Abstract
Surface plasmons (SPs) are surface charge density oscillations occuring at a metal/dieletric interface and are highly sensitive to refractive index variations adjacent to the surface. This sensitivity has been exploited successfully for chemical and biological assays. In these systems, a surface plasmon resonance (SPR)-based sensor detects temporal variations in the refractive index at a point. SPR has also been used in imaging systems where the spatial variations of refractive index in the sample provide the contrast mechanism. SPR imaging systems using high numerical aperture (NA) objective lenses have been designed to image adherent live cells with high magnification and near-diffraction limited spatial resolution. Addressing research questions in cell physiology and pharmacology often requires the development of a multimodal microscope where complementary information can be obtained.In this paper, we present the development of a multimodal microscope that combines SPR imaging with a number of additional imaging modalities including bright-field, epifluorescence, total internal reflection microscopy and SPR fluorescence microscopy. We used a high NA objective lens for SPR and TIR microscopy and the platform has been used to image live cell cultures demonstrating both fluorescent and label-free techniques. Both the SPR and TIR imaging systems feature a wide field of view (~300 µm) that allows measurements from multiple cells whilst maintaining a resolution sufficient to image fine cellular processes. The capability of the platform to perform label-free functional imaging of living cells was demonstrated by imaging the spatial variations in contractions from stem cell-derived cardiomyocytes. This technique shows promise for non-invasive imaging of cultured cells over very long periods of time during development.
Highlights
Excitable cells, such as cardiomyocytes and neurons, have been shown to produce fast optical signals that are the result of light scattering and birefringence changes associated with membrane depolarization [1,2,3]
We comment on the factors that affect the resolution of fine neuronal processes. (iii) We show that complementary information on the imaging resolution can be obtained from TIRM and demonstrate a combination of microscopy systems including bright-field, epi-fluorescence and SPR fluorescence microscopy (SPRF) that can be applied to cell physiology. (iv) We combine a number of the imaging techniques to demonstrate a typical experiment where structural and functional imaging is desired and in doing so we verify the ability of the system to study spatiotemporal cellular functions by imaging localized contractions of stem cell-derived cardiomyocytes. (v) We describe a detailed design of this platform to enable ease of implementation and characterization for use by the cell physiology community
We have shown that the system is capable of both structural and functional imaging of cultured cells
Summary
Excitable cells, such as cardiomyocytes and neurons, have been shown to produce fast optical signals that are the result of light scattering and birefringence changes associated with membrane depolarization [1,2,3]. SPR is very sensitive to perturbations of refractive index within this evanescent field; when there is a change in the refractive index of the dielectric medium, the characteristics of the light wave coupled to the surface plasmon changes and the resonance conditions are altered (figure 1(b)) This allows SPR to be used for label-free imaging of both structural (sensitive to local variations in density) [7,8,9,10], and functional (sensitive to movements of matter) [11,12,13,14] features. (iii) We show that complementary information on the imaging resolution can be obtained from TIRM and demonstrate a combination of microscopy systems including bright-field, epi-fluorescence and SPRF that can be applied to cell physiology. We comment on the factors that affect the resolution of fine neuronal processes. (iii) We show that complementary information on the imaging resolution can be obtained from TIRM and demonstrate a combination of microscopy systems including bright-field, epi-fluorescence and SPRF that can be applied to cell physiology. (iv) We combine a number of the imaging techniques to demonstrate a typical experiment where structural and functional imaging is desired and in doing so we verify the ability of the system to study spatiotemporal cellular functions by imaging localized contractions of stem cell-derived cardiomyocytes. (v) We describe a detailed design of this platform to enable ease of implementation and characterization for use by the cell physiology community
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