A micro-fabricated device (microICSI) improves porcine blastocyst development and procedural efficiency for both porcine intracytoplasmic sperm injection and human microinjection.

PURPOSE: Intracytoplasmic sperm injection (ICSI) imparts physical stress on the oolemma of the oocyte and remains among the most technically demanding skills to master, with success rates related to experience and expertise. ICSI is also time-consuming and requires workflow management in the laboratory. This study presents a device designed to reduce the pressure on the oocyte during injection and investigates if this improves embryo development in a porcine model. The impact of this device on laboratory workflow was also assessed. METHODS: Porcine oocytes were matured in vitro and injected with porcine sperm by conventional ICSI (C-ICSI) or with microICSI, an ICSI dish that supports up to 20 oocytes housed individually in microwells created through microfabrication. Data collected included set-up time, time to align the polar body, time to perform the injection, the number of hand adjustments between controllers, and degree of invagination at injection. Developmental parameters measured included cleavage and day 6 blastocyst rates. Blastocysts were differentially stained to assess cell numbers of the inner cell mass and trophectoderm. A pilot study with human donated MII oocytes injected with beads was also performed. RESULTS: A significant increase in porcine blastocyst rate for microICSI compared to C-ICSI was observed, while cleavage rates and blastocyst cell numbers were comparable between treatments. Procedural efficiency of microinjection was significantly improved with microICSI compared to C-ICSI in both species. CONCLUSION: The microICSI device demonstrated significant developmental and procedural benefits for porcine ICSI. A pilot study suggests human ICSI should benefit equally.

Breast cancer care and surgery in Australia and New Zealand: compliance of the BreastSurgANZ quality audit with international standards.

BACKGROUND: The Breast Surgeons of Australia and New Zealand (BreastSurgANZ) Quality Audit (BQA) of Breast Cancer Care is a prospective population-based database designed for annual audit of compliance with internally derived Quality Indicators (QI)s. While there is no international consensus for QIs, audit against an external international benchmark is possible through use of QIs defined by the 2017 European Society of Breast Cancer Specialists (EUSOMA) Guidelines. METHODS: BQA data from 29,088 female patients between 1/1/2018 and 31/12/2019 were stratified by the EUSOMA definition of low-volume hospitals (LVH <150 patients p.a.) and high-volume hospitals (HVH ≥150 patients p.a.), and percentage compliance (±95% CI) with 14 mandatory EUSOMA QI sub-parts were determined. RESULTS: ANZ LVH met the quality threshold for 10, and HVH for 8 EUSOMA QI that assessed MDT, surgical approach, adjuvant radiotherapy in the LVH setting, avoidance of overtreatment, and use of endocrine therapy. ANZ did not meet the quality thresholds for QIs assessing use of neoadjuvant chemotherapy, and adjuvant radiotherapy in the HVH setting. CONCLUSION: Breast cancer care in ANZ is comparable with an international standard. ANZ surgeons performed at a high standard in discussion of breast cancer patients by MDT, and appropriate use of adjuvant radiotherapy by LVH. Improvements can be made in completeness of data capture, and inclusion of genetic syndrome and Ki67% in data collection. Due to the rapid evolution of breast cancer treatments, there is need for contemporary update of QI relating to the use of neoadjuvant systemic therapies.

The use of neoadjuvant systemic therapies in breast cancer in Australia and New Zealand: Breast Surgeons of Australia and New Zealand quality audit.

BACKGROUND: Breast surgeons must maintain contemporary knowledge regarding appropriate referral for neoadjuvant chemotherapy (NACT) in breast cancer (BC) patients. To date, the greatest benefit is seen in stage II-III HER2-enriched and triple negative breast cancers (TNBC). This study is the first audit of use of NACT in Australia and New Zealand to stratify data by BC biological subtype. METHODS: Prospective data from 116,745 patients between 2010 and 2019 was provided by the Breast Surgeons of Australia and New Zealand (BreastSurgANZ) Quality Audit (BQA) of Breast Cancer Care. Annual rates of NACT use were determined and change across time analysed with fractional regression. Data from 2018 to 2019 were combined and stratified by biological subtype (LumA, LumB HER2-neg, LumB HER2-pos, HER2 enriched, TNBC, Other basal-like), and age (<50, 51-74, and ≥75 years) and compared using negative binomial regression. RESULTS: The use of NACT increased annually (OR 1.26, P < 0.001), and the use of additional adjuvant chemotherapy (ACT) decreased (OR 0.78, P < 0.001). A significantly greater use of NACT was noted in patients with TNBC and HER2+ BC, and in all patients aged <50 years compared with older ages (P < 0.001), regardless of biological subtype. CONCLUSION: Increased uptake of NACT and decreased use of additional ACT is in keeping with progressive change in practice in response to contemporary evidence. Expansion of BQA data fields related to use of NACT, and detailed audit of NACT rates in Stage II-III TNBC and HER2 enriched BC will allow accurate determination of quality of practice in ANZ.

Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery.

Polydimethylsiloxane (PDMS) has been the material of choice for microfluidic applications in cell biology for many years, with recent advances encompassing nano-scaffolds and surface modifications to enhance cell-surface interactions at nano-scale. However, PDMS has not previously been amenable to applications which require complex geometries in three dimensions for cell culture device fabrication in the absence of additional components. Further, PDMS microfluidic devices have limited capacity for cell retrieval following culture without severely compromising cell health. This study presents a designed and entirely 3D-printed microfluidic chip (8.8 mm × 8.2 mm × 3.6 mm) using two-photon polymerization (2PP). The 'nest' chip is composed of ten channels that deliver sub-microliter volume flowrates (to ~ 600 nL/min per channel) to 10 individual retrievable cell sample 'cradles' that interlock with the nest to create the microfluidic device. Computational fluid dynamics modelling predicted medium flow in the device, which was accurately validated by real-time microbead tracking. Functional capability of the device was assessed, and demonstrated the capability to deliver culture medium, dyes, and biological molecules to support cell growth, staining and cell phenotype changes, respectively. Therefore, 2PP 3D-printing provides the precision needed for nanoliter fluidic devices constructed from multiple interlocking parts for cell culture application.

Deletion of the Plasmodium falciparum exported protein PTP7 leads to Maurer's clefts vesiculation, host cell remodeling defects, and loss of surface presentation of EMP1.

Presentation of the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (EMP1), at knob-like protrusions on the surface of infected red blood cells, underpins the parasite's pathogenicity. Here we describe a protein PF3D7_0301700 (PTP7), that functions at the nexus between the intermediate trafficking organelle, the Maurer's cleft, and the infected red blood cell surface. Genetic disruption of PTP7 leads to accumulation of vesicles at the Maurer's clefts, grossly aberrant knob morphology, and failure to deliver EMP1 to the red blood cell surface. We show that an expanded low complexity sequence in the C-terminal region of PTP7, identified only in the Laverania clade of Plasmodium, is critical for efficient virulence protein trafficking.

Multimodal imaging reveals membrane skeleton reorganisation during reticulocyte maturation and differences in dimple and rim regions of mature erythrocytes.

The red blood cell (RBC) is remarkable in its ability to deform as it passages through the vasculature. Its deformability derives from a spectrin-actin protein network that supports the cell membrane and provides strength and flexibility, however questions remain regarding the assembly and maintenance of the skeletal network. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM) we have examined the nanoscale architecture of the cytoplasmic side of membrane discs prepared from reticulocytes and mature RBCs. Immunofluorescence microscopy was used to probe the distribution of spectrin and other membrane skeleton proteins. We found that the cell surface area decreases by up to 30% and the spectrin-actin network increases in density by approximately 20% as the reticulocyte matures. By contrast, the inter-junctional distance and junctional density increase only by 3-4% and 5-9%, respectively. This suggests that the maturation-associated reduction in surface area is accompanied by an increase in spectrin self-association to form higher order oligomers. We also examined the mature RBC membrane in the edge (rim) and face (dimple) regions of mature RBCs and found the rim contains about 1.5% more junctional complexes compared to the dimple region. A 2% increase in band 4.1 density in the rim supports these structural measurements.

Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine.

BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).

Surface area-to-volume ratio, not cellular viscoelasticity, is the major determinant of red blood cell traversal through small channels.

The remarkable deformability of red blood cells (RBCs) depends on the viscoelasticity of the plasma membrane and cell contents and the surface area to volume (SA:V) ratio; however, it remains unclear which of these factors is the key determinant for passage through small capillaries. We used a microfluidic device to examine the traversal of normal, stiffened, swollen, parasitised and immature RBCs. We show that dramatic stiffening of RBCs had no measurable effect on their ability to traverse small channels. By contrast, a moderate decrease in the SA:V ratio had a marked effect on the equivalent cylinder diameter that is traversable by RBCs of similar cellular viscoelasticity. We developed a finite element model that provides a coherent rationale for the experimental observations, based on the nonlinear mechanical behaviour of the RBC membrane skeleton. We conclude that the SA:V ratio should be given more prominence in studies of RBC pathologies.

The knob protein KAHRP assembles into a ring-shaped structure that underpins virulence complex assembly.

Plasmodium falciparum mediates adhesion of infected red blood cells (RBCs) to blood vessel walls by assembling a multi-protein complex at the RBC surface. This virulence-mediating structure, called the knob, acts as a scaffold for the presentation of the major virulence antigen, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1). In this work we developed correlative STochastic Optical Reconstruction Microscopy-Scanning Electron Microscopy (STORM-SEM) to spatially and temporally map the delivery of the knob-associated histidine-rich protein (KAHRP) and PfEMP1 to the RBC membrane skeleton. We show that KAHRP is delivered as individual modules that assemble in situ, giving a ring-shaped fluorescence profile around a dimpled disk that can be visualized by SEM. Electron tomography of negatively-stained membranes reveals a previously observed spiral scaffold underpinning the assembled knobs. Truncation of the C-terminal region of KAHRP leads to loss of the ring structures, disruption of the raised disks and aberrant formation of the spiral scaffold, pointing to a critical role for KAHRP in assembling the physical knob structure. We show that host cell actin remodeling plays an important role in assembly of the virulence complex, with cytochalasin D blocking knob assembly. Additionally, PfEMP1 appears to be delivered to the RBC membrane, then inserted laterally into knob structures.

Multimodal analysis of Plasmodium knowlesi-infected erythrocytes reveals large invaginations, swelling of the host cell, and rheological defects.

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block-face scanning electron microscopy to explore the topography of P. knowlesi-infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi-infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python-based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi-infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.

Improved Application of Carbon Nanotube Atomic Force Microscopy Probes Using PeakForce Tapping Mode.

In this work PeakForce tapping (PFT) imaging was demonstrated with carbon nanotube atomic force microscopy (CNT-AFM) probes; this imaging mode shows great promise for providing simple, stable imaging with CNT-AFM probes, which can be difficult to apply. The PFT mode is used with CNT-AFM probes to demonstrate high resolution imaging on samples with features in the nanometre range, including a Nioprobe calibration sample and gold nanoparticles on silicon, in order to demonstrate the modes imaging effectiveness, and to also aid in determining the diameter of very thin CNT-AFM probes. In addition to stable operation, the PFT mode is shown to eliminate "ringing" artefacts that often affect CNT-AFM probes in tapping mode near steep vertical step edges. This will allow for the characterization of high aspect ratio structures using CNT-AFM probes, an exercise which has previously been challenging with the standard tapping mode.

A new Python library to analyse skeleton images confirms malaria parasite remodelling of the red blood cell membrane skeleton.

We present Skan (Skeleton analysis), a Python library for the analysis of the skeleton structures of objects. It was inspired by the "analyse skeletons" plugin for the Fiji image analysis software, but its extensive Application Programming Interface (API) allows users to examine and manipulate any intermediate data structures produced during the analysis. Further, its use of common Python data structures such as SciPy sparse matrices and pandas data frames opens the results to analysis within the extensive ecosystem of scientific libraries available in Python. We demonstrate the validity of Skan's measurements by comparing its output to the established Analyze Skeletons Fiji plugin, and, with a new scanning electron microscopy (SEM)-based method, we confirm that the malaria parasite Plasmodium falciparum remodels the host red blood cell cytoskeleton, increasing the average distance between spectrin-actin junctions.

The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites.

Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.

Erythrocyte ß spectrin can be genetically targeted to protect mice from malaria.

The malaria parasite hijacks host erythrocytes to shield itself from the immune system and proliferate. Red blood cell abnormalities can provide protection from malaria by impeding parasite invasion and growth within the cell or by compromising the ability of parasites to avoid host clearance. Here, we describe 2 N-ethyl-N-nitrosourea-induced mouse lines, SptbMRI26194 and SptbMRI53426 , containing single-point mutations in the erythrocyte membrane skeleton gene, ß spectrin (Sptb), which exhibit microcytosis but retain a relatively normal ratio of erythrocyte surface area to volume and are highly resistant to rodent malaria. We propose the major factor responsible for malaria protection is the specific clearance of mutant erythrocytes, although an enhanced clearance of uninfected mutant erythrocytes was also observed (ie, the bystander effect). Using an in vivo erythrocyte tracking assay, we established that this phenomenon occurs irrespective of host environment, precluding the involvement of nonerythrocytic cells in the resistance mechanism. Furthermore, we recapitulated this phenotype by disrupting the interaction between ankyrin-1 and ß spectrin in vivo using CRISPR/Cas9 genome editing technology, thereby genetically validating a potential antimalarial target. This study sheds new light on the role of ß spectrin during Plasmodium infection and highlights how changes in the erythrocyte cytoskeleton can substantially influence malaria susceptibility with minimal adverse consequences for the host.

Aligned Carbon Nanotube Thin Films from Liquid Crystal Polyelectrolyte Inks.

Single walled carbon nanotube thin films are fabricated by solution shearing from high concentration sodium nanotubide polyelectrolyte inks. The solutions are produced by simple stirring of the nanotubes with elemental sodium in dimethylacetamide, and the nanotubes are thus not subject to any sonication-induced damage. At such elevated concentrations (∼4 mg mL(-1)), the solutions exist in the liquid crystal phase and during deposition this order is transferred to the films, which are well aligned in the direction of shear with a 2D nematic order parameter of ∼0.7 determined by polarized absorption measurements. Compared to similarly formed films made from superacids, the polyelectrolyte films contain smaller bundles and a much narrower distribution of bundle diameters. After p-doping with an organic oxidizer, the films exhibit a very high DC electrical to optical conductivity ratio of σ(DC)/σ(OP) ∼ 35, corresponding to a calculated DC conductivity of over 7000 S cm(-1). When very thin (T550 ∼ 96%), smooth (RMS roughness, R(q) ∼ 2.2 nm), and highly aligned films made via this new route are used as the front electrodes of carbon nanotube-silicon solar cells, the power conversion efficiency is almost an order of magnitude greater than that obtained when using the much rougher (R(q) ∼ 20-30 nm) and less conductive (peak σ(DC)/σ(OP) ∼ 2.5) films formed by common vacuum filtration of the same starting material, and having the same transmittance.

Simple and Rapid High-Yield Synthesis and Size Sorting of Multibranched Hollow Gold Nanoparticles with Highly Tunable NIR Plasmon Resonances.

Branched gold nanoparticles with sharp tips are considered excellent candidates for sensing and field enhancement applications. Here, a rapid and simple synthesis strategy is presented that generates highly branched gold nanoparticles with hollow cores and a ca.100% yield through a simple one-pot seedless reaction at room temperature in the presence of Triton X-100. It is shown that multibranched hollow gold nanoparticles of tunable dimensions, branch density and branch length can be obtained by adjusting the concentrations of the reactants. Insights into the formation mechanism point toward an aggregative type of growth involving hollow core formation first, and branching thereafter. The pronounced near-infrared (NIR) plasmon band of the nanoparticles is due to the combined contribution from hollowness and branching, and can be tuned over a wide range (≈700-2000 nm). It is also demonstrated that the high environmental sensitivity of colloidal dispersions based on multibranched hollow gold nanoparticles can be boosted even further by separating the nanoparticles into fractions of given sizes and improved monodispersity by means of a glycerol density gradient. The possibility to obtain highly monodisperse multibranched hollow gold nanoparticles with predictable dimensions (50-300 nm) and branching and, therefore, tailored NIR plasmonic properties, highlights their potential for theranostic applications.

Spring constant calibration techniques for next-generation fast-scanning atomic force microscope cantilevers.

As a recent technological development, high-speed atomic force microscopy (AFM) has provided unprecedented insights into dynamic processes on the nanoscale, and is capable of measuring material property variation over short timescales. Miniaturized cantilevers developed specifically for high-speed AFM differ greatly from standard cantilevers both in size and dynamic properties, and calibration of the cantilever spring constant is critical for accurate, quantitative measurement. This work investigates specifically, the calibration of these new-generation cantilevers for the first time. Existing techniques are tested and the challenges encountered are reported and the most effective approaches for calibrating fast-scanning cantilevers with high accuracy are identified, providing a resource for microscopists in this rapidly developing field. Not only do these cantilevers offer faster acquisition of images and force data but due to their high resonant frequencies (up to 2 MHz) they are also excellent mass sensors. Accurate measurement of deposited mass requires accurate calibration of the cantilever spring constant, therefore the results of this work will also be useful for mass-sensing applications.

Surfactant concentration dependent spectral effects of oxygen and depletion interactions in sodium dodecyl sulfate dispersions of carbon nanotubes.

Quenching of optical absorbance spectra for carbon nanotubes (CNTs) dispersed in sodium dodecyl sulfate (SDS) has been observed to be more pronounced at higher concentrations of the surfactant. The protonation-based quenching behavior displays wavelength dependence, affecting larger diameter nanotube species preferentially. Although absorbance may be recovered by hydroxide addition, pH measurements suggest that hydrolysis of SDS does not play a major role in the short term quenching behavior at high SDS concentrations. The degree of quenching is observed to correlate well with an increase in attractive depletion as SDS concentration is increased, while the extent of depletion is found to depend heavily on the concentration of preparation in comparison to the final SDS concentration. Attractive depletion in SDS is also found to be preferential for CNTs of larger diameter. It is proposed that depletion enhances the quenching effect due to close association of CNT-SDS complexes providing higher SDS densities on the CNT surface, leading to further oxidation. In addition, the quenching behavior in SDS is found to strongly suppress the optical and Raman signal from metallic nanotube species even at high pH. Displacement of SDS by sodium deoxycholate as a secondary surfactant is able to reverse the effects of protonation of metallic species, whereas hydroxide addition is only partially effective.

Efficient attachment of carbon nanotubes to conventional and high-frequency AFM probes enhanced by electron beam processes.

Carbon nanotubes are considered to be an ideal imaging tip for atomic force microscopy (AFM) applications, and a number of methods for fabricating these types of probe have been developed in recent years. This work reports the attachment of carbon nanotubes to AFM probes using a micromanipulator within a scanning electron microscope. Electron beam induced deposition and etching are used to enhance the quality and attachment of the carbon nanotube tip and improve the fabrication rate of the CNT AFM probes compared to existing techniques. The attachment process is also improved by using a mat of SWCNTs (buckypaper) as a CNT source, which simultaneously improves the ease of fabrication and rate of nanotube probe production. The aim of these improvements is to simplify and improve the attachment process such that these probes can be better and more widely used in applications that benefit from their unique properties. This improved process is then used to attach CNTs to the new generation of low-mass, high-frequency probes, which are designed for rapid AFM imaging. The ability of these probes to operate with CNT tips is demonstrated, and their wear-resistance properties were found to be significantly enhanced compared to unmodified probes. These wear-resistant probes imaging at high scan rates are proposed to be effective tools for increasing throughput in metrological analysis, particularly for imaging high-modulus surfaces with high roughness and high-aspect-ratio features.

Accurate measurement of Atomic Force Microscope cantilever deflection excluding tip-surface contact with application to force calibration.

Considerable attention has been given to the calibration of AFM cantilever spring constants in the last 20 years. Techniques that do not require tip-sample contact are considered advantageous since the imaging tip is not at risk of being damaged. Far less attention has been directed toward measuring the cantilever deflection or sensitivity, despite the fact that the primary means of determining this factor relies on the AFM tip being pressed against a hard surface, such as silicon or sapphire; which has the potential to significantly damage the tip. A recent method developed by Tourek et al. in 2010 involves deflecting the AFM cantilever a known distance from the imaging tip by pressing the cantilever against a sharpened tungsten wire. In this work a similar yet more precise method is described, whereby the deflection of the cantilever is achieved using an AFM probe with a spring constant much larger than the test cantilever, essentially a rigid cantilever. The exact position of loading on the test cantilever was determined by reverse AFM imaging small spatial markers that are milled into the test cantilever using a focussed ion beam. For V shaped cantilevers it is possible to reverse image the arm intersection in order to determine the exact loading point without necessarily requiring FIB milled spatial markers, albeit at the potential cost of additional uncertainty. The technique is applied to tip-less, beam shaped and V shaped cantilevers and compared to the hard surface contact technique with very good agreement (on average less than 5% difference). While the agreement with the hard surface contact technique was very good the error on the technique is dependent upon the assumptions inherent in the method, such as cantilever shape, loading point distance and ratio of test to rigid cantilever spring constants. The average error ranged between 2 to 5% for the majority of test cantilevers studied. The sensitivity derived with this technique can then be used to calibrate the cantilever spring constant using the thermal noise method, allowing complete force calibration to be accurately performed without tip-sample contact.