Saturday 13th August 2022

Wide-field multiphoton imaging through scattering media without correction

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Abstract

Optical approaches to fluorescent, spectroscopic, and morphological imaging have made exceptional advances in the last decade. Super-resolution imaging and wide-field multiphoton imaging are now underpinning major advances across the biomedical sciences. While the advances have been startling, the key unmet challenge to date in all forms of optical imaging is to penetrate deeper. A number of schemes implement aberration correction or the use of complex photonics to address this need. In contrast, we approach this challenge by implementing a scheme that requires no a priori information about the medium nor its properties. Exploiting temporal focusing and single-pixel detection in our innovative scheme, we obtain wide-field two-photon images through various turbid media including a scattering phantom and tissue reaching a depth of up to seven scattering mean free path lengths. Our results show that it competes favorably with standard point-scanning two-photon imaging, with up to a fivefold improvement in signal-to-background ratio while showing significantly lower photobleaching.

INTRODUCTION

A suite of powerful, disruptive optical imaging approaches across the physical and biomedical sciences has recently emerged. Super-resolution imaging led to new studies looking at nanometric features within cells that have revealed intricate aspects of subcellular processes (15). At the larger scale, methods such as optical coherence tomography (6) and light-sheet imaging (7) are taking hold in fields such as opthalmology, neuroscience, and developmental biology. In tandem with the requirement for a fast, wide-field visualization and super-resolved imaging across biomedicine, a grand challenge is to perform such imaging through highly scattering (turbid) media, namely, tissue. In particular, this is essential to move from superficial surface imaging to functional imaging at depth (811), which is crucial for biomedical areas including neuroscience. To address this area, aberration correction can be implemented (12). However, this does not readily take into account the properties of the medium, and actual retrieval of the emitted signal from depth in the medium can still be challenging. Key advances have emerged by a consideration of the propagation of light within a complex medium. In this field, a number of approaches use dynamic wavefront shaping for illumination of the sample with a calculated input complex wavefront (1316), which can focus light upon an embedded guide star. In essence, one determines the transmission matrix of the sample in this process (11, 17, 18). While this is powerful, the requirement of a guide star restricts the approach. Furthermore, it requires determination of the properties of the medium at one or more individual points, making it very challenging to implement for wide-field imaging.

An important advance would be the realization of a fast, wide-field imaging approach that would deliver and retrieve light from any given plane within a sample, even in the presence of turbidity. This would be without the requirement to characterize or even actively correct the aberrating effect of the turbid medium. Our approach to achieving this goal exploits temporal focusing (TF) microscopy (19, 20). By using the temporal rather than spatial degree of freedom, scanning of the optical axis for image reconstruction is avoided. Consequently, TF may record wide-field multiphoton images (19, 21). In addition, a little-recognized facet of TF is its ability to deliver light through scattering media. This ability has been used to project optical patterns for applications such as optogenetics, providing photostimulation at remarkable depths (9, 22, 23). Although TF can deliver light through a scattering medium very efficiently, collecting the emitted fluorescent light back through the same medium (i.e., truly achieving imaging) has not been accomplished to date. Separately, there has been the emergence of single-pixel detection, sometimes termed computational ghost imaging (24). In this form of imaging, known patterns illuminate an object, a single-element photodetector records the light intensity that is either transmitted or backscattered by the object, and images are reconstructed with the appropriate algorithm (25, 26).

However, while these studies in TF microscopy and in single-pixel detection have shown promise, none of them has addressed the challenge of correction-free wide-field imaging through turbid media. The scheme that we present here, which we call TempoRAl Focusing microscopy with single-pIXel detection (TRAFIX), uses a judicious combination of TF illumination with single-pixel imaging to obtain wide-field images of fluorescent microscopic samples within or even beyond biological tissues, in the presence of multiple scattering, without aberration correction or characterization of the turbid medium.

RESULTS

Principle of the technique

TF is based on decomposing an incident ultrashort pulsed light field into its constituent wavelengths with a diffraction grating. Each wavelength propagates along an individual path in the optical system, and these wavelengths constructively recombine to regain the original pulse duration only at the plane conjugate to the grating, generating axially confined mutiphoton excitation. In TRAFIX, orthonormal light patterns (in a Hadamard basis) are temporally focused through a turbid medium to illuminate a fluorescent microscopic sample of interest. The use of TF for this projection ensures the retention of the integrity of these patterns at any given plane within the turbid media (Fig. 1A). This can be regarded as due to the fact that ballistic photons remain unperturbed all the way to the object plane and arrive at the same time, contributing to the reconstitution of the pulse. In addition, the superposition of wavelets of slightly different wavelengths at the focal plane results in nearly speckle-free propagation through long distances in scattering media, as recognized by Papagiakoumou et al. (9). We confirm these aspects here with a numerical simulation. The same principle has been previously used for reducing out-of-focus excitation in line-scanning multiphoton microscopy (20). A scattering medium may affect the spatial and temporal degrees of freedom of an input field differently. In the time domain, the temporal profile of femtosecond pulses is not significantly distorted at substantial imaging depths such as 1-mm-thick brain tissue (27). As a consequence, TF may induce much more efficient multiphoton excitation when compared to standard point-focusing where spatial speckle greatly reduces the photon density at the focal spot. Consequently, TF is more robust than conventional focusing, resulting in a more intense fluorescence signal generated at large depths (28), which is a major attribute for our approach.

<a rel="nofollow" href="https://advances.sciencemag.org/content/advances/4/10/eaau1338/F1.large.jpg?width=800&height=600&carousel=1" title="Working principle of TRAFIX. (A) A femtosecond laser beam is expanded onto a spatial light modulator (SLM) that generates Hadamard patterns. Subsequently, the beam is diffracted from a grating, and the Hadamard patterns are projected onto a fluorescent sample after propagating through a scattering medium. Fluorescent light emitted by the sample is collected by the same objective after passing through the scattering medium a second time (epifluorescence geometry), and the total intensity is measured by a single-pixel detector. (B) A TF beam propagates through a turbid medium with minimal distortion, retaining the integrity of illumination patterns in the sample plane. Emitted fluorescent photons scatter as they propagate back through the tissue. In contrast to standard TF microscopy, TRAFIX tolerates scrambling of back-propagating light since only an intensity measurement is performed. In a single-pixel measurement, the fluorescent target is sequentially illuminated with Hadamard patterns (ψn), and the total intensity detected is stored as a coefficient (ωn). Gray background in the second column denotes regions of zero intensity. By adding up the Hadamard patterns weighted by their respective coefficients, an image of the fluorescent sample is reconstructed." class="fragment-images colorbox-load" rel="gallery-fragment-images-910963648" data-figure-caption="

Fig. 1 Working principle of TRAFIX.

(A) A femtosecond laser beam is expanded onto a spatial light modulator (SLM) that generates Hadamard patterns. Subsequently, the beam is diffracted from a grating, and the Hadamard patterns are projected onto a fluorescent sample after propagating through a scattering medium. Fluorescent light emitted by the sample is collected by the same objective after passing through the scattering medium a second time (epifluorescence geometry), and the total intensity is measured by a single-pixel detector. (B) A TF beam propagates through a turbid medium with minimal distortion, retaining the integrity of illumination patterns in the sample plane. Emitted fluorescent photons scatter as they propagate back through the tissue. In contrast to standard TF microscopy, TRAFIX tolerates scrambling of back-propagating light since only an intensity measurement is performed. In a single-pixel measurement, the fluorescent target is sequentially illuminated with Hadamard patterns (ψn), and the total intensity detected is stored as a coefficient (ωn). Gray background in the second column denotes regions of zero intensity. By adding up the Hadamard patterns weighted by their respective coefficients, an image of the fluorescent sample is reconstructed.

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Fig. 1 Working principle of TRAFIX.

(A) A femtosecond laser beam is expanded onto a spatial light modulator (SLM) that generates Hadamard patterns. Subsequently, the beam is diffracted from a grating, and the Hadamard patterns are projected…

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