Our goal is to develop novel correlative microscopy approaches for locating and characterizing engineered metal nanoparticles within complex cellular environments. This metrology will provide nanomedicine researchers with the tools to address critical questions with statistically relevant populations containing thousands of cells and will encourage the development of next-generation nanoparticle-based therapies.
Nanomedicine is a US $10B global market, but the big success stories to date are re-formulations of existing drugs into nano-sized delivery vehicles that target the appropriate tissue. Next-generation nanoparticle-based therapies are expected to target specific cell populations and sub-cellular locations; to date, much pre-clinical research has focused on functionalized gold nanoparticles. Such therapies have the potential to reduce the dangerous side effects of chemotherapy drugs (by targeted destruction of cancer cells) and combat diseases such as cystic fibrosis (by targeted delivery of genes to lung cells). Investigating how non-fluorescent engineered particles interact with, are internalized, and/or are trafficked through biological cells is critical to the success of these ventures. Individuals vary in their response to drugs due to genetic and other factors. Similarly, cell-to-cell variability makes it critical to quantify nanoparticle - cell interactions with statistically-relevant cell populations. To evaluate nanoparticle incorporation, existing techniques include inductively coupled plasma atomic emission spectroscopy (ICP-AES) or mass spectrometry (ICP-MS), which atomize entire samples (thousands of cells) and detect nanoparticles by quantifying the elemental composition. These bulk techniques provide general trends averaged over many cells, but cannot assess the inherent variability posed by individual cells. Transmission electron microscope (TEM) tomography provides nanometer resolution, however the ultimate volume analyzed is typically less than one cell, which is not statistically relevant. Our approach addresses this measurement gap by applying tools found within NIST Boulder's Precision Imaging Facility (PIF).
Correlative Microscopy of Gold Nanoparticles in Cells. Internalized gold nanoparticles are imaged within cross-sectioned cells (upper panel), whereas nanoparticles associated with the cell membrane are imaged with whole cells (lower panel). These images demonstrate consistent nanoparticle dimensions measured inside and outside cells by transmitted electrons (TEM), reflected electrons (SEM), or reflected ions (HIM).
Reconstruction of Gold Nanoparticle Clusters inside Cells. Focused ion beam (FIB) tomography alternates milling cellular material with a gallium ion beam and imaging cellular cross-sections with an electron beam. Specific whole cells within complex cultures can be sliced to reveal internalized metal nanoparticles and series of individual images used to construct cell models. Compared to TEM tomography, this approach decreases analysis times by increasing the total volume available during a single analysis.
Nanoparticle Distribution in Neurons and Glial Cells. Individually milling and imaging whole cells enables us to quantify nanoparticle distribution in cultures containing multiple cells. Possibilities include cancerous and non-cancerous cells or the cells that comprise a particular tissue. Here we examine neurons and glial cells derived from a single stem cell population. Differentiated neurons (circled in red, left image) can be distinguished morphologically from glial cells (circled in blue, left image) and this identification can be confirmed by standard immunocytochemical techniques (right image).
A.W. Sanders, A.N. Chiaramonti, A.E. Curtin, K.M. Jeerage, C.L. Schwartz. Multiscale correlative microscopy of the interaction of Au nanoparticles with rat cortex neural progenitor cells. Microscopy and Microanalysis (2013)
A.W. Sanders, K.M. Jeerage, C.L. Schwartz, A.E. Curtin, A.N. Chiaramonti. Correlating multiscale measurements of nanoparticles in primary cells. Microscopy and Microanalysis (2014)
K.M. Jeerage, T.L. Oreskovic, A.E. Curtin, A.W. Sanders, R.K. Schwindt, A.N. Chiaramonti. Citrate-stabilized gold nanoparticles as negative controls for measurements of neurite outgrowth. Toxicology In Vitro (2015)