spot of the ADS was sub-μm and the detection bandwidth was over 80 MHz. These advantages allow detection of high-frequency mechanical resonance of very small objects, which would have many important applications in nanotechnology. In this work, we optimized the operation conditions of ADS to achieve good sensitivity in aqueous solutions. We demonstrated good contrast and good spatial resolution of cancer cells in water with the optical profilometry mode. We also built an ADS-AFM (atomic force microscopy) for imaging in water. A novel cantilever holder was designed, and the spurious peaks were suppressed down to 26.0% of the real resonance peak. Most importantly, we demonstrated that the ADS-AFM could resolve single atomic steps on a graphite substrate and image soft DNA molecules on mica in water.

In this work, we present a design based on Lorentz force induction to excite pure torsional resonances of different types of cantilevers in air as well as in water. To demonstrate the atomic force microscopy imaging capability, the phase-modulation torsional resonance mode is employed to resolve fine features of purple membranes in a buffer solution. Most importantly, force-versus-distance curves using a relatively stiff cantilever can clearly detect the characteristic oscillatory profiles of hydration layers at a water-mica interface, indicating the high force sensitivity of the torsional mode. The high resonance frequencies and high quality-factors for the torsional mode may be of great potential for high-speed and high-sensitivity imaging in aqueous environment.

We performed experiments to measure the friction force against the relative orientation between the carbon nanotube probe and graphite substrate. As the sample stage revolved one full circle, six sharp peaks were registered and each separated by 60°. Furthermore, these six peaks formed into three pairs, exhibiting the threefold symmetry, which can be explained with contribution beyond the top layer of the graphite. Yet, due to the prominent layer structure of the graphite, the energy loss caused by friction was dissipated mostly through its top layer.

For calibrating the spring constant of a microcantilever, the thermal fluctuation method has been widely used in atomic force microscope (AFM). For this method, the force curve must be first acquired to correlate the voltage response of the photodiode with the known cantilever deflection. However, it usually takes a long time to move the cantilever tip close to the sample, and is not adapted to calibrate a tipless cantilever. To realize an efficient way of detecting photodiode responses, we developed a calibration routine employing an astigmatic detection system (ADS). In comparison with the technique of optical beam deflection (or optical lever), the ADS is more sensitive to the vertical and less sensitive to the angular displacement of a cantilever. Therefore, its photodiode response can be directly associated with a predetermined vertical movement of the cantilever, without the need of any subtract. Our experiments have verified that the derived spring constants by the ADS method are close to the typical values of the cantilevers, and this method can be applied to tipless cantilevers.

The diverse atomic configurations induce the anisotropic surface properties. For investigating anisotropic phenomena, we developed a rotational positioning system adapted to atomic force microscope (AFM). This rotational positioning system is applied to revolve the measured sample to defined angular direction, and it composed of an inertial rotational stepper and a visual angular measurement. The inertial rotational stepper with diameter 30 mm and height 7.6 mm can be easily attached to the AFM-system built in any general optical microscope. Based on a clearance less bearing and the inertial driving method, its bidirectional angular resolution reaches 0.005° per step. For realizing a close-loop controlled angular positioning function, the visual measurement method is utilized. Through the feedback control, the angular positioning error is less than 0.01°. For verifying the system performance, we used it to investigate the anisotropic surface properties of graphite. Through a modified cantilever tip, the atomic-scale stick-slip, and the anisotropic friction phenomena can be distinctly detected.