ABSTRACT
We automate the manipulation of genomic-length DNA in a nanofluidic device based on real-time analysis of fluorescence images. In our protocol, individual molecules are picked from a microchannel and stretched with pN forces using pressure driven flows. The millimeter-long DNA fragments free flowing in micro- and nanofluidics emit low fluorescence and change shape, thus challenging the image analysis for machine vision. We demonstrate a set of image processing steps that increase the intrinsically low signal-to-noise ratio associated with single-molecule fluorescence microscopy. Furthermore, we demonstrate how to estimate the length of molecules by continuous real-time image stitching and how to increase the effective resolution of a pressure controller by pulse width modulation. The sequence of image-processing steps addresses the challenges of genomic-length DNA visualization; however, they should also be general to other applications of fluorescence-based microfluidics.
Subject(s)
Automation, Laboratory/instrumentation , DNA , Microfluidics/instrumentation , Nanotechnology/instrumentation , Automation, Laboratory/methods , Equipment Design , Fluorescence , Microfluidics/methods , Nanotechnology/methods , Optical Imaging/instrumentation , Optical Imaging/methods , PressureABSTRACT
INTRODUCTION: Microfluidics systems usually consist of materials like PMMA--poly(methyl methacrylate) and PDMS--poly(dimethylsiloxane) and not polystyrene (PS), which is usually used for cell culture. Cellular and molecular responses in cells grown on PS are well characterized due to decades of accumulated research. In contrast, the experience base is limited for materials used in microfludics chip fabrication. METHODS: The effect of different materials (PS, PMMA and perforated PMMA with a piece of PDMS underneath) on the growth and differentiation of PC12 (adrenal phaeochromocytoma) cells into neuronal-like cells was investigated using cell viability, cell cycle distribution, morphology, and gene expression analysis. RESULTS/CONCLUSIONS: After differentiation, the morphology, viability and cell cycle distribution of PC12 cells grown on PS, PMMA with and without PDMS underneath was the same. By contrast, 41 genes showed different expression for PC12 cells differentiating on PMMA as compared to on PS. In contrast, 677 genes showed different expression on PMMA with PDMS underneath as compared with PC12 cells on PS. The differentially expressed genes are involved in neuronal cell development and function. However, there were also many markers for neuronal cell development and functions that were expressed similarly in cells differentiating on PS, PMMA and PMMA with PDMS underneath. In conclusion, it was shown that PMMA has a minor impact and PDMS a major impact on gene expression in PC12 cells.
Subject(s)
Dimethylpolysiloxanes/chemistry , Gene Expression/drug effects , Microfluidics/instrumentation , Neurons/metabolism , Animals , Biocompatible Materials , Cell Culture Techniques , Cell Cycle , Cell Survival , Equipment Design , G1 Phase , Gene Expression Profiling , Microfluidic Analytical Techniques/instrumentation , Oligonucleotide Array Sequence Analysis , PC12 Cells , Polymethyl Methacrylate/chemistry , Polystyrenes/chemistry , Predictive Value of Tests , RatsABSTRACT
Knowledge of cells' interactions with nanostructured materials is fundamental for bio-nanotechnology. We present results for how individual mouse fibroblasts from cell line NIH3T3 respond to highly spiked surfaces of silicon black that were fabricated by maskless reactive ion etching (RIE). We did standard measurements of cell viability, proliferation, and morphology on various surfaces. We also analyzed the motility of cells on the same surfaces, as recorded in time lapse movies of sparsely populated cell cultures. We find that motility and morphology vary strongly with nano-patterns, while viability and proliferation show little dependence on substrate type. We conclude that motility analysis can show a wide range of cell responses e.g. over a factor of two in cell speed to different nano-topographies, where standard assays, such as viability or proliferation, in the tested cases show much less variation of the order 10-20%.
Subject(s)
Nanotechnology , Silicon/chemistry , Animals , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Mice , NIH 3T3 Cells , Nanostructures/chemistry , Nanostructures/toxicity , Surface PropertiesABSTRACT
Motility studies of cells often rely on computer software that analyzes time-lapse recorded movies and establishes cell trajectories fully automatically. This raises the question of reproducibility of results, since different programs could yield significantly different results of such automated analysis. The fact that the segmentation routines of such programs are often challenged by nanostructured surfaces makes the question more pertinent. Here we illustrate how it is possible to track cells on bright field microscopy images with image analysis routines implemented in an open-source cell tracking program, PACT (Program for Automated Cell Tracking). We compare the automated motility analysis of three cell tracking programs, PACT, Autozell, and TLA, using the same movies as input for all three programs. We find that different programs track overlapping, but different subsets of cells due to different segmentation methods. Unfortunately, population averages based on such different cell populations, differ significantly in some cases. Thus, results obtained with one software package are not necessarily reproducible by other software.
