Assessing the imaging performance of light sheet microscopies in highly scattering tissues

Abstract

Light sheet microscopy (LSM) has emerged as an opticalimaging method for high spatiotemporal volumetric imaging of relatively transparent samples. While this capability has allowed the technique to be highly impactful in fields such as developmental biology, applications involving highly scattering thick tissues have been largely unexplored. Herein, we employ Monte Carlo simulations to explore the use of LSM for imaging turbid media. In particular, due to its similarity to dual-axis confocal (DAC) microscopy, we compare LSM performance to pointscanned (PS-DAC) and line-scanned (LS-DAC) dual-axis confocal microscopy techniques that have been previously shown to produce highquality images at round-trip optical lengths of ~9 -10 and ~3 -4 respectively. The results of this study indicate that LSM using widefield collection (WF-LSM) provides comparable performance to LS-DAC in thick tissues, due to the fact that they both utilize an illumination beam focused in one dimension (i.e. a line or sheet). On the other hand, LSM using confocal line detection (CL-LSM) is more analogous to PS-DAC microscopy, in which the illumination beam is focused in two dimensions to a point. The imaging depth of LSM is only slightly inferior to DAC (~2 -3 and ~6 -7 optical lengths for WF-LSM and CL-LSM respectively) due to the use of a lower numerical aperture (NA) illumination beam for extended imaging along the illumination axis. Therefore, we conclude that the ability to image deeply is dictated most by the confocality of the microscope technique. In addition, we find that imaging resolution is mostly dependent on the collection NA, and is relatively invariant to imaging depth in a homogeneous scattering medium. Our results indicate that superficial imaging of highly scattering tissues using light sheet microscopy is possible.

Enhancement of image quality and imaging depth with Airy light-sheet microscopy in cleared and non-cleared neural tissue

“…Analysis in the microscope coordinates, x or z , will show behaviour in the illumination or detection pathways separately; however, this is dependent on the orientation of the tissue. Following the convention set out by Glaser, Wang, and Liu [4], we perform this analysis along the depth axis of the tissue slice, z′ . This metric concisely encapsulates the performance of both the illumination and detection imaging sub-systems and is applicable for all areas imaged within the tissue.…”

“…While imaging fairly transparent specimens such as a zebrafish brain is achievable using current LSM systems [2], the ability to image larger less transparent turbid specimens, and in the presence of strong aberrations is desirable to advance neuroscience research. Few studies have investigated the performance of LSM in highly turbid media, and these are largely theoretical works (for example, [4,5]).…”

Fractal propagation method enables realistic optical microscopy simulations in biological tissues

“…6, the FPM is also able to provide 3D results, which are not computationally feasible with the FDTD method. These 3D beam profiles are a necessary input for common techniques such as the adjoint method for evaluating an optical microscope’s performance [50–53]. …”

Polarised light sheet tomography

“…The highly forward scattering nature of tissue increases the maximum depth at which satisfactory resolution can be achieved, which is in the range of 3-4 mean free paths, or about 300-400 μm for generic human tissue [26]. The test media used in the following experiments were designed to exploit this limitation to show the merits of polarised detection over conventional means.…”