Histological staining of tissue biopsies, especially hematoxylin and eosin (H&E) staining, serves as the benchmark for disease diagnosis and comprehensive clinical assessment of tissue. However, the typical formalin-fixation, paraffin-embedding (FFPE) process is laborious and time consuming, often limiting its usage in time-sensitive applications such as surgical margin assessment. To address these challenges, we combine an emerging 3D quantitative phase imaging technology, termed quantitative oblique back illumination microscopy (qOBM), with an unsupervised generative adversarial network pipeline to map qOBM phase images of unaltered thick tissues (i.e., label- and slide-free) to virtually stained H&E-like (vH&E) images. We demonstrate that the approach achieves high-fidelity conversions to H&E with subcellular detail using fresh tissue specimens from mouse liver, rat gliosarcoma, and human gliomas. We also show that the framework directly enables additional capabilities such as H&E-like contrast for volumetric imaging. The quality and fidelity of the vH&E images are validated using both a neural network classifier trained on real H&E images and tested on virtual H&E images, and a user study with neuropathologists. Given its simple and low-cost embodiment and ability to provide real-time feedback in vivo, this deep-learning-enabled qOBM approach could enable new workflows for histopathology with the potential to significantly save time, labor, and costs in cancer screening, detection, treatment guidance, and more.

The past 30 years have seen spectacular progress in the development of techniques for measuring the complete temporal field, and even the complete spatiotemporal field, of ultrashort laser pulses. The challenge has been to measure a pulse without the use of a shorter event or an independent known reference pulse, neither of which is typically available. We begin with autocorrelation, the first such “self-referenced” pulse-measurement method ever proposed, which measures only a rough pulse length, and we describe its limitations. One such limitation is the presence of a somewhat unintuitive “coherent artifact,” which occurs for complicated pulses and also when averaging over a train of pulses whose shapes vary from pulse to pulse. We then describe the most important modern techniques capable of measuring the complete temporal intensity and phase of even complicated ultrashort pulses, as well as their ability (or inability) to measure such unstable pulse trains. A pulse reliably measured with such a device can then be used as a reference pulse in conjunction with another technique, such as spectral interferometry or holography, to measure pulses otherwise unmeasurable by a self-referenced technique. Examples include techniques for measuring low-intensity pulse(s) and for measuring the complete spatiotemporal intensity and phase of arbitrary pulse(s). This Tutorial is limited to well-established, proven methods, but other methods whose description proves instructive will be discussed.

We further develop a simple device, called Spatially and Temporally Resolved Intensity and Phase Evaluation Device: Full Information from a Single Hologram (STRIPED FISH), for measuring the complete spatiotemporal intensity and phase, $I(x,y,t)$ and $\varphi (x,y,t)$, respectively, of an arbitrary ultrashort pulse on a single camera frame (and hence, on a single shot). We have increased the measurable bandwidth, eliminated most aberrations, and improved the uniformity of the multiple holograms in the device. We demonstrate these improvements by making single-camera-frame measurements of spatiotemporally complex subpicosecond crossed and chirped pulses from a Ti:sapphire oscillator. In order to display the massive resulting data files—pairs of four-dimensional intensity-and-phase data—we also develop a method for generating intuitive movies of the measured pulses. With these improvements, this device and its resulting movies should be able to perform and intuitively display true single-shot spatiotemporal measurements of most current ultrashort pulses.

Ultrashort pulses emerging from multimode optical fibers are spatiotemporally complex, because inside these fibers the modes have different spatial intensity patterns and experience different propagation velocities and dispersions. To determine the spatiotemporal field from multimode fibers, we applied a technique for the complete measurement of the output pulses called a spatially and temporally resolved intensity and phase evaluation device: full information from a single hologram. It yields the complete electric field over space and time from multiple digital holograms, simultaneously recorded at different frequencies on a single camera frame. Using femtosecond pulses from a Ti:sapphire laser, we measured the first few linearly polarized modes (${\mathrm{LP}}_{01}$, ${\mathrm{LP}}_{11}$, ${\mathrm{LP}}_{02}$, and ${\mathrm{LP}}_{21}$) inside several few-mode fibers. We also generate movies displaying the measured spatial, temporal, and spectral field features.

Multiple pulsing occurs in most ultrashort-pulse laser systems when pumped at excessively high powers, and small fluctuations in pump power in certain regimes can cause unusual variations in the temporal separations of sub-pulses. Unfortunately, the ability of modern intensity-and-phase pulse measurement techniques to measure such unstable multi-pulsing has not been studied. Here we report calculations and simulations finding that allowing variations in just the relative phase of a satellite pulse causes the second pulse to completely disappear from a spectral interferometry for direct electric field reconstruction (SPIDER) measurement. We find that, although neither frequency-resolved optical gating (FROG) nor autocorrelation can determine the precise properties of satellite pulses due to the presence of instability, they always succeed in, at least, seeing the satellite pulses. Also, additional post-processing of the measured FROG trace can determine the correct approximate relative height of the satellite pulse and definitively indicate the presence of unstable multiple-pulsing.

Quantitative oblique back-illumination microscopy (qOBM) is an emerging label-free optical imaging technology that enables 3D, tomographic quantitative phase imaging (QPI) with epi-illumination in thick scattering samples. In this work, we present a robust optimization of a flexible, fiber-optic-based qOBM system. Our approach enables in silico optimization of the phase signal-to-noise-ratio over a wide parameter space and obviates the need for tedious experimental optimization which could easily miss optimal conditions. Experimental validations of the simulations are also presented and sensitivity limits for the probe are assessed. The optimized probe is light-weight (∼40g) and compact (8mm in diameter) and achieves a 2µm lateral resolution, 6µm axial resolution, and a 300µm field of view, with near video-rate operation (10Hz, limited by the camera). The phase sensitivity is <20nm for a single qOBM acquisition (at 10Hz) and a lower limit of ∼3 nm via multi-frame averaging. Finally, to demonstrate the utility of the optimized probe, we image (1) thick, fixed rat brain samples from a 9L gliosarcoma tumor model and (2) freshly excised human brain tissues from neurosurgery. Acquired qOBM images using the flexible fiber-optic probe are in excellent agreement with those from a free-space qOBM system (both in-situ), as well as with gold-standard histopathology slices (after tissue processing).