Isotropic single-objective microscopy (ISO-microscopy) is a novel method developed by colleagues at the Fresnel Institute to overcome the limits set by diffraction in optical microscopy. With ISO-microscopy, light is focused into an isotropic spherical spot of about one wavelength dimension.
Watch this video to learn how ISO-microscopy works !
Two-photon excitation of single fluorescent molecules has generated much interest and expectations, especially for analytical sciences. However, current demonstrations struggle with low two-photon fluorescence rate per molecule and/or high background. In a recent publication in Biomedical Optics Express, we answer these challenges by using a single polystyrene microsphere under focused Gaussian illumination. This opens new opportunities to extend the applications of two-photon fluorescence detection.
1- We demonstrate a simple, robust, and low-cost solution to enhance the two-photon fluorescence signal per molecule up to one order of magnitude, without adding any significant photoluminescence noise. This goes significantly beyond the current state-of-the-art.
2- We perform a thorough characterization of the two-photon fluorescence enhancement in the vicinity of a single dielectric microsphere, and report 30x higher fluorescence enhancement factors than earlier work in the field.
3- The microspheres form an interesting structure to compare the gains in one- and two-photon excitation of fluorescence. Such comparison has been seldom reported in the litterature.
Following up with the story of the smallest single molecule detection apparatus ever, and first portable FCS system with true single molecule routine sensitivity, my team has recently published a technical note in the Spectra Analyse magazine. This is also an introduction as to why fluorescence correlation spectroscopy is a powerful method to extend standard fluorescence spetroscopies. A free reprint can be found here.
Summary: Fluorescence correlation spectroscopy (FCS) is a versatile method that would greatly benefit to remote optical fiber fluorescence sensors. However, the current state-of-the-art struggles with low detection sensitivities that prevent the extension of fiber-based FCS down to the single-molecule level. Here we describe a novel probe based on an optical fiber combined with a dielectric microsphere to perform FCS analysis down to the single fluorescent molecule level. This offers new opportunities for reducing the bulky microscope setup and extends FCS to remote or in vivo applications.
My team has released an article in the October 7th issue of the Journal of Physical Chemistry C.
A wide range of studies had been recently devoted to nanoaperture arrays as substrates to enhance the Raman scattering spectrocopy of adsorbed molecules. However, only partial information is known concerning the case of a single nanoaperture. Our paper fills this gap, and discusses the first quantitative evaluation of the SERS enhancement factors on single nanoapertures milled in optically thick gold films.
This work has three major aspects of interest to the SERS community:
1) Nanoapertures milled in noble metal films form interesting SERS substrates thanks to their rational and tunable design, controlled surface enhancement, and intrinsic robustness. We discuss the different design parameters yielding to optimum SERS enhancement already in a single aperture which can be used to further improve the SERS substrates with nanoaperture arrays.
2) We discuss different ways of expressing the SERS enhancement factor, and show excellent agreement between experimental results and numerical simulations.
3) Most experimental studies of controlled SERS substrates skip the issue of the adhesion layer between the gold nanostructure and the glass substrate. We experimentally demonstrate that the adhesion layer has a crucial effect on the SERS enhancement, and needs to be fully considered while designing nanosubstrates for high-sensitivity spectroscopy.
A reprint for personal use only is freely available here.