dissection of the labium and separation of the labrum&#39;s tip for fabricating an atomic force microscopy probe

Bio-Hybrid AFM Probe with Mosquito Labrum for Studying Biting Behaviors

World Health Organization (WHO) reported that vector-borne diseases (VBDs) account for over 17% of all infectious diseases, which cause more than 7,00,000 deaths per year globally. For example, as the deadliest animal in the world, mosquitoes spread numerous pathogens, such as dengue, malaria, and Zika, through blood-feeding arthropods, resulting in 700 million infections each year1. Explorations toward the development of effective measures to prevent VBDs are of crucial importance, including mimicking the penetration behaviors of mosquitoes to investigate their biting mechanisms and studies of potential barriers to prove their efficacy in preventing penetration. One key challenge is to develop proper approaches to perform such investigations. Efforts have been made in the literature, including the development of micro-scale needles that resemble the geometry of a mosquito stinger; however, many of the materials used to make these microneedles (i.e., viscoelastic materials2, silicon (Si), glass, ceramic3, etc.) have different mechanical properties than the biological material of the mosquito&#39;s proboscis. The engineered materials can be brittle and prone to fracture and buckling3,4, whereas the mosquito&#39;s proboscis can withstand fracture or buckling better4. The benefit of having a bio-hybrid probe using the labrum of a mosquito instead of engineered materials is that it can be a more accurate representation of the piercing mechanism of mosquitoes. Also, specialized tools must be integrated with micro needles to perform quantitative studies, such as the accurate measurement of force5, which is not easily achievable with customized setups using engineered microneedles.
Atomic force microscopy (AFM)-based approach is promising in that it operates by employing a cantilever with an ultra-fine tip that is carefully positioned close to a sample&#39;s surface. The tip can either scan across or be pressed towards/into a surface, experiencing varying attractive or repulsive forces due to its interactions with a sample6. These interactions lead to the cantilever&#39;s deflection, which is tracked by the reflection of a laser beam from the cantilever&#39;s top onto a photodetector6. The exceptional sensitivity to movement of the system enables AFM to conduct a diverse range of measurements, including but not limited to morphological mapping with picometer accuracy, force measurements ranging from piconewtons to micronewtons, and comprehensive multiphysics investigations7. For example, AFM indentations can be performed to precisely assess the response to the applied force of a sample and also to measure the hardness, elasticity, and other mechanical properties of a sample by coupling with appropriate analytical models8. The probe of the AFM is most commonly made of silicon (Si) or silicon nitride (Si3N4)8 with a length of 20-300 &#181;m9 and a tip radius on the order of several to tens of nanometers10. The nanometer scale tip radius can be ideal for applications such as high-resolution imaging; however, it does not possess the characteristics of biological stingers for studies that try to mimic penetration behaviors in terms of stiffness, radius, shape, and aspect ratio. For example, the microneedle structure of a mosquito is the fascicle, which has an aspect ratio of ~6011 (length ~1.5 mm to 2 mm; diameter ~30 &#181;m)12. While a conventional AFM probe can be assumed to resemble a biological stinger like a labrum, its distinct material properties and dimensions will not reflect the real situation during a bite.
To enable quantitative investigations of penetration behaviors mimicking biological bites of insects or other animals with stingers, here, a process to fabricate bio-hybrid AFM cantilevers with a biological stinger as its tip is developed. As a case study, an AFM cantilever with a mosquito labrum&#39;s tip attached was successfully demonstrated. Harnessing existing information from literature on the typical insertion forces that a mosquito uses to pierce through a victim&#39;s skin12,13, this bio-hybrid AFM cantilever can potentially allow near-real mimicry of mosquito bites under a regular AFM. The protocol of leveraging micro biological stingers to fabricate bio-hybrid AFM cantilevers can also be applied to the development of other sharp stinger-based biohybrid AFM cantilevers for quantitative investigations of a variety of biting mechanisms.
Terminologies 
A schematic of a proboscis and its components of interest are shown in Figure 1, and their definitions are (1) Proboscis: a body part from the mouth of a mosquito that allows the mosquito to feed itself, with a core-shell structure composed of the fascicle(core) and the labium(shell), (2) Labium: the dark and blunt outer cover of a proboscis2, (3) Fascicle: a group of slender needles contained inside of the labium, including two maxillae, two mandibles, a hypopharynx, and a labrum2, (4) Hypopharynx: responsible for secreting saliva into the host&#39;s bloodstream2, (5) Maxillae: serrated member assisting in the feeding mechanism2, (5) Mandibles: similar to the maxilla, they help the mosquito in the feeding mechanism and have a sharp tip2, (6) Labrum: the main member for penetrating the skin of a victim, which is much bigger than the maxillae, mandibles and hypopharynx. It also has sensory structures that allow it to find blood vessels and internal channels under the skin2, (7) Manipulator: an assembly with three degrees of freedom and micron-scale accuracy for positioning, allowing movement in XYZ directions, (8) Clamp assembly: a custom-made 2-part clamp mounted to the manipulator used to clamp the tipless AFM cantilever during the experiment.

Author Spotlight: Development of Bio-Hybrid AFM Cantilevers for Quantitative Analysis of Mosquito Biting Mechanisms

Entwicklung einer bio-hybriden Mosquito-Stinger-basierten Rasterkraftmikroskopie-Sonde

Präparieren des Labiums und Abtrennen der Labrumspitze zur Herstellung einer Rasterkraftmikroskopie-Sonde

Platzieren Sie zunächst eine tote Mücke auf dem Objektträger. Platzieren Sie dann vorsichtig eine Skalpellklinge über dem Labium in der Nähe des Kopfes der Mücke. Machen Sie einen Schnitt über die gesamte obere Hälfte des Labiums mit einer geringen Eindringtiefe.Halten Sie dann mit einer Pinzette den Kopf der Mücke fest und kneifen Sie mit einer weiteren Präzisionspinzette das Labium leicht zwischen der spitz zulaufenden Spitze und dem Schnitt zusammen. Ziehe dann die Pinzette, die das Labium hält, in Richtung der spitz zulaufenden Spitze. Lege die Mücke unter das Mikroskop, um zu überprüfen, ob die Spitze des Labrums vorhanden ist.Klemmen und schließen Sie nun die Spitzen einer Präzisionspinzette und platzieren Sie die Spitze der Pinzette direkt neben der Spitze des Labrums. Üben Sie mit der Pinzettenspitze eine sanfte Kraft senkrecht zur Länge des Faszikels auf das Labrum aus. Schieben Sie das Labrum weiter über den Objektträger, bis die Trennung von den anderen Faszikelgliedern erreicht ist.Untersuchen Sie abschließend die Probe unter einem Mikroskop, um die Trennung zu überprüfen.

Research

JoVE Journal

Engineering

Dissection of the Labium and Separation of the Labrum&#39;s Tip for Fabricating an Atomic Force Microscopy Probe

Development of a Bio-Hybrid Mosquito Stinger-Based Atomic Force Microscopy Probe

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The ...

Forschung

Ingenieurwesen

Isolation of the Desired Labrum Tip for Preparing the Atomic Force Microscopy Probe

Isolierung der gewünschten Labrumspitze zur Vorbereitung der Rasterkraftmikroskopie-Sonde

Mounting and Bonding the Labrum&#39;s Tip to the Atomic Force Microscopy (AFM) Tipless Cantilever Beam

Montieren und Verkleben der Spitze des Labrums mit dem spitzenlosen Cantilever-Strahl der Rasterkraftmikroskopie (AFM)