Metallic Nanofilm Enhanced Fluorescence Cell Imaging: A Study of Distance-Dependent Intensity and Lifetime by Optical Sectioning Microscopy

Abstract

Simple, stable, easily-fabricated smooth metallic nanofilm can improve the imaging intensity and imaging contrast. However, its application in micrometerscale cells has not been popularized due to the lack of full understanding of their related fluorescence properties. In this study, fluorescence enhancement of cell imaging on smooth Au nanofilm was investigated over a micrometer-scale range via employment of the optical sectioning method available with a laser scanning confocal fluorescence microscope. The fluorescence enhancement reduced with the distance away from the surface of metallic nanofilm, and this distance dependence was determined by the factors of numerical aperture, dye−substrate distance, and emission wavelength. In addition, distance-dependent fluorescence lifetime images of cells were also measured to study the interaction between fluorophores and metallic film. The enhancement effect of Au nanofilm on fluorescence cell imaging can be induced not only by the standing wave formed by the reflected light and exciting light but also by the interaction between fluorophore and surface plasmons on the metallic nanofilm. Our study on smooth metallic nanofilm should pave the way for utilizing its uniform fluorescence enhancement characteristic for biological imaging.

Nanoporous Metals: From Plasmonic Properties to Applications in Enhanced Spectroscopy and Photocatalysis

“…Plasmonic nanostructures have been widely exploited for the fluorescence emission enhancement, which has led to the widely explored field of the so-called metal-enhanced fluorescence (MEF) or plasmon-enhanced fluorescence (PEF) . PEF has attracted enormous research interest as it not only amplifies fluorescence emission intensity but also provides the opportunity to perform imaging with resolutions significantly beyond the diffraction limit . The plasmon-enhanced fluorescence benefits mainly from the local field enhancement (| E |) of the incident field and thus the fluorescence field enhancement is predicted to scale as square of the local field enhancement, i.e.…”

“…167 PEF has attracted enormous research interest 168 as it not only amplifies fluorescence emission intensity 169 but also provides the opportunity to perform imaging with resolutions significantly beyond the diffraction limit. 170 The plasmon-enhanced fluorescence benefits mainly from the local field enhancement (|E|) of the incident field and thus the fluorescence field enhancement is predicted to scale as square of the local field enhancement, i.e., |E| 2 . 171 Thus, tailoring the porosity and surface morphology of NPM platforms is found to give rise to strong local field enhancement and ultrahigh fluorescence enhancement.…”

Characterization of nanometric thin films with far-field light

“…The combination of nano-fabrication and -characterization techniques allowed us to assemble and validate controlled, smooth and uniform sandwiches featuring nm-thin uorescent transparent layers at precisely de ned axial distance and embedded in an environment having a RI close to that of a biological cell. Our sandwich samples can serve, e.g., as a nanoscopic axial ruler for axial super-resolution microscopies, based on astigmatic 36,37 , multi-focal 38,39 , or defocused imaging 40,41 but also on other point-spread-function engineering techniques 42,43 and they provide a valuable reference for interpreting the axial dynamics of intracellular processes in the near-membrane space of live cells, cultured on a glass coverslip 44,45 . We demonstrate that combined sample-plane and quantitative BFP imaging provides detailed insight into uorophore properties and axial dynamics.…”

“…As a simple and reliable far-eld optical technique, the SAF-based uorophore-height measurement is an interesting alternative and complement to other axial nanoscopies. For the sub-20-nm range where our technique lacks sensitivity, quenching-based optical rulers, based on plasmonics 45 , distance-dependent uorescence-lifetime modulation 46 , metal-induced energy transfer (MIET) [47][48][49] , or transparent conductive oxide (TCO-) based transparent rulers 50 can close the gap, offering nm sensitivity.…”

Derivative Matrix-Isopotential Synchronous Spectrofluorimetry and Hantzsch Reaction: A Direct Route to Simultaneous Determination of Urinary δ-Aminolevulinic Acid and Porphobilinogen

“…Modified Hantzsch reaction is reckoned as one of the most frequently employed derivatization agent for δ-ALA, as it entails the detection based on fluorescence ( Tomokuni et al, 1992 ; Oishi et al, 1996 ; Donnelly et al, 2006 ; Kanto et al, 2013 ). Fluorescence spectrometry has been a powerful tool for the identification, monitoring, and quantification of various analytes in a broad range of environments, on account of high selectivity and sensitivity ( Ou-Yang et al, 2018 ; Tsumura et al, 2018 ; Cheng et al, 2019 ; Zhai et al, 2020 ; Ding et al, 2022 ; Remolina et al, 2022 ; Wang et al, 2022 ). Despite the extensive use, the considerable background fluorescence and the spectral overlap contributed by the interfering components are liable to pose a formidable diagnostic challenge for conventional fluorescence spectrometry in case of complex multi-component analysis.…”