Improvement of Biomolecule Quantification Precision and Use of a Single-Element Aspheric Objective Lens in Fluorescence Correlation Spectroscopy

Abstract

We found a way to increase the precision with which biomolecules present at concentrations below 10(-10) M can be quantified by fluorescence correlation spectroscopy (FCS). The effectiveness of the way was demonstrated experimentally by using a single-element aspheric objective lens, which was newly developed to reduce the cost of FCS instruments. In the first part of this paper, the relative standard deviation (RSD) of FCS-based concentration measurements is estimated theoretically by an analytical approximation assuming the detection volume profiles in FCS setups to be Gaussian and by molecular simulations in which more realistic profiles are calculated from physical parameters of the measurement setups. In a limit of infinitely bright molecules and zero background emission, the analytical approximation predicts that the RSD at a concentration is minimized when the mean number of molecules in a detection volume is approximately 0.5. A detection volume of the order of 10(-13) L thus gives smaller RSD values for concentrations from 10(-11) to 10(-10) M than does one of the order of 10(-15) L, which is widely used in FCS. This prediction is supported by the molecular simulations, taking into account the finite molecule brightness and background emission. In the second part of the paper, the RSD is evaluated experimentally with an FCS setup with a detection volume of 1.1 x 10(-13) L. The newly developed objective lens, serving as the bottom of the sample cell in this setup, has a large numerical aperture (0.9) without using immersion liquid. When a calibration line was made by 30-s FCS measurements of Cy3-labeled, 112-mer single-stranded DNA solutions, the RSD roughly agreed with the simulation result and was less than 0.1 for DNA concentrations from 2 x 10(-11) to 10(-10) M.

Disposable Microscope Objective Lenses for Fluorescence Correlation Spectroscopy Using Latex Microspheres

“…Looking for simpler and more cost-efficient systems, the use of a single-element aspheric lens for FCS has been investigated in ref . The specifically designed aspheric lens has 0.9 NA, which allows for efficiently detecting the fluorescence of a single molecule.…”

Dual-wavelength metalens enables Epi-fluorescence detection from single molecules

“…MDE represents a metric of single molecule fluorescence intensity the system can detect and amounts to a product of the laser power density, i.e. the rate of pumping the molecules to their singlet excited state, and the collection efficiency of the system (CEF) 52 . Under the diffraction limit conditions, the laser power density is proportional to with FE exc being the metalens focusing efficiency (transmittance in case of conventional objective lens) at the laser wavelength.…”

“…Under the diffraction limit conditions, the laser power density is proportional to with FE exc being the metalens focusing efficiency (transmittance in case of conventional objective lens) at the laser wavelength. On the other hand, for a correctly set pinhole size, CEF is proportional to 52 , where FE em is the metalens focusing efficiency (transmittance in case of conventional objective lens) at the emission wavelength, n is the refractive index of the aqueous solution. Figure 2e displays the MDE plot as a function of NA at four exemplary metalens focusing efficiencies.…”

Molecular Fluorescence, Phosphorescence, and Chemiluminescence Spectrometry

“…Examples of FCS using a subwavelength sized aperture in a surface plasmon coupled emission microscope and utilizing single-mode optical fiber were reported during the review period. Additional advances included a newly developed single-element aspheric objective lens that reduced the cost of FCS instruments and improved biomolecule quantification precision and a prism-based multicolor instrument that offered high optical stability and no focal volume mismatch for the multicolor detection of molecular dynamics . A new distributed algorithm for multi-τ autocorrelation was also reported to reduce the memory requirements associated with accessing the large time-domain data records generated in FCS experiments .…”