Abstract
We present an approach for fabrication of reproducible, chemically and mechanically robust functionalized layers based on MgF2 thin films on thin glass substrates. These show great advantages for use in super-resolution microscopy as well as for multi-electrode-array fabrication and are especially suited for combination of these techniques. The transparency of the coated substrates with the low refractive index material is adjustable by the layer thickness and can be increased above 92%. Due to the hydrophobic and lipophilic properties of the thin crystalline MgF2 layers, the temporal stable adhesion needed for fixation of thin tissue, e.g. cryogenic brain slices is given. This has been tested using localization-based super-resolution microscopy with currently highest spatial resolution in light microscopy. We demonstrated that direct stochastic optical reconstruction microscopy revealed in reliable imaging of structures of central synapses by use of double immunostaining of post- (homer1 and GluA2) and presynaptic (bassoon) marker structure in a 10 µm brain slice without additional fixing of the slices. Due to the proven additional electrical insulating effect of MgF2 layers, surfaces of multi-electrode-arrays were coated with this material and tested by voltage-current-measurements. MgF2 coated multi-electrode-arrays can be used as a functionalized microscope cover slip for combination with live-cell super-resolution microscopy.
Highlights
We present an approach for fabrication of reproducible, chemically and mechanically robust functionalized layers based on MgF2 thin films on thin glass substrates
The most diverse approaches or algorithms used in the techniques of stimulated emission depletion microscopy (STED)[1,2,3], of direct stochastic optical reconstruction microscopy[4,5,6,7] or of structured illumination microscopy (SIM)[8,9] require adapted surfaces and functionalization
We found that a small layer thickness of about 110 nm on ultra-thin cover glasses and a roughness of less than 5 nm prevent aberrations and are excellently suited for the use in Super-resolution microscopy (SRM)
Summary
We present an approach for fabrication of reproducible, chemically and mechanically robust functionalized layers based on MgF2 thin films on thin glass substrates These show great advantages for use in super-resolution microscopy as well as for multi-electrode-array fabrication and are especially suited for combination of these techniques. Magnesium fluoride (MgF2) investigated in this publication is known as a standard material for coatings in optics, since it has interesting physical and chemical properties such as high hardness, good stability in hostile environments, and a very low refractive index (n = 1.38)[11,12,13,14,15]. We show the properties of the robust, functionalized surfaces based on MgF2 thin films prepared by electron beam evaporation (EBE) as a method for producing an electrically isolating and optically antireflective adhesion layer on very thin Borofloat glass substrates for use in biological microscopic and physiological investigations. A technology flowchart for ITO/MgF2-MEAs was developed and resulting properties are tested
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