Optical microscopy based on structured optical beams

Abstract

Nowadays, two-photon microscopy (TPM) is a gold standard tool for the neuro and bio-imaging community owing to providing high-resolution images of cells buried deep inside the sample with possible low background noise. Those properties are accomplished by the localized two-photon excitation restricted at the tiny focal point and using the near-infrared (NIR) excitation in the therapeutic window, having less absorption and scattering in any bio-sample. Therefore, the low absorption and scattering of the excitation beam and the localized fluorescent emission property make it widely used and distinct from other imaging modalities.

Although TPM can provide a high-resolution image with tremendous penetration depth, still, it suffers from the resolution constraint imposed by diffraction and limited penetration depth owing to the severe scattering and absorption, which restricts light from focusing inside any specimen. Over time, several approaches have been adopted to enhance the TPM's spatial resolution, imaging speed, and penetration depth. Among them, structured optical beam illumination is one of the well-established and popular methods due to its robust, low-cost, easy, and compact setup design. To accomplish all the shortcomings of the TPM, a structured optical beam illumination strategy was utilized.

To overcome the imaging resolution limitation, a doughnut-shaped hollow Gaussian beam (HGB), having order-dependent lateral spot size proposed as an illumination beam in TPM. This allows seeing structures beyond the diffraction limit by harnessing the features of lateral spot size squeezes with increasing its mode order. Also, the HGB illumination enhances the penetration depth by improving the signal-to-background ratio (SBR) by redistributing the overall excitation power from the center with the help of its doughnut-like shape. Additionally, a translating lens-based module was also proposed to provide more flexibility in the system by modulating the effective numerical aperture (NA) by controlling the power distribution into the objective lens.

Next, demonstrated a dual-Bessel beam module-based TPM to overcome the imaging speed limitation suffered by the HGB-based TPM system. The module enhances the imaging speed by several factors compared to the standard TPM by leveraging the volumetric imaging technique and combining spatially separated Bessel beams, one followed by another, and producing an axially continued huge elongated profile with maintaining the lateral spot size throughout the illumination profile. Moreover, an axicon displacement module was also implemented to accommodate the axial profile tuning by modulating the separation between the axicons.

After that, a flexible focus TPM was presented to provide additional flexibility in the dual-Bessel beam module-based TPM system and to eliminate the artifact contributed by the on-axis oscillation from the Gaussian-based Bessel beam. The on-axis oscillation-free flexible Bessel profile was furnished by illuminating the axicon with an HGB, and the generated Bessel focus can be easily tuned by alternating the HGB order. The lateral and axial profile of the HGB-Based Bessel profile shrinks as the HGB order increases. Additionally, the dual-Bessel and axicon displace modules were also explored to achieve an extensively elongated with a widely tunable illumination profile to image any desirable thick sample.