Wavefront shaping is increasingly being used in modern microscopy to obtain high-resolution images deep inside inhomogeneous media. Wavefront shaping methods typically rely on the presence of a “guide star” to find the optimal wavefront to mitigate the scattering of light. However, the use of guide stars poses severe limitations. Notably, only objects in the close vicinity of the guide star can be imaged. Here, we introduce a guide-star-free wavefront shaping method in which the optimal wavefront is computed using a digital model of the sample. The refractive index model of the sample, that serves as the input for the computation, is constructed in situ by the microscope itself. In a proof of principle imaging experiment, we demonstrate a large improvement in the two-photon fluorescence signal through a diffuse medium, outperforming state-of-the-art wavefront shaping by a factor of two in imaging depth.

Enabling handheld perfusion imaging would drastically improve the feasibility of perfusion imaging in clinical practice. Therefore, we examine the performance of handheld laser speckle contrast imaging (LSCI) measurements compared to mounted measurements, demonstrated in psoriatic skin. A pipeline is introduced to process, analyze and compare data of 11 measurement pairs (mounted-handheld LSCI modes) operated on 5 patients and various skin locations. The on-surface speeds (i.e. speed of light beam movements on the surface) are quantified employing mean separation (MS) segmentation and enhanced correlation coefficient maximization (ECC). The average on-surface speeds are found to be 8.5 times greater in handheld mode compared to mounted mode. Frame alignment sharpens temporally averaged perfusion maps, especially in the handheld case. The results show that after proper post-processing, the handheld measurements are in agreement with the corresponding mounted measurements on a visual basis. The absolute movement-induced difference between mounted-handheld pairs after the background correction is $$16.4\pm 9.3~\%$$ 16.4 ± 9.3 % (mean ± std, $$n=11$$ n = 11 ), with an absolute median difference of $$23.8\%$$ 23.8 % . Realization of handheld LSCI facilitates measurements on a wide range of skin areas bringing more convenience for both patients and medical staff.

Laser speckle contrast imaging (LSCI) is a non-invasive and affordable technique to visualize skin perfusion. Handheld use of the system facilitates measurements on various skin areas in a flexible manner. However, movement artefacts caused by handheld operation or test subject movements hamper its performance. In this work, we study the influence of the laser beam type in handheld-LSCI by evaluating the speckle contrast on static objects for beams with planar, spherical or scrambled wavefronts, and for movement artefacts caused by tilting or translation of wavefronts. We show that the scrambled waves made by often-used engineered diffusers lead to significantly larger movement artefacts than planar or spherical waves.

Functional performance of handheld laser speckle contrast imaging (LSCI) is compromised by movement artefacts. Here we quantify the movements of a handheld LSCI system employing electromagnetic (EM) tracking and measure the applied translational, tilt and on-surface laser beam speeds. By observing speckle contrast on static objects, the magnitudes of translation and tilt of wavefronts are explored for various scattering levels of the objects. We conclude that for tissue mimicking static phantoms, on-surface speeds play a dominant role to wavefront tilt speed in creation of movement artefacts. The ratio depends on the optical properties of the phantom. Furthermore, with the same applied speed, the drop in the speckle contrast increases with decreasing reduced scattering coefficient, and hence the related movement artefact increases.

Fluid flow shear stresses are strong regulators for directing the organization of vascular networks. Knowledge of structural and flow dynamics information within complex vasculature is essential for tuning the vascular organization within engineered tissues, by manipulating flows. However, reported investigations of vascular organization and their associated flow dynamics within complex vasculature over time are limited, due to limitations in the available physiological pre-clinical models, and the optical inaccessibility and aseptic nature of these models. Here, we developed laser speckle contrast imaging (LSCI) and side-stream dark field microscopy (SDF) systems to map the vascular organization, spatio-temporal blood flow fluctuations as well as erythrocytes movements within individual blood vessels of developing chick embryo, cultured within an artificial eggshell system. By combining imaging data and computational simulations, we estimated fluid flow shear stresses within multiscale vasculature of varying complexity. Furthermore, we demonstrated the LSCI compatibility with bioengineered perfusable muscle tissue constructs, fabricated via molding techniques. The presented application of LSCI and SDF on perfusable tissues enables us to study the flow perfusion effects in a non-invasive fashion. The gained knowledge can help to use fluid perfusion in order to tune and control multiscale vascular organization within engineered tissues.