Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Abstract

Techniques available for micro-and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe "pokes" the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Nicotine induces morphological and functional changes in astrocytes via nicotinic receptor activity

“…In order to visualize these fine processes, we utilized a label free approach with high‐resolution AFM that allowed us to determine the impact of nicotine exposure. AFM is a scanning probe microscopic technique which enables the formation of topographic images of a sample by measuring the force between an AFM probe and the sample, and is responsive to changes in the contour of the surface resulting in extremely high resolution images of sample morphology (Sandin, Aryal, Wilkop, Richards, & Grady, 2020). This technique allowed us to characterize the fine processes present in astrocytes (Gadegaard, 2006).…”

Macrophage-Engineered Vesicles for Therapeutic Delivery and Bidirectional Reprogramming of Immune Cell Polarization

“…Thus, the obtained images were analyzed with Nikon image processing software. 59 MEV Uptake. 100 million M1 and 110 million M2 macrophages were used to prepare M1EVs and M2EVs, respectively, for the study of MEV uptake by M1 or M2 macrophages.…”

Current Trends of Food Analysis, Safety, and Packaging

“…The presence of various toxigenic fungi or their secondary metabolites as mycotoxins in human food and livestock feed is a recurring food safety problem so the mycotoxin-producing species and the particular strain must be authentically identified by adopting recently developed techniques (like PCR-based techniques, laser biospeckle technique, apta-sensor, immune-sensor, enzymatic-sensors, and others) and by the development of new fungal selective agar media for the isolation of toxigenic fungal strains only. Similarly, several innovative methods such as attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), enzyme-linked immunosorbent assay (ELISA), liquid chromatography-tandem mass spectrometry (LC-MS/MS) via a multiple antibody immunoaffinity column, and suspension array technology can be applied for detection of mycotoxins in commodity [ 199 , 200 ]. As shown by recent reports, “Surface Active Maghemite Nanoparticles” is indicated as a stable and good magnetic nanocarrier for mycotoxin removal; the potential of which can be tapped by the food industries [ 201 ].…”