Saturated absorption competition microscopy

Abstract

ABSTRACT:We introduce the concept of saturated absorption competition (SAC) microscopy as a means of providing sub-diffraction spatial resolution in fluorescence imaging. Unlike the post-competition process between stimulated and spontaneous emission that is used in stimulated emission depletion (STED) microscopy, SAC microscopy breaks the diffraction limit by emphasizing a pre-competition process that occurs in the fluorescence absorption stage in a manner that shares similarities with ground-state depletion (GSD) microscopy. Moreover, unlike both STED and GSD microscopy, SAC microscopy offers a reduction in complexity and cost by utilizing only a single continuous-wave laser diode and an illumination intensity that is ~ 20x smaller than that used in STED. Our approach can be physically implemented in a confocal microscope by dividing the input laser source into a time-modulated primary excitation beam and a doughnut-shaped saturation beam, and subsequently employing a homodyne detection scheme to select the modulated fluorescence signal. Herein, we provide both a physico-chemical model of SAC and experimentally demonstrate by way of a proof-of-concept experiment a transverse spatial resolution of ~λ/6.Owing to the advantage of being non-invasive in observing living samples, far-field optical

Heterochromatic Nonlinear Optical Responses in Upconversion Nanoparticles for Super‐Resolution Nanoscopy

“…Instead of creating the contrast from dual‐beam (de‐) excitation patterns, the emission in different color bands can have distinct power‐dependent responses to a single doughnut excitation, so that to display the different emission PSF patterns, e.g., red PSF Gau (Gaussian PSF) and green PSF Dou (doughnut PSF), as shown in Figure 2a. Due to the emission saturation effect, [ 22 ] the emission doughnut PSF contains more information at high spatial frequency. This first offers the spatial domain opportunity in PSF engineering by simply subtracting the image of PSF Dou from the one of PSF Gau with an appropriate normalizing coefficient, following PSF FED = PSF Gau − r × PSF Dou , so that the sub‐diffraction image can be obtained by a single beam scanning super‐resolution microscopy (Figure 2b and Figure S3, Supporting Information).…”

“…1A. Due to the emission saturation effect (22), the emission doughnut PSF contains more information at high spatial frequency. This first offer the spatial domain opportunity in PSF engineering by simply subtracting the image of 𝑃𝑆𝐹 from the one of 𝑃𝑆𝐹 with an appropriate normalising coefficient, following 𝑃𝑆𝐹 = 𝑃𝑆𝐹 − 𝑟 * 𝑃𝑆𝐹 , so that the sub-diffraction image can be obtained by a single beam scanning super-resolution microscopy (Fig.…”

“…In the latest development, using upconversion nanoparticles (UCNPs), [ 12 ] single scan using two synchronized laser beams has resulted in a fluorescence emission difference microscopy approach that uses the two‐color PSF engineering. Explorations of the nonlinear properties of fluorescent probes, either inducing binary stochastic methods, [ 13,14 ] quantum coherent control, [ 15 ] or under saturated conditions, [ 16–19 ] have resulted in the new approaches of stimulated emission, [ 6,20 ] ground state depletion, [ 9 ] absorption, [ 21,22 ] or fluorescence photoswitching. [ 23 ] Nevertheless, as a prerequisite to extract the broader coverage of spatial information to enhance the quality of super‐resolution images, these advances still require specialized optics to meet the particular excitation conditions to produce images with multiplexed PSF.…”

“…In the latest development, using upconversion nanoparticles (UCNPs) (12), single scan using two synchronised laser beams has resulted in a fluorescence emission difference (FED) microscopy approach that uses the two-colour PSF engineering. Explorations of the nonlinear properties of fluorescent probes, either inducing binary stochastic methods (13), (14), quantum coherent control (15) or under saturated conditions (16), (17), (18), (19), have resulted in the new approaches of stimulated emission (6), (20), ground state depletion (9), absorption (21), (22) or fluorescence photo-switching (23).…”

High resolution imaging with anomalous saturated excitation

“…The key is to simultaneously record two or more images with different spatial frequency distributions. It can be realized by changing the shape of excitation laser beam [17,[34][35][36]. Here, we modulated the power of the excitation laser to obtain images with different spatial frequency distributions.…”