Room-Temperature Detection of a Single Molecule’s Absorption by Photothermal Contrast

Abstract

So far, single-molecule imaging has predominantly relied on fluorescence detection. We imaged single nonfluorescent azo dye molecules in room-temperature glycerol by the refractive effect of the heat that they release in their environment upon intense illumination. This photothermal technique provides contrast for the absorbing objects only, irrespective of scattering by defects or roughness, with a signal-to-noise ratio of ~10 for a single molecule in an integration time of 300 milliseconds. In the absence of oxygen, virtually no bleaching event was observed, even after more than 10 minutes of illumination. In a solution saturated with oxygen, the average bleaching time was of the order of 1 minute. No blinking was observed in the absorption signal. On the basis of bleaching steps, we obtained an average absorption cross section of 4 angstroms(2) for a single chromophore.

Physics of Nanomechanical Spectrometry of Viruses

“…We envision that the mass, position and orientation-dependent stiffness can be obtained by tracking the frequency of multiple vibration modes and solving the inverse problem 8,[10][11] . In addition, the implementation of imaging techniques that can approximately resolve the position of the adsorbate on the cantilever can simplify the problem and reduce the error in the calculations [31][32] .…”

Quantitative absorption spectroscopy of nano-objects

“…14 In 2010, Gaiduk, Orrit and coworkers optimized the sensitivity of this heterodyne technique and managed to detect single dye molecules, with an ACS as weak as 1 nm 2 . 5 This series of reported experiments is closely related to our QWLSI technique since it is based on the same physics: a photothermal induced variation of the refractive index surrounding the absorbing structure. The fundamental differences is that we do not modulate the heating and we do not perform the same kind of measurements: While the PTI techniques measure either a modulated scattered intensity, 5,13 or a combination of both the modulated intensity and phase gradient by DIC, 11 the QWLSI technique measures a quantitative 2D phase image.…”

“…5, Gaiduk et al overcame this problem by previously measuring the photothermal signal of a reference gold spherical nanoparticle of a given size, in the same environment, and computing the theoretical ACS using Mie theory. 5 This gave them a calibration coefficient linking ACS and photothermal signal, assuming a linearity of the signal. Then, they performed measurements on single molecules, measured the photothermal signal, and used the calibration coefficient to indirectly retrieve an absolute value of the ACS of the molecule.…”

“…Several techniques have been developed recently to estimate ACSs of metal NPs. [3][4][5] However, they present major limitations: i) They rely on the assumption that ACS can be assimilated to extinction cross section. This assumption is valid only for NPs much smaller than the illumination wavelength.…”

Heterodyne detected nonlinear optical imaging in a lock‐in free manner

“…Several heterodyne detected optical imaging techniques based on interferometric CARS [1][2][3], stimulated Raman scattering (SRS) [4], transient absorption [5], and photothermal effect [6] have shown great potential for label-free imaging, e.g., SRS microscopy [7,[8][9][10][11] has been used for vibrational imaging of biomass [12], pharmaceutical samples [13,14], and lipid bodies [10,15]. All these techniques require the extraction of a small AC signal at the sub-microvolt level from a noisy environment.…”