Quantitative Phase Microscopy System Research Articles

Single-shot quantitative phase microscopy: a multi-functional tool for cell analysis.

This study showcases the multifunctionality of a single-shot quantitative phase microscopy (QPM) system for comprehensive cell analysis. The system captures four high-contrast images in one shot, enabling tasks like cell segmentation, measuring cell confluence, and estimating cell mass. We demonstrate the usability of the QPM system in routine biological workflows, showing how its integration with computational algorithms enables automated, precise analysis, achieving accuracy scores between 85% and 97% across samples with varying cell densities, even those with low signal-to-noise ratios. This cost-effective tool operates under low-intensity light and resists vibrations, making it highly versatile for researchers in both optical and biological fields.

Dynamic quantitative phase microscopy: a single-shot approach using geometric phase interferometry

There is a significant gap in cost-effective quantitative phase microscopy (QPM) systems for studying dynamic cellular processes while maintaining accuracy for long-term cellular monitoring. Current QPM systems often rely on complex and expensive voltage-controllable components like Spatial Light Modulators or two-beam interferometry. To address this, we introduce a QPM system optimized for time-varying phase samples using azobenzene liquid crystal as a Zernike filter with a polarization-sensing camera. This system operates without input voltage or moving components, reducing complexity and cost. Optimized for gentle illumination to minimize phototoxicity, it achieves a 1 Hz frame rate for prolonged monitoring. The system demonstrated accuracy with a maximum standard deviation of ±42 nm and low noise fluctuations of ±2.5 nm. Designed for simplicity and single-shot operations, our QPM system is efficient, robust, and precisely calibrated for reliable measurements. Using inexpensive optical components, it offers an economical solution for long-term, noninvasive biological monitoring and research applications.

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By adopting the polarization phase shifting and space division multiplexing in digital holographic microscopy, a reflectance quantitative phase microscopy system capable of dynamic quantitative phase imaging and depth‐resolved imaging is developed. The compact design of on‐axis near common‐path Fizeau polarization interferometry offers a single‐shot microscopy system with robust phase stability in comparison to other techniques. The proposed system has the advantages of label‐free, singleshot, and depth‐selective imaging features, which will be a prospective tool in the quantified investigations in medical diagnosis and in vivo imaging in optical phase microscopy.Further details can be found in the article by Hanzi Liu, Xiaoyan Wu, Guodong Liu, Hongliang Ren, Vinu R. V., Ziyang Chen, and Jixiong Pu (e202100400)image

Label‐free single‐shot imaging with on‐axis phase‐shifting holographic reflectance quantitative phase microscopy

Quantitative phase microscopy (QPM) has been emerged as an indispensable diagnostic and characterization tool in biomedical imaging with its characteristic nature of label-free, noninvasive, and real time imaging modality. The integration of holography to the conventional microscopy opens new advancements in QPM featuring high-resolution and quantitative three-dimensional image reconstruction. However, the holography schemes suffer in space-bandwidth and time-bandwidth issues in the off-axis and phase-shifting configuration, respectively. Here, we introduce an on-axis phase-shifting holography based QPM system with single-shot imaging capability. The technique utilizes the Fizeau interferometry scheme in combination with polarization phase-shifting and space-division multiplexing to achieve the single-shot recording of the multiple phase-shifted holograms. Moreover, the high-speed imaging capability with instantaneous recording of spatially phase shifted holograms offers the flexible utilization of the approach in dynamic quantitative phase imaging with robust phase stability. We experimentally demonstrated the validity of the approach by quantitative phase imaging and depth-resolved imaging of paramecium cells. Furthermore, the technique is applied to the phase imaging and quantitative parameter estimation of red blood cells. This integration of a Fizeau-based phase-shifting scheme to the optical microscopy enables a simple and robust tool for the investigations of engineered and biological specimen with real-time quantitative analysis.

High space-bandwidth in quantitative phase imaging using partially spatially coherent digital holographic microscopy and a deep neural network.

Quantitative phase microscopy (QPM) is a label-free technique that enables monitoring of morphological changes at the subcellular level. The performance of the QPM system in terms of spatial sensitivity and resolution depends on the coherence properties of the light source and the numerical aperture (NA) of objective lenses. Here, we propose high space-bandwidth quantitative phase imaging using partially spatially coherent digital holographic microscopy (PSC-DHM) assisted with a deep neural network. The PSC source synthesized to improve the spatial sensitivity of the reconstructed phase map from the interferometric images. Further, compatible generative adversarial network (GAN) is used and trained with paired low-resolution (LR) and high-resolution (HR) datasets acquired from the PSC-DHM system. The training of the network is performed on two different types of samples, i.e. mostly homogenous human red blood cells (RBC), and on highly heterogeneous macrophages. The performance is evaluated by predicting the HR images from the datasets captured with a low NA lens and compared with the actual HR phase images. An improvement of 9× in the space-bandwidth product is demonstrated for both RBC and macrophages datasets. We believe that the PSC-DHM + GAN approach would be applicable in single-shot label free tissue imaging, disease classification and other high-resolution tomography applications by utilizing the longitudinal spatial coherence properties of the light source.

Highly temporal stable, wavelength-independent, and scalable field-of-view common-path quantitative phase microscope.

.Significance: High temporal stability, wavelength independency, and scalable field of view (FOV) are the primary requirements of a quantitative phase microscopy (QPM) system. The high temporal stability of the system provides accurate measurement of minute membrane fluctuations of the biological cells that can be an indicator of disease diagnosis.Aim: The main aim of this work is to develop a high temporal stable technique that can accurately quantify the cell’s dynamics such as membrane fluctuations of human erythrocytes. Further, the technique should be capable of acquiring scalable FOV and resolution at multiple wavelengths to make it viable for various biological applications.Approach: We developed a single-element nearly common path, wavelength-independent, and scalable resolution/FOV QPM system to obtain temporally stable holograms/interferograms of the biological specimens.Results: With the proposed system, the temporal stability is obtained without using any vibration isolation table. The capability of the proposed system is first demonstrated on USAF resolution chart and polystyrene spheres ( diameter). Further, the system is implemented for single shot, wavelength-independent quantitative phase imaging of human red blood cells (RBCs) with scalable resolution using color CCD camera. The membrane fluctuation of healthy human RBCs is also measured and was found to be around 47 nm.Conclusions: Contrary to its optical counterparts, the present system offers an energy efficient, cost effective, and simple way of generating object and reference beam for the development of common-path QPM. The present system provides the flexibility to the user to acquire multi-wavelength quantitative phase images at scalable FOV and resolution.

Portable quantitative phase microscope for material metrology and biological imaging

Quantitative phase microscopy (QPM) has emerged as an important tool for material metrology and biological imaging. For broader adoption in those applications, we have proposed and demonstrated a new portable off-axis QPM method, which works in both transmission and reflection modes to meet different sample measurement requirements. The temporal and spatial sensitivities of our system, as quantified by optical path-length difference values, are 0.65 nm and 1.04 nm, respectively. To demonstrate its applicability for a wide range of applications, we deployed our system for profiling transistor gold electrode samples, observing red blood cell membrane fluctuations, imaging living cells flowing in a microfluidic chip, etc. Our portable QPM system has a low-cost design and involves a simple and robust phase-retrieval algorithm that we envision will allow for broader deployment at different environmental settings, including in resource-limited sites and integration with other metrology or imaging modalities.

Integration of diffraction phase microscopy and Raman imaging for label-free morpho-molecular assessment of live cells.

Label-free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label-free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho-molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr-path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi-wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label-free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.

Combined confocal Raman and quantitative phase microscopy system for biomedical diagnosis

We have developed a novel multimodal microscopy system that incorporates confocal Raman, confocal reflectance, and quantitative phase microscopy (QPM) into a single imaging entity. Confocal Raman microscopy provides detailed chemical information from the sample, while confocal reflectance and quantitative phase microscopy show detailed morphology. Combining these intrinsic contrast imaging modalities makes it possible to obtain quantitative morphological and chemical information without exogenous staining. For validation and characterization, we have used this multi-modal system to investigate healthy and diseased blood samples. We first show that the thickness of a healthy red blood cell (RBC) shows good correlation with its hemoglobin distribution. Further, in malaria infected RBCs, we successfully image the distribution of hemozoin (malaria pigment) inside the cell. Our observations lead us to propose morphological screening by QPM and subsequent chemical imaging by Raman for investigating blood disorders. This new approach allows monitoring cell development and cell-drug interactions with minimal perturbation of the biological system of interest.

The recognition of biological cells utilizing quantitative phase microscopy system

Single ion bombardment of biological cells using an ion microprobe is a promising technique. However, for microprobe investigations of biological processes at a cellular level the precise recognition of a single cell and particularly of its nucleus is required. Moreover, cells must be examined in their natural state and environment (i.e. without previously being killed, preferentially not fixed nor stained) and, also, the use of ultraviolet light for cells observation should be avoided. Additionally, in order to obtain statistically significant results of irradiation, the possibility of fast automatic recognition of thousands of objects must be provided.Because computer recognition strongly depends on the quality of an image, the optical imaging system is of crucial importance. For this purpose one of the best solutions could be the Quantitative Phase microscopy (QPm) technique. QPm is the recent digital technique of phase contrast microscopy, providing quantitative phase and intensity data obtained from a series of defocused images. The following phase contrast modalities may be generated digitally from the computed QPm phase data with a greater degree of flexibility: Pure phase images (intensity-free), standard phase contrast (e.g. Zernike phase contrast), differential interference contrast (DIC), Hoffman modulation contrast, and simulated darkfield.