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Register a new userScattering oblique plane microscopy for in-vivo blood cell imaging
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Oblique plane microscopy (OPM) enables high speed, volumetric fluorescence imaging through a single-objective geometry. While these advantages have positioned OPM as a valuable tool to probe biological questions in animal models, its potential for in vivo human imaging is largely unexplored due to its typical use with exogenous fluorescent dyes. Here we introduce a scattering-contrast oblique plane microscope (sOPM) and demonstrate label-free imaging of blood cells flowing through human capillaries in vivo. The sOPM illuminates a capillary bed in the ventral tongue with an oblique light sheet, and images side- and back- scattered signal from blood cells. By synchronizing sOPM with a conventional capillaroscope, we acquire paired widefield and axial images of blood cells flowing through a capillary loop. The widefield capillaroscope image provides absorption contrast and confirms the presence of red blood cells (RBCs), while the sOPM image may aid in determining whether optical absorption gaps (OAGs) between RBCs have cellular or acellular composition. Further, we demonstrate consequential differences between fluorescence and scatteri
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Multifocal plane microscopy (MUM) allows three dimensional objects to be imaged in a single camera frame. Our approach uses dual orthogonal diffraction phase gratings with a quadratic distortion of the lines to apply defocus to the first diffraction orders which, when paired with a relay lens, allows for 9 focal planes to be imaged on a single camera chip. This approach requires a strong signal level to ensure sufficient intensity in the diffracted light, but has the advantage of being compact and straightforward to implement. As the microscope begins to focus deeper into the sample, aberrations caused by refractive index mismatch and inhomogeneity in the samples media have an adverse effect on the signals quality. In this paper, we investigate the image quality improvement brought by applying adaptive optics (AO) to multifocal plane microscopy. A single correction device (an 8x8 deformable mirror (DM)) is combined with an image-based AO control strategy to perform the correction of optical aberrations. We compare full end-to-end modelling results using an established numerical modelling system adapted for microscopy to laboratory results both on a test sample and on a number of biological samples. Finally, we will demonstrate that combining AO and MUM, we are able to improve the image quality of biological samples and provide a good correction throughout the volume of the biological sample.
Multiphoton microscopy (MPM) has gained enormous popularity over the years for its capacity to provide high resolution images from deep within scattering samples1. However, MPM is generally based on single-point laser-focus scanning, which is intrinsically slow. While imaging speeds as fast as video rate have become routine for 2D planar imaging, such speeds have so far been unattainable for 3D volumetric imaging without severely compromising microscope performance. We demonstrate here 3D volumetric (multiplane) imaging at the same speed as 2D planar (single plane) imaging, with minimal compromise in performance. Specifically, multiple planes are acquired by near-instantaneous axial scanning while maintaining 3D micron-scale resolution. Our technique, called reverberation MPM, is well adapted for large-scale imaging in scattering media with low repetition-rate lasers, and can be implemented with conventional MPM as a simple add-on.
We report both sub-diffraction-limited quantum metrology and quantum enhanced spatial resolution for the first time in a biological context. Nanoparticles are tracked with quantum correlated light as they diffuse through an extended region of a living cell in a quantum enhanced photonic force microscope. This allows spatial structure within the cell to be mapped at length scales down to 10 nm. Control experiments in water show a 14% resolution enhancement compared to experiments with coherent light. Our results confirm the longstanding prediction that quantum correlated light can enhance spatial resolution at the nanoscale and in biology. Combined with state-of-the-art quantum light sources, this technique provides a path towards an order of magnitude improvement in resolution over similar classical imaging techniques.
We present a technically simple implementation of quantitative phase imaging in confocal microscopy based on synthetic optical holography with sinusoidal-phase reference waves. Using a Mirau interference objective and low-amplitude vertical sample vibration with a piezo-controlled stage, we record synthetic holograms on commercial confocal microscopes (Nikon, model: A1R; Zeiss: model: LSM-880), from which quantitative phase images are reconstructed. We demonstrate our technique by stain-free imaging of cervical (HeLa) and ovarian (ES-2) cancer cells and stem cell (mHAT9a) samples. Our technique has the potential to extend fluorescence imaging applications in confocal microscopy by providing label-free cell finding, monitoring cell morphology, as well as non-perturbing long-time observation of live cells based on quantitative phase contrast.
Thick biological tissues give rise to not only the scattering of incoming light waves, but also aberrations of the remaining unscattered waves. Due to the inability of existing optical imaging methodologies to overcome both of these problems simultaneously, imaging depth at the sub- micron spatial resolution has remained extremely shallow. Here we present an experimental approach for identifying and eliminating aberrations even in the presence of strong multiple light scattering. For time-gated complex-field maps of reflected waves taken over various illumination channels, we identify two sets of aberration correction maps, one for the illumination path and one for the reflection path, that can preferentially accumulate the unscattered signal waves over the multiple-scattered waves. By performing closed-loop optimization for forward and phase- conjugation processes, we demonstrated a spatial resolution of 600 nm up to the unprecedented imaging depth of 7 scattering mean free paths.
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