Annular Beam Shaping in Multiphoton Microscopy to Reduce Out-of-Focus Background

Abstract

Despite the inherent spatial confinement of multiphoton processes that arises from focusing through an objective, the maximum imaging depth in conventional multiphoton microscopy is ultimately limited by noise from out-of-focus fluorescence. This is particularly evident when imaging beyond shallow depths in highly scattering tissue as increased laser powers are necessary. The out-of-focus signal originates from multiphoton processes taking place primarily at shallow depths and deteriorates contrast and limits imaging depth. In this paper, annular laser beams are explored as a concept to reduce this background signal in multiphoton microscopy. The approach is theoretically verified by data from simulations and proof of principle is demonstrated on a custom-built experimental multiphoton microscopy platform. Annular laser beams were created by adopting wavefront control using a spatial light modulator and implemented for imaging tissue phantoms simulating turbid media and human skin ex vivo. The signal-to-background ratios were calculated and compared to images acquired with a traditional, filled-aperture Gaussian beam. Experiments in tissue phantom show an improvement in signal-to-background ratio of about 30% when using annular beam illumination in comparison to Gaussian illumination at specific depths. When laser power is not the limiting factor, this approach is expected to provide even greater benefits.

Application of a reflective microscope objective for multiphoton microscopy

“…However, the throughput is significantly improved if an annular-shaped beam is adopted (<10% reflection) and hence, we choose to use it for the RO in this simulation and all subsequent experiments. Annular beams have previously been demonstrated to provide an approximately 20% improvement in lateral resolution (Hell et al, 1995) and reduce out-of-focus background signal (Borglin et al, 2017). However, in this paper we utilise the annular beam to primarily improve the signal throughput of the RO, rather than for resolution enhancement.…”

Adaptive optics-based wavefront-enhanced laser-induced fluorescence (WELIF) for improved analytical performance

“…5–9 As a result of the significant progress in beam shaping and delivery methodologies, various studies have been developed to investigate their impact on therapeutic and diagnostic procedures, 10,11 tissue imaging, and microscopy. 12–14…”

“…[5][6][7][8][9] As a result of the signicant progress in beam shaping and delivery methodologies, various studies have been developed to investigate their impact on therapeutic and diagnostic procedures, 10,11 tissue imaging, and microscopy. [12][13][14] A DM incorporated with a Shack-Hartmann wavefront sensor (SHWFS) is used in a closed-loop conguration called adaptive optics (AO) for rapid compensation of wavefront aberrations. AO has been rst proposed to compensate for timevarying disturbances in the atmosphere, which cause blurring in astronomical images in telescopes.…”

Azimuthally Polarized and Unidirectional Excitonic Emission from Deep Subwavelength Transition Metal Dichalcogenide Annular Heterostructures

“…In addition to this, monolayer TMDs can be freely transferred between different substrates through interlayer van der Waals forces, forming heterostructures that can potentially serve as ultrathin light sources in various photonic platforms. − However, the atomically thin nature of the monolayer TMDs also leads to relatively weak light–matter interaction, such that it is challenging for the monolayer TMDs alone to manipulate the 2D excitonic emission properties. As a result, the 2D excitons in the atomically thin TMDs lack emission directionality, which limits their practical applications in free space communications, optical sensors, optical microscopy, and so on. Recent progresses on dielectric photonic crystal (PhC) slabs and metasurfaces have shown that such photonic nanostructures can produce photonic modes with specific angular momentum dispersion, which can be utilized to effectively redistribute the excitonic angular emission of monolayer TMDs. , However, due to the low refractive index nature of conventional dielectric materials, these nanostructures usually require a large thickness to support the photonic modes, increasing the thickness of the entire device to hundreds of nanometers despite the atomically thin nature of the monolayer TMDs. ,− …”

“…4−9 However, the atomically thin nature of the monolayer TMDs also leads to relatively weak light−matter interaction, such that it is challenging for the monolayer TMDs alone to manipulate the 2D excitonic emission properties. As a result, the 2D excitons in the atomically thin TMDs lack emission directionality, 6 which limits their practical applications in free space communications, 10 optical sensors, 11 optical microscopy, 12 and so on. Recent progresses on dielectric photonic crystal (PhC) slabs and metasurfaces have shown that such photonic nanostructures can produce photonic modes with specific angular momentum dispersion, which can be utilized to effectively redistribute the excitonic angular emission of monolayer TMDs.…”