Holographic virtual staining of individual biological cells.

Many medical and biological protocols for analyzing individual biological cells involve morphological evaluation based on cell staining, designed to enhance imaging contrast and enable clinicians and biologists to differentiate between various cell organelles. However, cell staining is not always allowed in certain medical procedures. In other cases, staining may be time-consuming or expensive to implement. Staining protocols may be operator-sensitive, and hence may lead to varying analytical results, as well as cause artificial imaging artifacts or false heterogeneity. We present a deep-learning approach, called HoloStain, which converts images of isolated biological cells acquired without staining by holographic microscopy to their virtually stained images. We demonstrate this approach for human sperm cells, as there is a well-established protocol and global standardization for characterizing the morphology of stained human sperm cells for fertility evaluation, but, on the other hand, staining might be cytotoxic and thus is not allowed during human in vitro fertilization (IVF). After a training process, the deep neural network can take images of unseen sperm cells retrieved from holograms acquired without staining and convert them to their stainlike images. We obtained a fivefold recall improvement in the analysis results, demonstrating the advantage of using virtual staining for sperm cell analysis. With the introduction of simple holographic imaging methods in clinical settings, the proposed method has a great potential to become a common practice in human IVF procedures, as well as to significantly simplify and radically change other cell analyses and techniques such as imaging flow cytometry.

Optimal spatial bandwidth capacity in multiplexed off-axis holography for rapid quantitative phase reconstruction and visualization: erratum.

We correct a typo that repeated itself in several equations. Our previous results and conclusions are unchanged.

Single-exposure full-field multi-depth imaging using low-coherence holographic multiplexing.

We present a new interferometric imaging approach that allows for multiple-depth imaging in a single acquisition, using off-axis low-coherence holographic multiplexing. This technique enables sectioned imaging of multiple slices within a thick sample, in a single image acquisition. Each slice has a distinct off-axis interference fringe orientation indicative of its axial location, and the camera acquires the multiplexed hologram containing the different slices at once. We demonstrate the proposed technique for amplitude and phase imaging of optically thick samples.

Simultaneous three-wavelength unwrapping using external digital holographic multiplexing module.

We present an external interferometric setup that is able to simultaneously acquire three wavelengths of the same sample instance without scanning or multiple exposures. This setup projects onto the monochrome digital camera three off-axis holograms with rotated fringe orientations, each from a different wavelength channel, without overlap in the spatial-frequency domain, and thus allows the full reconstruction of the three complex wavefronts from the three wavelength channels. We use this new setup for three-wavelength phase unwrapping, allowing phase imaging of thicker objects than possible with a single wavelength, but without the increased level of noise. We demonstrate the proposed technique for micro-channel profiling and label-free cell imaging.

Flipping interferometry with doubled imaging area.

We present a new external off-axis holographic module that doubles the acquired complex wavefront field of view, based on using both holographic flipping and multiplexing. In contrast to previous designs, this design does not require spatial filtering (no pinhole or lenses) to create the reference beam externally. In addition, the overlap area between the fields of view, as well as the off-axis angle between the sample and reference beams, can be fully controlled. As we demonstrate experimentally, this approach is useful for quantitative phase microscopy of extended stationary and dynamic samples, such as cancer cells during rapid flow and beating cardiomyocytes.

Simultaneous two-wavelength phase unwrapping using an external module for multiplexing off-axis holography.

We present a dual-wavelength external holographic microscopy module for quantitative phase imaging of 3D structures with extended thickness range. This is done by simultaneous acquisition of two off-axis interferograms, each at a different wavelength, and generation of a synthetic wavelength, which is larger than the sample optical thickness, allowing two-wavelength unwrapping. The simultaneous acquisition is carried out by using optical multiplexing of the two interferograms onto the camera, where each of them has orthogonal off-axis interference fringe direction in relation to the other one. We used the system to quantitatively image a 7.96 µm step target and 30.5 µm circular copper pillars.

Six-pack off-axis holography.

We present a new holographic concept, named six-pack holography (6PH), in which we compress six off-axis holograms into a single multiplexed off-axis hologram without loss of magnification or resolution. The multiplexed hologram contains straight off-axis fringes with six different orientations, and can be generated optically or digitally. We show that since the six different complex wavefronts do not overlap in the spatial frequency domain, they can be fully reconstructed. 6PH allows more than 50% improvement in the spatial bandwidth consumption when compared to the best multiplexing method proposed so far. We expect the 6PH concept to be useful for a variety of applications, such as field-of-view multiplexing, wavelength multiplexing, temporal multiplexing, multiplexing for super-resolution imaging, and others.

Flipping interferometry and its application for quantitative phase microscopy in a micro-channel.

We present a portable, off-axis interferometric module for quantitative phase microscopy of live cells, positioned at the exit port of a coherently illuminated inverted microscope. The module creates on the digital camera an interference pattern between the image of the sample and its flipped version. The proposed simplified module is based on a retro-reflector modification in an external Michelson interferometer. The module does not contain any lenses, pinholes, or gratings and its alignment is straightforward. Still, it allows full control of the off-axis angle and does not suffer from ghost images. As experimentally demonstrated, the module is useful for quantitative phase microscopy of live cells rapidly flowing in a micro-channel.

Erythrocyte volumetric measurements in imaging flow cytometry using simultaneous three-wavelength digital holographic microscopy.

We report a cross-talk free simultaneous three-wavelength digital holographic microscopy setup for spectroscopic imaging of biological cells during flow. The feasibility of the proposed measurement technique is demonstrated on erythrocytes, due to their unique morphology and dependency of hemoglobin (Hb) molecule absorption on wavelength. From the spectroscopic quantitative phase profiles of cells acquired during flow in a microfluidic device, we decoupled the refractive index and the physical thickness. We then used our quantitative phase imaging results to dynamically calculate the mean cell volume (MCV), mean corpuscular Hb concentration (MCHC), mean corpuscular Hb content (MCH) and sphericity index.

PhUn-Net: ready-to-use neural network for unwrapping quantitative phase images of biological cells.

We present a deep-learning approach for solving the problem of 2π phase ambiguities in two-dimensional quantitative phase maps of biological cells, using a multi-layer encoder-decoder residual convolutional neural network. We test the trained network, PhUn-Net, on various types of biological cells, captured with various interferometric setups, as well as on simulated phantoms. These tests demonstrate the robustness and generality of the network, even for cells of different morphologies or different illumination conditions than PhUn-Net has been trained on. In this paper, for the first time, we make the trained network publicly available in a global format, such that it can be easily deployed on every platform, to yield fast and robust phase unwrapping, not requiring prior knowledge or complex implementation. By this, we expect our phase unwrapping approach to be widely used, substituting conventional and more time-consuming phase unwrapping algorithms.

TOP-GAN: Stain-free cancer cell classification using deep learning with a small training set.

We propose a new deep learning approach for medical imaging that copes with the problem of a small training set, the main bottleneck of deep learning, and apply it for classification of healthy and cancer cell lines acquired by quantitative phase imaging. The proposed method, called transferring of pre-trained generative adversarial network (TOP-GAN), is hybridization between transfer learning and generative adversarial networks (GANs). Healthy cells and cancer cells of different metastatic potential have been imaged by low-coherence off-axis holography. After the acquisition, the optical path delay maps of the cells are extracted and directly used as inputs to the networks. In order to cope with the small number of classified images, we use GANs to train a large number of unclassified images from another cell type (sperm cells). After this preliminary training, we change the last layers of the network and design automatic classifiers for the correct cell type (healthy/primary cancer/metastatic cancer) with 90-99% accuracies, although small training sets of down to several images are used. These results are better in comparison to other classic methods that aim at coping with the same problem of a small training set. We believe that our approach makes the combination of holographic microscopy and deep learning networks more accessible to the medical field by enabling a rapid, automatic and accurate classification in stain-free imaging flow cytometry. Furthermore, our approach is expected to be applicable to many other medical image classification tasks, suffering from a small training set.

Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy.

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.

Dynamic measurements of flowing cells labeled by gold nanoparticles using full-field photothermal interferometric imaging.

We present highly dynamic photothermal interferometric phase microscopy for quantitative, selective contrast imaging of live cells during flow. Gold nanoparticles can be biofunctionalized to bind to specific cells, and stimulated for local temperature increase due to plasmon resonance, causing a rapid change of the optical phase. These phase changes can be recorded by interferometric phase microscopy and analyzed to form an image of the binding sites of the nanoparticles in the cells, gaining molecular specificity. Since the nanoparticle excitation frequency might overlap with the sample dynamics frequencies, photothermal phase imaging was performed on stationary or slowly dynamic samples. Furthermore, the computational analysis of the photothermal signals is time consuming. This makes photothermal imaging unsuitable for applications requiring dynamic imaging or real-time analysis, such as analyzing and sorting cells during fast flow. To overcome these drawbacks, we utilized an external interferometric module and developed new algorithms, based on discrete Fourier transform variants, enabling fast analysis of photothermal signals in highly dynamic live cells. Due to the self-interference module, the cells are imaged with and without excitation in video-rate, effectively increasing signal-to-noise ratio. Our approach holds potential for using photothermal cell imaging and depletion in flow cytometry.

Linear and nonlinear optical waveguiding in bio-inspired peptide nanotubes.

Unique linear and nonlinear optical properties of bioinspired peptide nanostructures such as wideband transparency and high second-order nonlinear optical response, combined with elongated tubular shape of variable size and rapid self-assembly fabrication process, make them promising for diverse bio-nano-photonic applications. This new generation of nanomaterials of biological origin possess physical properties similar to those of biological structures. Here, we focus on new specific functionality of ultrashort peptide nanotubes to guide light at fundamental and second-harmonic generation (SHG) frequency in horizontal and vertical peptide nanotubes configurations. Conducted simulations and experimental data show that these self-assembled linear and nonlinear optical bio-waveguides provide strong optical power confinement factor, demonstrate pronounced directionality of SHG and high conversion efficiency of SHG ∼10(-5). Our study gives new insight on physics of light propagation in nanostructures of biological origin and opens the avenue towards new and unexpected applications of these waveguiding effects in bio-nanomaterials both for biomedical nonlinear microscopy imaging recognition and development of novel integrated nanophotonic devices.

Quantitative study of optical and mechanical bone status using multispectral photoacoustics.

Osteoporosis is a major public health problem worldwide. Here, we present a quantitative multispectral photoacoustic method for the evaluation of bone pathologies which has significant advantages over pure ultrasonic or pure optical methods as it provides both molecular information and bone mechanical status. This is enabled via a simultaneous measurement of the bone's optical properties as well as the speed of sound and ultrasonic attenuation in the bone. To test the method's quantitative predictions, a combined ultrasonic and photoacoustic system was developed. Excitation was performed optically via a portable triple laser-diode system and acoustically via a single element transducer. Additional dual transducers were used for detecting the acoustic waves that were generated by the two modalities. Both temporal and spectral parameters were compared between different excitation wavelengths and measurement modalities. Short photoacoustic excitation wavelengths allowed sensing of the cortical layer while longer wavelengths produced results which were compatible with the quantitative ultrasound measurements.

Detection and controlled depletion of cancer cells using photothermal phase microscopy.

We present a dual-modality technique based on wide-field photothermal (PT) interferometric phase imaging and simultaneous PT ablation to selectively deplete specific cell populations labelled by plasmonic nanoparticles. This combined technique utilizes the plasmonic reaction of gold nanoparticles under optical excitation to produce PT imaging contrast by inducing local phase changes when the excitation power is weak, or ablation of selected cells when increasing the excitation power. Controlling the entire process is carried out by dynamic quantitative phase imaging of all cells (labelled and unlabelled). We demonstrate our ability to detect and specifically ablate in vitro cancer cells over-expressing epidermal growth factor receptors (EGFRs), labelled with plasmonic nanoparticles, in the presence of either EGFR under-expressing cancer cells or white blood cells. The latter demonstration establishes an initial model for depletion of circulating tumour cells in blood. The proposed system is able to image in wide field the label-free quantitative phase profile together with the PT phase profile of the sample, and provides the ability of both detection and selective cell ablation in a controlled environment. Quantitative phase imaging with molecular specificity and specific cell depletion. (a) Label-free quantitative phase profiles of mixed population of EGFR(+) /EGFR(-) cancer cells. (b) When weak modulated PT excitation is applied, selective phase contrast is generated in the modulation frequency only for the EGFR(+) cancer cells labelled with plasmonic nanoparticles. (c) When stronger modulated PT excitation is applied, selective ablation of the EGFR(+) cancer cells labelled with plasmonic nanoparticles occurs. White scalebars represent 10 µm upon sample.

Wide-field interferometric phase microscopy with molecular specificity using plasmonic nanoparticles.

We present a method for adding molecular specificity to wide-field interferometric phase microscopy (IPM) by recording the phase signatures of gold nanoparticles (AuNPs) labeling targets of interest in biological cells. The AuNPs are excited by time-modulated light at a wavelength corresponding to their absorption spectral peak, evoking a photothermal (PT) effect due to their plasmonic resonance. This effect induces a local temperature rise, resulting in local refractive index and phase changes that can be detected optically. Using a wide-field interferometric phase microscope, we acquired an image sequence of the AuNP sample phase profile without requiring lateral scanning, and analyzed the time-dependent profile of the entire field of view using a Fourier analysis, creating a map of the locations of AuNPs in the sample. The system can image a wide-field PT phase signal from a cluster containing down to 16 isolated AuNPs. AuNPs are then conjugated to epidermal growth factor receptor (EGFR) antibodies and inserted to an EGFR-overexpressing cancer cell culture, which is imaged using IPM and verified by confocal microscopy. To the best of our knowledge, this is the first time wide-field interferometric PT imaging is performed at the subcellular level without the need for total internal reflection effects or scanning.