Alex de Marco

Monash University (Australia) · Department of Biochemistry and Molecular Biology

This report introduces Juno, a modular Python package for optical design and simulation. Juno consists of a complete library that includes a graphical user interface to design and visualise arbitrary optical elements, set up wave propagation simulations and visualise their results. To ensure an efficient visualisation of the results, all simulation...

Automation in microscopy is the key to success in long and complex experiments. Most microscopy manufacturers provide Application Programming Interfaces (API) to enable communication between a user-defined program and the hardware. Although APIs effectively allow the development of complex routines involving hardware control, the developers need to...

The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acu...

The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. The latter activity is furthermore important...

Piezoelectrics interconvert mechanical energy and electric charge and are widely used in actuators and sensors. The best performing materials are ferroelectrics at a morphotropic phase boundary, where several phases coexist. Switching between these phases by electric field produces a large electromechanical response. In ferroelectric BiFeO3, strain...

Refractive axicons are conically shaped optical devices that are capable of generating nondiffracting Bessel-like beams over extended depths-of-focus (DOFs). In addition to the substantially longer DOF compared to those produced by parabolic focusing lenses, the axicons can generate beams with better resolution for the same form-factor of the optic...

Volumetric, sub-micron to micron level resolution imaging is necessary to assay phenotypes or characteristics at the sub-cellular/organelle scale. However, three-dimensional fluorescence imaging of cells is typically low throughput or compromises on the achievable resolution in space and time. Here, we capitalise on the flow control capabilities of...

The analysis of spermatozoa morphology is fundamental to understand male fertility and the etiology of infertility. Traditionally scanning electron microscopy (SEM) has been used to define surface topology. Recently, however, it has become a critical tool for three-dimensional analysis of internal cellular ultrastructure. Modern SEM provides nanome...

The analysis of spermatozoa morphology is fundamental to understand male fertility and the aetiology of infertility. Traditionally scanning electron microscopy (SEM) has been used to define surface topology. Recently, however, it has become a critical tool for three dimensionally analyse of internal cellular ultrastructure. Modern SEM provides nano...

We report on the experimental demonstration of refractive micro-optical elements with arbitrary exponential surface profiles. Refractive optical elements such as lenses and axicons have parabolic (power-exponent of two) or conical (power-exponent of one) surface profiles, respectively. Here, we analyze micro-optical elements with non-parabolic surf...

Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure...

Human Cytomegalovirus (HCMV) infects over half the world's population, is a leading cause of congenital birth defects, and poses serious risks for immuno-compromised individuals. To expand the molecular knowledge governing virion maturation, we analysed HCMV virions using proteomics, and identified a significant proportion of host exosome constitue...

Human Cytomegalovirus (HCMV) infects over half the world's population, is a leading cause of congenital birth defects, and poses serious risks for immuno-compromised individuals. To expand the molecular knowledge governing virion maturation, we analysed HCMV virions using proteomics, and identified a significant proportion of host exosome constitue...

Human Cytomegalovirus (HCMV) infects over half the world's population, is a leading cause of congenital birth defects, and poses serious risks for immuno-compromised individuals. To expand the molecular knowledge governing virion maturation, we analysed HCMV virions using proteomics, and identified a significant proportion of host exosome constitue...

Exosomes, a form of extracellular vesicle, are an important precursor in regenerative medicine. Microfluidic methods exist to capture these sub-micrometer sized objects from small quantities of sample, ideal for multiple diagnostic applications. To address the challenge of extraction from large volumes, we use the visual access offered by microflui...

The development of protocols for bio/chemical reaction requires alternate dispensing and mixing steps. Whilst most microfluidic systems use the opening of additional parts of the channel to allow the ingress of fixed volumes of fluid, this requires knowledge of the protocol before the design of the chip. Our approach of using a microfluidic valve t...

Optical elements rely on refraction, diffraction, or reflection for light manipulation. Fusing diffractive and refractive functions in a single element provides an extra layer of control over the wave propagation, allowing complex beam shaping through self-aligned, monolithic and miniaturized optics. Using gray-scale lithography with high-current f...

Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a typical thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET im...

Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryopreserved cells are milled and a lamella with a thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cr...

The combination of refractive and diffractive components in a single optical element provides miniaturization of optical systems and enhancement of their performance. Thus, hybrid singlet lenses with diffractive structures added on top of the refractive curved surface were shown to have reduced chromatic and spherical aberration. Optical systems ba...

Patterning of aligned structures on supporting thin membranes is required in many applications, such as fabrication of stacked zone plates and realization of observation windows in nanofluidic channels. Here, the authors present a process flow to fabricate self-aligned structures on both sides of a membrane stack by means of a single-step through-m...

Ion Induced Bidirectional Bending for Controlled Manipulation at Nanoscale - Volume 25 Supplement - Vivek Garg, Rakesh G. Mote, Tsengming Chou, Amelia Liu, Alex De Marco, Bhaveshkumar Kamaliya, Shi Qiu, Jing Fu

Cryo-Electron Tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the ev...

Site-specific ion-irradiation is a promising tool fostering strain-engineering of freestanding nanostructures to realize 3D-configurations towards various functionalities. We first develop a novel approach of fabricating freestanding 3D silicon nanostructures by low dose ion-implantation followed with chemical-etching. The fabricated nanostructures...

Diffractive x-ray lenses suffer from limited focusing efficiency, which can be improved by replacing the binary nanostructures of the lenses with kinoforms. Here we present the first example of kinoform lenses for x rays and compare their efficiency with those of binary Fresnel zone plates (FZPs), realized through gray-scale-focused Xe ion beam lit...

Surface Acoustic Wave atomisation has been widely explored for use in pharmacological delivery, hence performance is characterised predominately in terms of droplet size and maximum delivery of fluid, to ensure suffcient dosage is delivered to the right location. For the application of Cryo Electro Microscopy grid preparation, however, what is requ...

We present a device capable of combining nanofluidics and cryogenic transmission electron microscopy (cryo-TEM) to allow inspection of water-soluble samples under near-native conditions. The devices can be produced in a multitude of designs, but as a general rule, they consist of channels or chambers enclosed between two electron-transparent silico...

We present and demonstrate a dextrous microfluidic device which features a reaction chamber with volume flexibility. This feature is critical for developing protocols directly on the chip when the exact reaction is not yet defined, enabling bio/chemical reactions on chip to be performed without volumetric restrictions. This is achieved by the integ...

The high refractive index of lithium niobate crystal (n = 2.2) and the highly transparent range (300-5000 nm), makes it a perfect material for refractive lenses and other types of micro-optical elements. This material already finds extensive use in waveguides and photonic crystals, however, little work has been done on producing refractive optical...

Over the past decade, gallium Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) has been established as a key technology for cellular tomography. The utility of this approach, however, is severely limited both by throughput and the limited selection of compatible sample preparation protocols. Here, we address these limitations and present oxy...

Focused ion beam (FIB) systems based on high brightness plasma ion sources are becoming largely diffuse in material and semiconductor research, thanks to the higher current densities and milling rates provided by noble gas ions (e.g., Xe) compared with traditional liquid metal Ga FIBs. In this paper, we demonstrate the feasibility of a rapid, direc...

Cryo-Electron Microscopy (cryo-EM) has become an invaluable tool for structural biology. Over the past decade, the advent of direct electron detectors and automated data acquisition has established cryo-EM as a central method in structural biology. However, challenges remain in the reliable and efficient preparation of samples in a manner which is...

Cryo-electron microscopy has undergone a revolution in recent years and it has contributed significantly to a number of different areas in biological research. In this manuscript, we will describe some of the recent advancements in cryo-electron microscopy focussing on the advantages that this technique can bring rather than on the technology. We w...

Cryo-electron tomography (cryo-ET) is a three-dimensional imaging technique that makes it possible to analyse the structure of complex and dynamic biological assemblies in their native conditions. The latest technological and image processing developments demonstrate that it is possible to obtain structural information at nanometre resolution. The...

Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach i...

The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic s...

Actin and actin-binding proteins are incorporated into HIV-1 particles, and F-actin has been suggested to bind the NC domain in HIV-1 Gag. Furthermore, F-actin has been frequently observed in the vicinity of HIV-1 budding sites by cryo-electron tomography (cET). Filamentous structures emanating from viral buds and suggested to correspond to actin f...

Cryo-electron tomography combined with image processing by sub-tomogram averaging is unique in its power to resolve the structures of proteins and macromolecular complexes in situ. Limitations of the method, including the low signal-to-noise ratio within individual images from cryo-tomographic datasets and difficulties in determining the defocus at...

Poring Over the Nuclear Pore The nuclear pore is a macromolecular complex that traverses the paired membranes of the nuclear envelope through which a variety of nuclear protein and RNA cargoes must traffic. Szymborska et al. (p. 655 , published online 11 July) combined super-resolution microscopy with single-particle averaging to localize the prote...

During its replication cycle, HIV-1 assembles an immature virus particle by accumulation of viral proteins underneath the cellular membrane. Upon budding of the assembled proteins through the membrane to release an enveloped, immature virus particle, a set of proteolytic cleavage events promotes dramatic changes in the structural organization of th...

Retroviruses are a family of membrane-enveloped RNA viruses that can retrotranscribe and integrate their genome in the host cell chromatin. During the active production of virions the structural polyprotein Gag assembles together with the genomic RNA to form immature particles, which bud out from the host cell. After budding the Gag protein undergo...

HIV-1 buds as an immature, non-infectious virion. Proteolysis of its main structural component Gag is required for morphological maturation and infectivity, and leads to release of four functional domains and the spacer peptides SP1 and SP2. The N-terminal cleavages of Gag and the separation of SP1 from CA are all essential for viral infectivity, w...

The assembly of retroviruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid). Assembly of an infectious virion proceeds in two stages. In the first stage, Gag oligomerization into a hexameric p...

Averaged estimates of RNA polymerase II (RNAPII) elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb min(-1). In this work, nascent RNAs from an integrated human immunodeficiency virus type 1-derived vector were detectable at the single living cell level by fluorescent RNA tagging. At steady state, a constant number...

Immature retroviral particles are assembled by self-association of the structural polyprotein precursor Gag. During maturation the Gag polyprotein is proteolytically cleaved, yielding mature structural proteins, matrix (MA), capsid (CA), and nucleocapsid (NC), that reassemble into a mature viral particle. Proteolytic cleavage causes the N terminus...

Supporting image analysis procedures and additional cryo electron tomography images. (1.45 MB PDF)

Radius-angle-frequency plots. A) 3D view of the radius-angle-frequency plot calculated from the immature HIV data, illustrating the relationship between the three axes. Three perpendicular sections are shown intersecting at the point: 53 nm radius, 60° angle and 7 nm frequency. The colour bar represents the value of autocorrelation and is common to...

Fourier shell correlation. A) Fourier shell correlation plots for all the variants. The resolution was determined as the frequency at which the FSC curve drops below 0.5 correlation which is highlighted with the dashed line. B) Plot showing the variation in resolution according to radius. At each radius the resolution was determined by Fourier shel...

The morphology of virus variants. Central sections of tomographic reconstructions of the HIV-1 variants analyzed acquired at different defocuses (df), and coming from 3 different preparations, to illustrate consistency of virus morphology between preparations. A gaussian filter was applied the tomograms (8 kernel, 0.4 sigma). The scale bar is 50 nm...

Supplementary methods. (0.05 MB PDF)

The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the struc...

HIV-1 buds form infected cells in an immature, non-infectious form. Maturation into an infectious virion requires proteolytic cleavage of the Gag polyprotein at five positions, leading to a dramatic change in virus morphology. Immature virions contain an incomplete spherical shell where Gag is arranged with the N-terminal MA domain adjacent to the...

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three differ...

The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the struc...

The histone chaperone nucleosome assembly protein, hNAP-1, is a host cofactor for the activity of the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. The interaction between these two proteins has been shown to be important for Tat-mediated transcriptional activation and for efficient viral infection. Visualization of HIV-1 transcri...

A system for Low Energy X-ray Fluorescence (LEXRF) has been implemented in the TwinMic [1] microscope station operating at Elettra synchrotron facility. The integrated LEXRF is coupled with the STXM operation mode. This allows simultaneous collection of XRF maps, brightfield images and phase contrast images [2], providing complementary information...

Novel low-energy X-ray fluorescence (LEXRF) system based on a multiple Si drift detector (SDD) configuration has been developed and implemented in the European TwinMic X-ray microspectroscopy station operating at the Italian synchrotron radiation facility ELETTRA. The setup, hosting up to eight large-area SDDs with specially adapted readout electro...

We report on a low-energy x-ray fluorescence (LEXRF) spectrometer system implemented in the European TwinMic x-ray spectromicroscope,1 which is operated in the 280–2200 eV energy range and allows sub-100 nm optical resolution by means of focusing with diffractive optics. The LEXRF setup is a modular system based on the use of multiple windowless la...

The human immunodeficiency virus type 1 (HIV-1) favors integration in active genes of host chromatin. It is believed that transcriptional interference of the viral promoter over the endogenous gene or vice versa might occur with implications in HIV-1 post-integrative transcriptional latency. In this work a cell line has been transduced with a HIV-b...