Fluorescence imaging is used widely in almost all the biosciences. Examples include imaging of microscope slides with spots of DNA or proteins, multiplexed assays of classes of molecules like cytokines or capture immunoassays. Fluorescence occurs in all directions and it is necessary to collect as much of the emission as possible and to focus the emission onto a detector. The instruments for these measurements all contain multiple lens, mirrors and filters for directing the emission towards the detector. The optical components and principles have not changed in over 100 years. A modern microscope has the same shape and configuration as those over 100 years old. In contrast there has been enormous progress in electronics where the cost per GB of memory has decreased 1 million-fold. We propose a widely applicable approach to fluorescence imaging that can provide high spatial resolution over large areas, easily up to the frame size of 35 mm film. This will be accomplished using Tamm structures which consist of multiple layers of dielectrics and a top metallic layer; which are called Tamm structures (TS). These structures contain no nanoscale features in the x-y plane and vapor deposition can produce large area structures at low cost. Tamm structures support optical modes which are perpendicular to the surface, and we have recently shown fluorophores close to the surface couple to these modes and also radiate perpendicular to the surface. In this project the Tamm structures will be placed directly onto CMOS imaging detectors (CID) for imaging. We refer to this method as coupled-emission microscopy (CEM). Practical CEM devices require improved z-axis confinement and reduced sample-to-detector distances. We propose development of CEM to obtain a spatial resolution of 1 ?m or better. This will be accomplished by optical simulations and refined methods for preparation of Tamm structures and other multi- layer structures (MLS). Two independent methods will be used to test for improved z-axis confinement and improved coupling efficiency. Commercial CIDs contain protective cover slips which prevent close contact with the Tamm structures. Methods will be developed to place the TS directly onto the CID surface and for higher spatial resolution fabrication of the Tamm structure directly on the CID surface. Various methods of illumination and/or thin-film filters will be developed to reject incident light. Spatial resolution will be measured using fluorescent nanobeads (NBs) as point sources will also be used to determine the number of independent measurable locations. The effects of surface topology will be examined for effects on spatial resolution and sensitivity by increases in fluorophore-TS coupling efficiency. We will also examine alternative structures which are known to display perpendicular modes such as the three layer metal-dielectric-metal (MDM) structures and structures which do not contain any metal and display optical Tamm states (OTS). Replacement of even a small fraction of existing instruments with CEM devices would have a large impact on biology and medicine.
We propose a widely applicable approach to fluorescence imaging that can provide high spatial resolution over large areas, easily up to the frame size of 35 nm film. There exists a large infrastructure of optical instrumentation for research and diagnostic testing. Replacement of even a small fraction of existing instruments with the proposed (coupled-emission microscopy) CEM devices would have a large impact on biology and medicine.
|Javmen, Artur; Szmacinski, Henryk; Lakowicz, Joseph R et al. (2018) Blocking TIR Domain Interactions in TLR9 Signaling. J Immunol 201:995-1006|