Focusing ultrashort laser pulses in time in tandem with single-pixel detection images at depth with no sample characterization.
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.
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.
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.
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.
Attenuation of optical fields owing to scattering and absorption limits the penetration depth for imaging. Whilst aberration correction may be used, this is difficult to implement over a large field-ofview in heterogeneous tissue. Attenuation-compensation allows tailoring of the maximum lobe of a propagation-invariant light field and promises an increase in depth penetration for imaging. Here we show this promising approach may be implemented in multi-photon (two-photon) light-sheet fluorescence microscopy and, furthermore, can be achieved in a facile manner utilizing a graded neutral density filter, circumventing the need for complex beam shaping apparatus. A "gold standard" system utilizing a spatial light modulator for beam shaping is used to benchmark our implementation. The approach will open up enhanced depth penetration in light-sheet imaging to a wide range of end users. Light-sheet fluorescence microscopy (LSFM) has transformed the field of imaging in recent years, owing to its optical sectioning capabilities, resulting in fast, highly resolved images with significantly reduced photo-bleaching and photo-toxicity 1,2. Propagation-invariant light fields, such as Airy and Bessel beams, have been employed in LSFM not only because of their pseudo-nondiffracting properties which enables them to retain their transverse profile on propagation, but also due to their self-healing capabilities on interaction with obstacles during propagation 3-7. However, attenuation due to scattering and absorption results in an exponential decay of intensity of any given optical field as it penetrates deep into tissue and limits the penetration depth achievable for deep tissue imaging. Recently, the capability to shape the envelope profile of a light field arbitrarily 8-11 has been demonstrated to counteract the attenuation-induced exponential decrease in intensity, by tailoring an exponential rise in intensity along the direction of propagation 10. Building upon this, LSFM exploiting attenuation-compensated Airy beams in single-photon imaging has demonstrated improved image quality at depth in attenuating biological specimens without any increase in the peak intensity of the illuminating light-sheet 12. This is achieved by the selective delivery of additional intensity to greater depths within the attenuating medium, potentially minimizing photo-damage across the specimen. Our previous work utilized a spatial light modulator (SLM) for generation of attenuation-compensated Airy beams solely for single-photon imaging. While this approach offers excellent beam quality and has the flexibility to dynamically adjust the beam shape to optimally counteract the specimen attenuation, it adds cost and complexity to such a system, limiting the potential uptake of the method. In this work, we show that attenuation-compensation of propagation invariant Airy fields can be achieved for multi-photon (two-photon) LSFM, and can be implemented in an inexpensive and facile manner. This is achieved by exploiting readily-available graded neutral densi...
Optical approaches have broadened their impact in recent years with innovations in both wide-field and superresolution 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 significantly reducing photobleaching 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.
Attenuation of optical fields owing to scattering and absorption limits the penetration depth for imaging. Whilst aberration correction may be used, this is difficult to implement over a large field-of-view in heterogeneous tissue. Attenuation-compensation allows tailoring of the maximum lobe of a propagation-invariant light field and promises an increase in depth penetration for imaging. Here we show this promising approach may be implemented in multi-photon (twophoton) light-sheet fluorescence microscopy and, furthermore, be achieved in a facile manner utilizing a graded neutral density filter, circumventing the need for complex beam shaping apparatus. A "gold standard" system utilizing a spatial light modulator for beam shaping is used to benchmark our implementation. The approach will open up enhanced depth penetration in light-sheet imaging to a wide range of end users.
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