The characterization of ultrathin transparent films is paramount for various optoelectronic materials, coatings, and photonics. However, characterizing such thin layers is difficult and it requires specialized clean-room equipment and trained personnel. Here, a contact-less, all-optical method is introduced and validated for characterizing nanometric transparent films using far-field optics. A series of nanometric, smooth, and homogeneous layered samples are fabricated first, alternating transparent spacer and fluorescent layers in a controlled manner. Fluorescence radiation pattern originating from the thin fluorophore layers is then recorded and analyzed and quantitative image analysis is used to perform in operando measurements of the refractive index, film homogeneity and to estimate axial fluorophore distances at a sub-wavelength scale with a precision of a few of nanometers. The results compare favorably to measurements obtained through more complicated and involved techniques. Applications in nanometrology and biological axial super-resolution imaging are presented. It is demonstrated in live cells the precise axial localization of single organelles in cortical astrocytes, an important type of brain cell. The approach is cheap, versatile and it will have applications in various fields of photonics.

Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities can expect a homogenous and time‐invariant illumination and an uniform detection sensitivity, which are prerequisites for imaging analysis, tracking or fluorimetric pH or Ca2+‐concentration measurements. Similarly, confirming the three‐dimensional (3‐D) resolution of optical sectioning microscopes calls for a regular calibration with a standardized point source. The test samples required for such measurements are typically different ones, they are often expensive and they depend much on the very microscope technique used. Similarly, the ever‐increasing choice among microscope techniques and geometries increases the demand for comparison across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3‐D test sample that can complement existing and much more expensive calibration samples: commercial tissue paper labeled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub‐μm to cm scale, acquired on epifluorescence, confocal, image scanning, two‐photon (2P) and light‐sheet microscopes.

The fabrication and characterisation of ultra-thin, transparent films is paramount for protective layers on semiconductors, solar cells, as well as for nano-composite materials and optical coatings. Similarly, the probe volume of nano-sensors, as well the calibration of axial distances in super-resolution microscopies, all require the metrology of axial fluorophore distances. However, the reliable production and precise characterisation of such nanometric thin layers are difficult and labor-intense and they require specialized equipment and trained personnel. In our present work, we describe a simple, non-invasive, all-optical technique for simultaneously measuring the refractive index, thickness, and homogeneity of such thin films. We assemble transparent layers from My-133-MC, a biomimetic transparent polymer with a refractive index of 1.33, amenable for applications in the life sciences. All parameters characterising the films are obtained in a single measurement from the analysis of supercritical angle fluorescence radiation patterns acquired on a minimally modified inverted microscope. Results compare favorably to those obtained through a combination of atomic force and electron microscopy, surface-plasmon resonance spectroscopy and ellipsometry. To illustrate the utility of our technique, we present two applications, one in metrology and one in bio-imaging; (i), the calibration of axial fluorophore distance in a total internal reflection fluorescence geometry; and, (ii), live-cell super-resolution imaging of organelle dynamics in cortical astrocytes, an important type of brain cell. Our approach is cheap, versatile and it has obvious applications in profilometry, biophotonics, photonic devices, and optical nano-metrology.

Ultra-thin, transparent films are being used as protective layers on semiconductors, solar cells, as well as for nano-composite materials and optical coatings. Nano-sensors, photonic devices and calibration tools for axial super-resolution microscopies, all rely on the controlled fabrication and analysis of ultra-thin layers. Here, we describe a simple, non-invasive, optical technique for simultaneously characterizing the refractive index, thickness, and homogeneity of nanometric transparent films. In our case, these layers are made of the biomimetic polymer, My-133-MC, having a refractive index of 1.33, so as to approach the cytosol for biological applications. Our technique is based on the detection in the far field and the analysis of supercritical angle fluorescence (SAF), i.e., near-field emission from molecular dipoles located very close to the dielectric interface. SAF emanates from a 5-nm J-aggregate emitter layer deposited on and in contact with the inspected polymer film. Our results compare favorably to that obtained through a combination of atomic force and electron microscopy, surface-plasmon resonance spectroscopy and ellipsometry. We illustrate the value of the approach in two applications, (i), the measurement of axial fluorophore distance in a total internal reflection fluorescence geometry; and, (ii), axial super-resolution imaging of organelle dynamics in a living biological sample, cortical astrocytes, an important type of brain cell. In the later case, our approach removes uncertainties in the interpretation of the nanometric axial dynamics of fluorescently labeled vesicles. Our technique is cheap, versatile and it has obvious applications in microscopies, profilometry and optical nano-metrology.

Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities expect a homogenous and time-invariant illumination and a uniform detection sensitivity, which are prerequisites for quantitative imaging analysis, particle tracking or fluorometric pH or Ca2+-concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning micro-scopes prior to volumetric reconstructions calls for a regular calibration with a standardised point source. Typically, the test samples required for such calibration measurements are different ones, and they depend much on the very microscope technique used. Also, the ever-increasing choice among these techniques increases the demand for comparison and metrology across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: simple commercial tissue paper labelled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-µm to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes.Graphical abstractPyranine-labeled tissue paper, imaged upon 405-nm epifluorescence excitation through a 455LP LP dichroic and 465LP emission filter. Objective ×20/NA0.25. Overlaid are the normalised absorbance (dashed) and emission spectra (through line), respectively. In the present work we show that this “primitive” and inexpensive three-dimensional (3-D) test sample is a surprisingly versatile and powerful tool for quality assessment, comparison across microscopes as well as routine metrology for optical sectioning techniques, both for research labs and imaging core facilities.Research highlights-highlighter-pen marked tissue paper is a surprisingly powerful and versatile test sample for 3-D fluorescence microscopies-standard tissue paper presents features ranging from 400 nm to centimetres-our sample can simultaneously be used for testing intensity, field homogeneity, resolution, optical sectioning and image contrast-it is easy to prepare, versatile, photostable and inexpensive