Label-free imaging of cell attachment with photonic crystal enhanced microscopy

Abstract

We introduce photonic crystal enhanced microscopy (PCEM) as a label-free biosensor imaging technique capable of measuring cell surface attachment and attachment modulation. The approach uses a photonic crystal optical resonator surface incorporated into conventional microplate wells and a microscope-based detection instrument that measures shifts in the resonant coupling conditions caused by localized changes in dielectric permittivity at the cell-sensor interface. Four model systems are demonstrated for studying cancer cells, primary cardiac muscle cells, and stem cells. First, HepG2/C3 hepatic carcinoma cells were cultured and observed via PCEM in order to characterize cell adhesion in the context of growth and locomotion. Second, Panc-1 pancreatic cancer cells were used to verify that cell attachment density decreases in response to staurosporine, a drug that induces apoptosis. Third, we used PCEM to confirm the influence of integrin-mediated signaling on primary neonatal cardiomyocyte growth and development. Rounded cardiomyocytes consistently showed decreased cell attachment density as recorded via PCEM, while spreading cells exhibited greater attachment strength as well as increased contractility. Finally, PCEM was used to monitor the morphological changes and extracellular matrix remodeling of porcine adipose-derived stem cells subjected to a forced differentiation protocol. Each of these experiments yielded information regarding cell attachment density without the use of potentially cytotoxic labels, enabling study of the same cells for up to several days.

Photonic crystals: a versatile platform for optics-based biological detection

“…PC biosensors consist of a low refractive index sub-wavelength periodic grating structure that functions as a highly reflective optical resonator. While previously reported PC surfaces have been comprised of a rectangular grating profile with a period of 550 nm and a grating depth of approximately 150 nm [15], the biosensors used for cell attachment biosensor imaging have been modified to reduce possible effects of sensor morphology on the formation of cell attachments [9]. To reduce the occurrence of such effects, the PC biosensors used in this study possess a period of 360 nm with a grating depth of approximately 30 nm.…”

“…This talk will summarize recent technical advancements and life science applications for photonic crystal surfaces and related detection instrumentation. The development of label-free imaging systems for quantification of biomolecule binding density in array formats and for monitoring the interaction of live cells with surfaces will be described [9], with applications for DNA microarray quality control [10] and stem cell differentiation imaging. Photonic Crystal Enhanced Fluorescence (PCEF) utilizes optical resonances at multiple wavelengths in the visible region of the spectrum to simultaneously magnify the electric field intensity exposed to surface-bound fluorophores and to increase the collection efficiency of fluorescence emission [11,12], resulting in substantially increased detection sensitivity for gene expression analysis and cancer biomarker detection in serum [13].…”

Planar Photonic Crystal Biosensor for Quantitative Label‐Free Cell Attachment Microscopy

“…By measuring changes in PWV on a pixel-by-pixel basis on a PC surface with ≈0.5 µm pixel resolution, PCEM has been demonstrated as a powerful tool for quantification and imaging of cell–surface interactions under the circumstances of complex cellular activities such as chemotaxis and drug-induced apoptosis. [16,24] By virtue of the surface-confined evanescent waves generated by the PC biosensor at the resonant condition, PCEM can probe cell activity that is localized to the interface between the cells and their substrate. The spatial resolution of PCEM label-free imaging is dictated by the lateral propagation of the resonant electromagnetic field standing wave.…”

“…Dynamic information about evolution of cell focal adhesions, cell–surface attachment morphology, and cell attachment gradients within individual cells can be provided over biologically relevant time scales without the use of fluorescent labels or colored stains. [16,24,26–28] …”

High frame rate multi-resonance imaging refractometry with distributed feedback dye laser sensor

“…2,3 Several optical sensors allow spatially resolving the refractive index on surfaces in microscope like set-ups. This imaging refractometry has e.g., been applied to detect nanoparticles, 4,5 proteins, [6][7][8] viruses [9][10][11] and cells [12][13][14][15][16] and has enabled imaging as well as multiplexed sensing. 10,17 In this context, label-free signal transduction is based on surface plasmon resonance, 9,12,13,17,18 plasmonic hole arrays, [19][20][21][22][23] photonic crystals 5,[14][15][16] and interferometry.…”