Characterizing the Nano‐Bio Interface Using Microscopic Techniques: Imaging the Cell System is Just as Important as Imaging the Nanoparticle System

Christie M. Sayes1, Henry Lujan1

1 Department of Environmental Science, Baylor University, Waco, Texas
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.26
Online Posting Date:  September, 2017
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The rapid growth of nanotechnology and its industries has elevated the need to understand the risks associated with handling, using, and disposing of nanomaterials. These risks can be assessed through exposure measurement and hazard identification. One of the common challenges associated with quantifying nanomaterials in products, waste, humans, or the environment is the lack of tools available to measure concentration. The ability of refined tools and techniques to qualitatively detect nanoparticles in complex matrices has been demonstrated. For biological and ecological tests systems, dose can be represented as initial concentration in the applied matrix, concentration administered during the route of exposure, concentration at the target organ, and intake concentration at the cellular level. Each of these concentration measurements requires different sets of tools to perform accurate analyses. Advances in microscopy techniques provide new opportunities for reporting observations occurring at the interaction of a nanoparticle with a biomolecular entity of similar size within a biological test(s) system. This protocol outlines the steps to image nanomaterials within cell‐based systems. © 2017 by John Wiley & Sons, Inc.

Keywords: nano‐bio interface; electron microscopy; fluorescence microscopy; atomic force microcopy

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Characterizing the Nano‐Bio Interface via Fluorescence Microscopy
  • Basic Protocol 2: Characterizing the Nano‐Bio Interface via Transmission Electron Microscopy
  • Basic Protocol 3: Characterizing the Nano‐Bio Interface via Atomic Force Microscopy
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Characterizing the Nano‐Bio Interface via Fluorescence Microscopy

  • Cells of interest
  • Cell culture medium
  • Medium growth supplements
  • Phenol red‐free cell culture medium
  • Fluorescent dyes (i.e., nucleus, mitochondria, membrane, or other stains to highlight cell structures of interest)
  • Nanoparticles (i.e., carbonaceous, zerovalent metals, metal oxides, polymeric)
  • Anti‐fade reagent (e.g., SlowFade)
  • Microscope slides (1 × 3–mm plain glass slide)
  • Cover slips (0.17 mm thickness)
  • Immersion oil
  • Optical lens cleaner or wipes for cleaning lens
  • Biological safety cabinet (or laminar flow hood)
  • Cell/tissue incubator maintained at 37°C and 5% CO 2
  • Fluorescence microscope with a 10× objective (for low magnification) and 40×, 60×, or 100× objective (for increasingly higher magnifications)
  • Filter cubes matched to fluorescent dyes being used; examples include 4′,6‐diamidino‐2‐phenylindole, dihydrochloride (DAPI)‐stained nuclei with a WU near‐ultraviolet fluorescence cube and antibody labeling with fluorescein isothiocyanate (FITC) using the WIB green long‐pass fluorescence cube
  • Camera
  • Light source (fluorescence microscopy requires monochromatic illumination; three main types of light sources include lamp, i.e., xenon arc or mercury‐vapor; laser; and diode, i.e., light‐emitting)

Basic Protocol 2: Characterizing the Nano‐Bio Interface via Transmission Electron Microscopy

  • Cells of interest
  • Cell culture medium
  • Medium growth supplements
  • Ultrapure water
  • Phosphate‐buffered saline (PBS)
  • Glutaraldehyde
  • Epoxy reagents (resin, adhesive, curing agent, and accelerant)
  • Stains (osmium tetroxide, uranyl acetate, and lead citrate)
  • Nanoparticles (i.e., carbonaceous, zerovalent metals, metal oxides, polymeric)
  • Biological safety cabinet (or laminar flow hood)
  • Cell/tissue incubator maintained at 37°C and 5% CO 2
  • Grids (for example, Ted Pella,, produces TEM mesh grids in different pitch length, hole width, and bar width; grids can be made out of copper, nickel, or gold)
  • Ultramicrotome
  • Transmission electron microscope (TEM)
  • Energy dispersive X‐ray spectrometer

Basic Protocol 3: Characterizing the Nano‐Bio Interface via Atomic Force Microscopy

  • Cells of interest
  • Cell culture medium
  • Medium growth supplements
  • Phosphate‐buffered saline (PBS)
  • Ultrapure water
  • Nanoparticles (i.e., carbonaceous, zerovalent metals, metal oxides, polymeric)
  • Biological safety cabinet (or laminar flow hood)
  • Coverslips
  • Cell/tissue incubator maintained at 37°C and 5% CO 2
  • Atomic force microscope (AFM) with liquid (or fluid) cell
  • Sharp tip (or probe; typically made of silicon or silicon nitride, or with a radius of curvature on the order of nanometers; images can be enhanced depending on the tip's material, size, and mode used while imaging)
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Literature Cited

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