Homogeneous fluorescent thin films as long-term stable microscopy reference layers

摘要

Calibration and validation of fluorescence microscopy devices and components require a high level of stability and repeatability in their fluorescent properties, both spatially and temporally. In order to establish a dependable reference point, from which all variations within the microscope and peripheral devices can be tested, an exceedingly homogeneous fluorescence response must be provided through a calibration tool. We present material system optimization and microfabrication process development, as well as long-term stability considerations for such a calibration tool. Stringent specifications for film thickness (< 1μm ±0.1% over 1.5x1.5 mm) and for fluorescence response distribution (within 1%) apply, and should hold for up to 100 hours under continuous white irradiation. Low conversion efficiency demands high pick up efficiency and therefor reduces focal depth by high NA of applied fluorescence microscope lens. High spatial resolutions demands use of high quality lenses that typically show low field curvatures and good chromatic corrections. Therefore, the focal plane is flat and well defined in the z-plane. Fluorescent, ligand capped core-shell quantum dots (SMQDs) were embedded in diluted PMMA at low concentrations. The formulations were spin-coated on silicon and glass wafers to obtain films with thicknesses under 1 μm and low variations on a 100 mm wafer. Fluorescence properties of the SMQD were preserved in the matrix material, and agglomerations were not detectable in the fluorescence response nor in SEM images. Gradual degradation of the fluorescence response due to film aging was managed through robust packaging solutions.