Two-dimensional (2D) scanning acoustic microscopy (SAM) is a useful tool for the direct observation of biological tissue using high-frequency ultrasonic waves [1,2,3,4,5,6,7,8]. One of the advantages of this microscopy method is observing the elastic properties of living cells without undergoing the histochemical staining process [6]. In other words, the ability to observe living cells without fixation or antibodies. This is important as fixation causes the cells to die, hence disabling the observation of dynamic biological processes. Acoustic observation has demonstrated that the application of drugs, such as anticancer drugs, to cells can also reduce and change the trends of acoustic impedance [3]. SAM allows intracellular observation along the depth direction (B-mode). Hozumi et al. [1] reported having converted the reflection intensity from the internal region of glial cells into the distribution of acoustic impedance via deconvolution in the frequency domain, thus obtaining a three-dimensional (3D) cross-sectional view of the cell.

The present study investigated 3D observation and estimation of aggression of living microglia (MG) treated with an insecticide prenatally. MG, the target cell of this research, are a type of neuroglia, others being astrocytes and oligodendrocytes. MG are the primary immune cell in our central nervous system (CNS), accounting for 10–15% of cells in the brain [9]. Their primary function is to maintain the delicate homeostasis of the brain. As the brain's primary immune cells, they are actively involved when neuroinflammation occurs. MG express a variety of motilities to carry out their function under physiological and pathological environments. In the physiological environment, they are actively scavenging and surveying the brain for abnormalities. MG are conventionally assigned as M0, M1, or M2 MG [10,11,12,13,14,15,16]. The M0 phenotype or “resting state” is actively scavenging and surveying the brain for abnormalities. Alternatively, in a pathological environment, it is activated and migrates toward the trauma site, turning into phagocytotic cells to engulf damaged cells. The M1 phenotype is a common way to characterize this proinflammatory function of MG. Another morphology of MG in between these two environments is the M2 phenotype, in which MG are anti-inflammatory. They protect surrounding cells from inflammation. However, the M0, M1, and M2 phenotypes and the term “activated” MG are oversimplified and may no longer be viable ways to classify these complicated and varied MG morphologies [17]. Therefore, using SAM with C-mode and B-mode acoustic imaging paired with confocal microscopy may elucidate how to study and classify various living MG motilities.

For MG to carry out their function, they express a lot of protein on their surface, acting as receptors to detect even small changes in the brain condition. Receptors that detect purines, such as adenosine triphosphate (ATP), are the focus of this research. ATP is released into the extracellular environment by inflamed cells [18, 19]. When MG detect this distressed cell signal in ATP, they respond to it to perform their function [9].

Aside from ATP as stimulation for inflammation, acetamiprid (ACE) was also used. ACE, a type of insecticide from the group neonicotinoid, mimics the nicotine chemical structure to bind to the nicotinic acetylcholine receptors. Neonicotinoids have been linked to the colony collapse disorder of bees [20, 21], while the effects of neonicotinoids on humans are considered safe as they are thought to be highly specific to targeted pests; however, some reports suggest otherwise [22,23,24,25,26,27,28,29]. ACE may have the potential to cause damage to humans, especially to the developing brain. Therefore, further research on the usage of ACE is vital.

Although extensive research is performed on the function of MG, studies on its morphology are still lacking and difficult to achieve with the current observation method. The conventional methods of observing MG are via optical and confocal microscopes. Although these methods are widely used, they are insufficient to study the dynamicity of MG. An optical microscope using phase-contrast imaging can only provide information on the shape of the MG without further internal cytoskeleton distribution details. A confocal microscope requires a fixing and staining process, causing cells to no longer be viable. Observing chemotaxis in MG as immune cells in the brain is crucial for a better understanding of neurological diseases. This paper proposed observing living MG and their morphological changes and dynamicity in response to inflammation via the scanning acoustic microscope. We further confirmed MG observation and dispersion of MG-specific markers Iba-1 and P2Y12 via a confocal microscope.