RESUMO
Enveloped viruses assemble and bud from the host cell membranes. Any role of cortical actin in these processes have often been a source of debate. Here, we assessed if cortical actin was involved in HIV-1 assembly in infected CD4 T lymphocytes. Our results show that preventing actin branching not only increases HIV-1 particle release but also the number of individual HIV-1 Gag assembly clusters at the T cell plasma membrane. Indeed, in infected T lymphocytes and in in vitro quantitative model systems, we show that HIV-1 Gag protein prefers areas deficient in F-actin for assembling. Finally, we found that the host factor Arpin, an inhibitor of Arp2/3 branched actin, is recruited at the membrane of infected T cells and it can associate with the viral Gag protein. Altogether, our data show that, for virus assembly and particle release, HIV-1 prefers low density of cortical actin and may favor local actin debranching by subverting Arpin.
Assuntos
Actinas , HIV-1 , Actinas/metabolismo , HIV-1/metabolismo , Montagem de Vírus , Produtos do Gene gag/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Membrana Celular/metabolismo , Proteínas Virais/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Glycan-glycan interactions between viral particles and host cells may lengthen the dwell time of the virus on the cell surface to facilitate cellular receptor engagement. Here, we present a protocol for visualizing glycan-mediated binding between virus or virus-like-particles (VLPs) and human peripheral blood mononuclear cells using transmission electron microscopy (TEM). We describe steps for virus and VLP production, isolation of human peripheral blood mononuclear cells, and sample preparation. We then detail procedures for thin-section TEM. For complete details on the use and execution of this protocol, please refer to Spillings et al.1.
Assuntos
Leucócitos Mononucleares , Vírion , Humanos , Microscopia Eletrônica de Transmissão , Linhagem Celular , Membrana CelularRESUMO
Prior to receptor engagement, a specific, non-electrostatic glycan-glycan interaction between viral particles and host cells may lengthen the dwell time of the virus at the cellular surface, thereby facilitating subsequent virus entry. Here, we present a protocol for quantifying the level of glycan-mediated binding between virus or virus-like-particles and human peripheral blood mononuclear cells (PBMCs) using a nanoluciferase reporter system. We describe steps for virus production, isolation of PBMCs, and performing a nanoluciferase binding assay. For complete details on the use and execution of this protocol, please refer to Spillings et al.1.
Assuntos
Leucócitos Mononucleares , Vírion , Humanos , Bioensaio , Membrana Celular , Internalização do VírusRESUMO
Many transient processes in cells arise from the binding of cytosolic proteins to membranes. Quantifying this membrane binding and its associated diffusion in the living cell is therefore of primary importance. Dynamic photonic microscopies, e.g., single/multiple particle tracking, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy (FCS), enable non-invasive measurement of molecular mobility in living cells and their plasma membranes. However, FCS with a single beam waist is of limited applicability with complex, non-Brownian, motions. Recently, the development of FCS diffusion laws methods has given access to the characterization of these complex motions, although none of them is applicable to the membrane binding case at the moment. In this study, we combined computer simulations and FCS experiments to propose an FCS diffusion law for membrane binding. First, we generated computer simulations of spot-variation FCS (svFCS) measurements for a membrane binding process combined to 2D and 3D diffusion at the membrane and in the bulk/cytosol, respectively. Then, using these simulations as a learning set, we derived an empirical diffusion law with three free parameters: the apparent binding constant KD, the diffusion coefficient on the membrane D2D, and the diffusion coefficient in the cytosol, D3D. Finally, we monitored, using svFCS, the dynamics of retroviral Gag proteins and associated mutants during their binding to supported lipid bilayers of different lipid composition or at plasma membranes of living cells, and we quantified KD and D2D in these conditions using our empirical diffusion law. Based on these experiments and numerical simulations, we conclude that this new approach enables correct estimation of membrane partitioning and membrane diffusion properties (KD and D2D) for peripheral membrane molecules.
Assuntos
Bicamadas Lipídicas , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Membranas , Espectrometria de Fluorescência/métodos , DifusãoRESUMO
Polarized or precision targeting of protein complexes to their destinations is fundamental to cellular homeostasis, but the mechanism underpinning directional protein delivery is poorly understood. Here, we use the uropod targeting HIV synapse as a model system to show that the viral assembly machinery Gag is copolarized with the intracellular calcium (Ca2+) gradient and binds specifically with Ca2+. Conserved glutamic/aspartic acids flanking endosomal sorting complexes required for transport binding motifs are major Ca2+ binding sites. Deletion or mutation of these Ca2+ binding residues resulted in altered protein trafficking phenotypes, including (i) changes in the Ca2+-Gag distribution relationship during uropod targeting and/or (ii) defects in homo/hetero-oligomerization with Gag. Mutation of Ca2+ binding amino acids is associated with enhanced ubiquitination and a decline in virion release via uropod protein complex delivery. Our data that show Ca2+-protein binding, via the intracellular Ca2+ gradient, represents a mechanism that regulates intracellular protein trafficking.
RESUMO
Here, we present ultrastructural analyses showing that incoming HIV are captured near the lymphocyte surface in a virion-glycan-dependent manner. Biophysical analyses show that removal of either virion- or cell-associated N-glycans impairs virus-cell binding, and a similar glycan-dependent relationship is observed between purified HIV envelope (Env) and primary T cells. Trimming of N-glycans from either HIV or Env does not inhibit protein-protein interactions. Glycan arrays reveal HIV preferentially binds to N-acetylglucosamine and mannose. Interfering with these glycan-based interactions reduces HIV infectivity. These glycan interactions are distinct from previously reported glycan-lectin and non-specific electrostatic charge-based interactions. Specific glycan-glycan-mediated attachment occurs prior to virus entry and enhances efficiency of infection. Binding and fluorescent imaging data support glycan-glycan interactions as being responsible, at least in part, for initiating contact between HIV and the host cell, prior to viral Env-cellular CD4 engagement.
Assuntos
Anticorpos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Polissacarídeos/metabolismo , Internalização do Vírus/efeitos dos fármacos , Anticorpos Neutralizantes/metabolismo , Membrana Celular/metabolismo , Glicosilação/efeitos dos fármacos , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Vírion/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/químicaRESUMO
Transmission of HIV across the mucosal surface of the female reproductive tract to engage subepithelial CD4-positive T cells is not fully understood. Cervical epithelial cells express complement receptor 3 (CR3) (integrin αMß2 or CD11b/CD18). In women, the bacterium Neisseria gonorrhoeae uses CR3 to invade the cervical epithelia to cause cervicitis. We hypothesized that HIV may also use CR3 to transcytose across the cervical epithelia. Here, we show that HIV-1 strains bound with high affinity to recombinant CR3 in biophysical assays. HIV-1 bound CR3 via the I-domain region of the CR3 alpha subunit, CD11b, and binding was dependent on HIV-1 N-linked glycans. Mannosylated glycans on the HIV surface were a high-affinity ligand for the I-domain. Man5 pentasaccharide, representative of HIV N-glycans, could compete with HIV-1 for CR3 binding. Using cellular assays, we show that HIV bound to CHO cells by a CR3-dependent mechanism. Antibodies to the CR3 I-domain or to the HIV-1 envelope glycoprotein blocked the binding of HIV-1 to primary human cervical epithelial (Pex) cells, indicating that CR3 was necessary and sufficient for HIV-1 adherence to Pex cells. Using Pex cells in a Transwell model system, we show that, following transcytosis across an intact Pex cell monolayer, HIV-1 is able to infect TZM-bl reporter cells. Targeting the HIV-CR3 interaction using antibodies, mannose-binding lectins, or CR3-binding small-molecule drugs blocked HIV transcytosis. These studies indicate that CR3/Pex may constitute an efficient pathway for HIV-1 transmission in women and also demonstrate strategies that may prevent transmission via this pathway. IMPORTANCE In women, the lower female reproductive tract is the primary site for HIV infection. How HIV traverses the epithelium to infect CD4 T cells in the submucosa is ill-defined. Cervical epithelial cells have a protein called CR3 on their surface. We show that HIV-1 binds to CR3 with high affinity and that this interaction is necessary and sufficient for HIV adherence to, and transcytosis across, polarized, human primary cervical epithelial cells. This suggests a unique role for CR3 on epithelial cells in dually facilitating HIV-1 attachment and entry. The HIV-CR3 interaction may constitute an efficient pathway for HIV delivery to subepithelial lymphocytes following virus transmission across an intact cervical epithelial barrier. Strategies with potential to prevent transmission via this pathway are presented.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Cricetinae , Animais , Humanos , Feminino , Antígeno de Macrófago 1/metabolismo , HIV-1/metabolismo , Cricetulus , Células Epiteliais/microbiologia , Células CHO , Transcitose , Polissacarídeos/metabolismoRESUMO
Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55Gag precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55Gag or its maturation products (NCp15, NCp9 and NCp7) and the 5' gRNA region and their structural consequences, in vitro. We show that Pr55Gag and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55Gag maturation products on the gRNA structural maturation.
Assuntos
Regiões 5' não Traduzidas , Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , HIV-1/fisiologia , RNA Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Viral/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus , Replicação ViralRESUMO
During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. siRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single-molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.
Assuntos
HIV-1/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vírion/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Membrana Celular/metabolismo , Células HEK293 , HIV-1/fisiologia , Humanos , Células Jurkat , Imagem Individual de Molécula/métodosRESUMO
Persistence of HIV through integration into host DNA in CD4+ T cells presents a major barrier to virus eradication. Viral integration may be curtailed when CD8+ T cells are triggered to kill infected CD4+ T cells through recognition of histocompatibility leukocyte antigen (HLA) class I-bound peptides derived from incoming virions. However, this has been reported only in individuals with "beneficial" HLA alleles that are associated with superior HIV control. Through interrogation of the pre-integration immunopeptidome, we obtain proof of early presentation of a virion-derived HLA-A∗02:01-restricted epitope, FLGKIWPSH (FH9), located in Gag Spacer Peptide 2 (SP2). FH9-specific CD8+ T cell responses are detectable in individuals with primary HIV infection and eliminate HIV-infected CD4+ T cells prior to virus production in vitro. Our data show that non-beneficial HLA class I alleles can elicit an effective antiviral response through early presentation of HIV virion-derived epitopes and also demonstrate the importance of SP2 as an immune target.
Assuntos
Antivirais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Peptídeos/metabolismo , Vírion/imunologia , Antivirais/farmacologia , HumanosRESUMO
OBJECTIVES: A major COVID-19 vaccine strategy is to induce antibodies that prevent interaction between the Spike protein's receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2). These vaccines will also induce T-cell responses. However, concerns were raised that aberrant vaccine-induced immune responses may exacerbate disease. We aimed to identify minimal epitopes on the RBD that would induce antibody responses that block the interaction of the RBD and ACE2 as a strategy leading to an effective vaccine with reduced risk of inducing immunopathology. METHODS: We procured a series of overlapping 20-amino acid peptides spanning the RBD and asked which were recognised by plasma from COVID-19 convalescent patients. Identified epitopes were conjugated to diphtheria-toxoid and used to vaccinate mice. Immune sera were tested for binding to the RBD and for their ability to block the interaction of the RBD and ACE2. RESULTS: Seven putative vaccine epitopes were identified. Memory B-cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains. CONCLUSION: COVID-19 convalescent patients have SARS-CoV-2-specific antibodies and MBCs, the specificities of which can be defined with short peptides. Epitope-specific antibodies synergistically block RBD-ACE2 interaction.
RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged virus that causes coronavirus infectious disease 2019 (COVID-19). SARS-CoV-2 spike protein, like SARS-CoV-1, uses the angiotensin converting enzyme 2 (ACE2) as a cellular receptor to initiate infection. Compounds that interfere with the SARS-CoV-2 spike protein receptor binding domain protein (RBD)-ACE2 receptor interaction may function as entry inhibitors. Here, we used a dual strategy of molecular docking and surface plasmon resonance (SPR) screening of compound libraries to identify those that bind to human ACE2 or the SARS-CoV-2 spike protein receptor binding domain (RBD). Molecular modeling screening interrogated 57,641 compounds and focused on the region of ACE2 that is engaged by RBD of the SARS-CoV-2 spike glycoprotein and vice versa. SPR screening used immobilized human ACE2 and SARS-CoV-2 Spike protein to evaluate the binding of these proteins to a library of 3,141 compounds. These combined screens identified compounds from these libraries that bind at KD (equilibrium dissociation constant) <3 µM affinity to their respective targets, 17 for ACE2 and 6 for SARS-CoV-2 RBD. Twelve ACE2 binders and six of the RBD binders compete with the RBD-ACE2 interaction in an SPR-based competition assay. These compounds included registered drugs and dyes used in biomedical applications. A Vero-E6 cell-based SARS-CoV-2 infection assay was used to evaluate infection blockade by candidate entry inhibitors. Three compounds demonstrated dose-dependent antiviral in vitro potency-Evans blue, sodium lifitegrast, and lumacaftor. This study has identified potential drugs for repurposing as SARS-CoV-2 entry inhibitors or as chemical scaffolds for drug development.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, has caused more than 60 million cases worldwide with almost 1.5 million deaths as of November 2020. Repurposing existing drugs is the most rapid path to clinical intervention for emerging diseases. Using an in silico screen of 57,641 compounds and a biophysical screen of 3,141 compounds, we identified 22 compounds that bound to either the angiotensin converting enzyme 2 (ACE2) and/or the SARS-CoV-2 spike protein receptor binding domain (SARS-CoV-2 spike protein RBD). Nine of these drugs were identified by both screening methods. Three of the identified compounds, Evans blue, sodium lifitegrast, and lumacaftor, were found to inhibit viral replication in a Vero-E6 cell-based SARS-CoV-2 infection assay and may have utility as repurposed therapeutics. All 22 identified compounds provide scaffolds for the development of new chemical entities for the treatment of COVID-19.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Aminopiridinas/farmacologia , Animais , Benzodioxóis/farmacologia , Linhagem Celular , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Azul Evans/farmacologia , Humanos , Simulação de Acoplamento Molecular , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Ligação Proteica/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Sulfonas/farmacologia , Ressonância de Plasmônio de Superfície , Células VeroRESUMO
The central dogma of molecular biology describes the flow of genetic information from DNA to protein via an RNA intermediate. For many years, RNA has been considered simply as a messenger relaying information between DNA and proteins. Recent advances in next generation sequencing technology, bioinformatics, and non-coding RNA biology have highlighted the many important roles of RNA in virtually every biological process. Our understanding of RNA biology has been further enriched by a number of significant advances in probing RNA structures. It is now appreciated that many cellular and viral biological processes are highly dependent on specific RNA structures and/or sequences, and such reliance will undoubtedly impact on the evolution of both hosts and viruses. As a contribution to this special issue on host immunity and virus evolution, it is timely to consider how RNA sequences and structures could directly influence the co-evolution between hosts and viruses. In this manuscript, we begin by stating some of the basic principles of RNA structures, followed by describing some of the critical RNA structures in both viruses and hosts. More importantly, we highlight a number of available new tools to predict and to evaluate novel RNA structures, pointing out some of the limitations readers should be aware of in their own analyses.
Assuntos
Conformação de Ácido Nucleico , RNA/genética , Viroses/imunologia , Vírus/genética , Animais , Sequência de Bases , Evolução Molecular , Interações Hospedeiro-Patógeno/imunologia , Humanos , RNA/química , Virulência/genética , Virulência/imunologia , Viroses/virologia , Vírus/patogenicidadeRESUMO
The Pr55Gag precursor specifically selects the HIV-1 genomic RNA (gRNA) from a large excess of cellular and partially or fully spliced viral RNAs and drives the virus assembly at the plasma membrane. During these processes, the NC domain of Pr55Gag interacts with the gRNA, while its C-terminal p6 domain binds cellular and viral factors and orchestrates viral particle release. Gag∆p6 is a truncated form of Pr55Gag lacking the p6 domain usually used as a default surrogate for wild type Pr55Gag for in vitro analysis. With recent advance in production of full-length recombinant Pr55Gag, here, we tested whether the p6 domain also contributes to the RNA binding specificity of Pr55Gag by systematically comparing binding of Pr55Gag and Gag∆p6 to a panel of viral and cellular RNAs. Unexpectedly, our fluorescence data reveal that the p6 domain is absolutely required for specific binding of Pr55Gag to the HIV-1 gRNA. Its deletion resulted not only in a decreased affinity for gRNA, but also in an increased affinity for spliced viral and cellular RNAs. In contrast Gag∆p6 displayed a similar affinity for all tested RNAs. Removal of the C-terminal His-tag from Pr55Gag and Gag∆p6 uniformly increased the Kd values of the RNA-protein complexes by ~ 2.5 fold but did not affect the binding specificities of these proteins. Altogether, our results demonstrate a novel role of the p6 domain in the specificity of Pr55Gag-RNA interactions, and strongly suggest that the p6 domain contributes to the discrimination of HIV-1 gRNA from cellular and spliced viral mRNAs, which is necessary for its selective encapsidation.
Assuntos
Genoma Viral/genética , HIV-1/fisiologia , Precursores de Proteínas/metabolismo , RNA Viral/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , HIV-1/genética , Humanos , Mutação , Plasmídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/genética , RNA Viral/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
Induction of persistent antibody responses by vaccination is generally thought to depend on efficient help by T follicular helper cells. Since the T helper cell response to HIV Env may not be optimal, we explored the possibility of improving the HIV Env antibody response to virus-like particle (VLP) vaccines by recruiting T helper cells induced by commonly used licensed vaccines to provide help for Env-specific B cells. B cells specific for the surface protein of a VLP can internalize the entire VLP and thus present peptides derived from the surface and core proteins on their major histocompatibility complex class II (MHC-II) molecules. This allows T helper cells specific for the core protein to provide intrastructural help for B cells recognizing the surface protein. Consistently, priming mice with an adjuvanted Gag protein vaccine enhanced the HIV Env antibody response to subsequent booster immunizations with HIV VLPs. To harness T helper cells induced by the licensed Tetanolpur vaccines, HIV VLPs that contained T helper cell epitopes of tetanus toxoid were generated. Tetanol-immunized mice raised stronger antibody responses to immunizations with VLPs containing tetanus toxoid T helper cell epitopes but not to VLPs lacking these epitopes. Depending on the priming immunization, the IgG subtype response to HIV Env after the VLP immunization could also be modified. Thus, harnessing T helper cells induced by other vaccines appears to be a promising approach to improve the HIV Env antibody response to VLP vaccines.IMPORTANCE Induction of HIV Env antibodies at sufficient levels with optimal Fc effector functions for durable protection remains a challenge. Efficient T cell help may be essential to induce such a desirable antibody response. Here, we provide proof of concept that T helper cells induced by a licensed vaccine can be harnessed to provide help for HIV Env-specific B cells and to modulate the Env-specific IgG subtype response.
Assuntos
Formação de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Células HEK293 , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Humanos , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , VacinaçãoRESUMO
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 compatible with high time resolution. The delivery of sample onto the grid is recognized as a critical step in the workflow as it is a source of variability and loss of material due to the blotting which is usually required. Here, we present a method for sample delivery and plunge freezing based on the use of Surface Acoustic Waves to deploy 6-8⯵m droplets to the EM grid. This method minimises the sample dead volume and ensures vitrification within 52.6â¯ms from the moment the sample leaves the microfluidics chip. We demonstrate a working protocol to minimize the atomised volume and apply it to plunge freeze three different samples and provide proof that no damage occurs due to the interaction between the sample and the acoustic waves.