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Attenuation of optical fields owing to scattering and absorption limits the penetration depth into tissue. Whilst aberration correction may be used this is difficult to implement over a large field of view in heterogeneous tissue. Recently, the novel approach of attenuation-compensation of propagation-invariant light fields has shown increase in depth penetration for light-sheet microscopy. Here we show this powerful approach may be implemented in a facile manner utilizing a graded neutral density filter circumventing the need for expensive beam shaping apparatus. A ‘gold standard’ system utilizing a spatial light modulator for beam shaping is used to benchmark our low-cost implementation.

This work is supported by the UK Engineering and Physical Sciences Research Council for funding through grants EP/P030017/1 and EP/M000869/1, and has received funding from the European Union's Horizon 2020 Programme through the project Advanced BiomEdical OPTICAL Imaging and Data Analysis (BE-OPTICAL) under grant agreement no. 675512. Optical approaches have broadened their impact in recent years with innovations in both wide-field and super-resolution imaging, which now underpin biological and medical sciences. Whilst these advances have been remarkable, to date, the ongoing challenge in optical imaging is to penetrate deeper. TRAFIX is an innovative approach that combines temporal focusing illumination with single-pixel detection to obtain wide-field multiphoton images of fluorescent microscopic samples deep through scattering media without correction. It has been shown that it can image through biological samples such as rat brain or human colon tissue up to a depth of seven scattering mean-free-path lengths. Comparisons of TRAFIX with standard point-scanning two-photon microscopy show that the former can yield a five-fold higher signal-to-background ratio while signifcantly reducing photo bleaching of the specimen. Here, we show the first preliminary demonstration of TRAFIX with three-photon excitation imaging dielectric beads. We discuss the advantages of the TRAFIX approach combined with compressive sensing for biomedicine. Publisher PDF

Funding: UK Engineering and Physical Sciences Research Council (EPSRC) - EP/P030017/1. We demonstrate the thermal advantages of using low repetition rate, high peak power lasers for imaging in two-photon light sheet microscopy using a Bessel light beam. We compare the use of two ultrashort pulsed lasers in such an imaging system: a high repetition rate source operating at 80 MHz and a low repetition rate source operating at 1 MHz. The low repetition rate laser requires approximately one order of magnitude lower average power than the high repetition rate source to yield the same fluorescent signal. These lasers are used to image Zebrafish larvae and record their heart rates. The data show heart rate values 30% in excess of the ground truth baseline value when imaged with the high repetition rate source due to deleterious heating, whereas the low repetition rate source yields data only a few percent above this ground truth value. Publisher PDF

We describe a new strategy for wide field multiphoton imaging through turbulent media by projecting orthonormal patterns using temporal focusing and recording fluorescent signals with single pixel detection.

This work has received funding from the European Union’s Horizon 2020 Programme through the project Advanced BiomEdical OPTICAL Imaging and Data Analysis (BEOPTICAL) under grant agreement no. 675512 and the UK Engineering and Physical Sciences Research Council (grants EP/P030017/1 and EP/R004854/1). We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 μm diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams. Postprint

We demonstrate a new approach, temporal focusing microscopy with single-pixel detection (TEMPPIX), for wide-field multiphoton imaging through scattering media without any a priori knowledge or requirement to determine the properties of the media.

Compressive sensing can overcome the Nyquist criterion and record images with a fraction of the usual number of measurements required. However, conventional measurement bases are susceptible to diffraction and scattering, prevalent in high-resolution microscopy. Here, we explore the random Morlet basis as an optimal set for compressive multiphoton imaging, based on its ability to minimise the space-frequency uncertainty. We implement this approach for the newly developed method of wide-field multiphoton microscopy with single-pixel detection (TRAFIX), which allows imaging through turbid media without correction. The Morlet basis is well-suited to TRAFIX at depth, and promises a route for rapid acquisition with low photodamage.