No Arabic abstract
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 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.
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
High-resolution optical microscopy suffers from a low contrast in scattering media where a multiply scattered wave obscures a ballistic wave used for image formation. To extend the imaging depth, various gating operations - confocal, coherence, and polarization gating - have been devised to filter out the multiply scattered wave. However, these gating methods are imperfect as they all act on the detection plane located outside a scattering medium. Here, we present a new gating scheme, called space gating, that rejects the multiply scattered wave directly at the object plane inside a scattering medium. Specifically, we introduced a 30 $mu$m-wide acoustic focus to the object plane and reconstructed a coherent image only with the ballistic wave modulated by acousto-optic interaction. This method allows us to reject the multiply scattered wave that the existing gating methods cannot filter out and improves the ratio of the ballistic wave to the multiply scattered wave by more than 100 times for a scattering medium more than 20 times thicker than its scattering mean free path. Using the coherent imaging technique based on space gating, we demonstrate the unprecedented imaging capability - phase imaging of optically transparent biological cells fully embedded within a scattering medium - with a spatial resolution of 1.5 $mu$m.
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.
We introduce a new modality for dynamic phase imaging in confocal microscopy based on synthetic optical holography. By temporal demultiplexing of the detector signal into a series of holograms, we record time-resolved phase images directly in the time domain at a bandwidth as determined by the photo detector and digitizer. We demonstrate our method by optical imaging of transient vibrations in an atomic force microscope cantilever with 100 ns time resolution, and observe the dynamic deformation of the cantilever surface after excitation with broadband mechanical pulses. Temporal Fourier transform of a single data set acquired in 4.2 minutes yields frequency and mode profile of all excited out-of-plane vibration modes with sub-picometer vertical sensitivity and sub-micrometer lateral resolution. Our method has the potential for transient and spectroscopic vibration imaging of micromechanical systems at nano- and picosecond scale time resolution.