Microscopy For Quantitative Phase Imaging Research Articles

Antiproliferative and Morphological Effects of Fenretinide Lipid Nanosystems in Colon Adenocarcinoma Cells

Objective: Colon adenocarcinoma is characterized by the downregulation of the retinoic acid receptor, making natural retinoids such as all-trans retinoic acid, 9-cis retinoic acid and 13-cis retinoic acid effective in treatment and chemoprevention due to their ability to increase RARβ expression. However, major limitations to their use include tolerability and acquired resistance. In this study, we evaluated fenretinide, a semisynthetic derivative of all-trans retinoic acid, in an HT-29 cell line. Fenretinide was evaluated both as a free drug and encapsulated in self-assembling phosphatidylcholine nanosystems with the aim of increasing the aqueous solubility and cell availability of the drug. Methods: Fenretinide was encapsulated in lipid nanosystems obtained in water by the dispersion of an amphiphilic mixture of phospholipids, glyceryl tributyrate and polysorbate 80. The physico-chemical characterization of the nanosystems was carried out by dynamic light scattering and spectrophotometry. The biological activity was evaluated by quantitative phase imaging microscopy, MTT assay, flow cytometry and confocal laser-scanning fluorescence microscopy. Results: Fenretinide in phosphatidylcholine nanosystems was more active than free fenretinide in inhibiting HT-29 cells’ proliferation, as indicated by quantitative phase imaging data. Indeed, encapsulated fenretinide increased duplication time, decreased dry mass and decreased the rate of cell growth more efficiently than fenretinide. Moreover, encapsulated fenretinide effectively decreased the motility of the cells that survived the treatment. Conclusions: The results indicate that the proposed nanosystems can be considered a valuable alternative to natural retinoids in the chemoprevention and treatment of colorectal cancer. This is due to the favorable pharmacologic characteristics of fenretinide in colorectal cancer and the improved drug activity provided by nanoencapsulation.

Comparative analysis of digital holographic microscopy and digital lensless holographic microscopy for quantitative phase imaging

This study provides a detailed comparison of two widely used quantitative phase imaging (QPI) techniques: single-shot off-axis digital holographic microscopy (DHM) and digital lensless holographic microscopy (DLHM). The primary aim is to evaluate and contrast critical aspects of their imaging performance, including spatial phase sensitivity, phase measurement accuracy, and spatial lateral resolution. Employing typical configurations for both DHM and DLHM, the study utilizes a customized phase test target featuring linear phase changes introduced by a specially designed linear density attenuation filter. Ground truth data from an atomic force microscope is incorporated to validate the experimental findings. The comparative analysis reveals that DHM and DLHM exhibit nearly identical spatial phase sensitivity, with DHM demonstrating a minimal 3.2% measurement error compared to DLHM's 4% in height measurement accuracy. Notably, DHM achieves a finer spatial lateral resolution down to 3.1 µm, surpassing DLHM's 5.52 µm. While DHM outperforms DLHM in precision and resolution, the latter offers advantages in terms of portability and cost-effectiveness. These findings provide valuable insights for researchers and practitioners, aiding in the informed selection of QPI methods based on specific application requirements.

Abstract 3759: Impact of individual and combined deregulation of miR-143 and miR-506 on cytokinetic and morphometric parameters in A549 lung cancer cells

Abstract Lung cancer (LC) is among the leading causes of death from malignancies worldwide. miRNAs (miRs), small noncoding RNAs capable of regulating gene expression through the cell’s RNAi mechanism, have emerged as promising molecules for cancer treatment. In our previous work, we identified two miRs, miR-143 and miR-506, whose transient transfection induced apoptosis, cell cycle inhibition, and cyclin-dependent kinase (CDK) 1, 4, and 6 downregulations. Here, we developed stable deregulations of the miRs’ expressions in A549 cells, individually or in combination, to evaluate their effect on LC cell behavior. To establish homogeneous populations of deregulated cells, A549 cells with stable up and downregulated miR-143 and miR-506, alone or in combination, were sorted according to marker expression using fluorescence-activated cell sorting (FACS). miR deregulations were confirmed with quantitative real-time polymerase chain reaction (qRT -PCR) analysis, and the cells were analyzed for changes in cell proliferation through cell cycle analysis using flow cytometry and cell doubling time using Quantitative Phase Imaging (QPI). With the same method, morphometric characteristics were quantified. Finally, we evaluated the motility of the cells using a scratch assay and determined the gene expression of CDKs. Taqman qPCR analysis indicated a >5-fold increase of the respective miR’s basal expression due to the stable transfections. The cell cycle analysis indicated a complex behavior for the individual miRs, while, in contrast, the combined upregulation of the two miRs indicated a G2 inhibition, representing a well-defined behavior in comparison to both the control and downregulation groups. Furthermore, the combined miR upregulation increased the cell doubling time compared to the control and respective downregulations. Similarly, using QPI microscopy, we observed that the miR combination upregulation had higher cellular sphericity, perimeter, and area compared to the control group. The wound healing assay did not indicate statistically significant differences among the different groups, with miR-506 upregulation demonstrating the slowest closure rate, followed by miR -143. In contrast, the differences between the miR-combination and control group were small, although the anti-miR-combination indicated an accelerated closure. In conclusion, the combined upregulation of both miRs inhibited cell proliferation through a G2 arrest, while the deregulation of the individual miRs demonstrated a complex behavior. Thus, the miR-combination’s consistent, promising behavior would indicate potential benefits using the two miRs against LC that merits further evaluation. Citation Format: Archana Shrestha, Behnaz Lahooti, Mahboubeh Madadi, Constantinos M. Mikelis, George Mattheolabakis. Impact of individual and combined deregulation of miR-143 and miR-506 on cytokinetic and morphometric parameters in A549 lung cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3759.

High-speed Fourier ptychographic microscopy for quantitative phase imaging.

Fourier ptychographic microscopy (FPM), as an emerging computational imaging method, has been applied to quantitative phase imaging with resolution bypassing the physical limit of the detection objective. Due to the weak illumination intensity and long image acquisition time, the achieved imaging speed in current FPM methods is still low, making them unsuitable for real-time imaging applications. We propose and demonstrate a high-speed FPM method based on using laser illumination and digital micro-mirror devices for illumination angle scanning. In this new, to the best of our knowledge, FPM method, we realized quantitative phase imaging and intensity imaging at over 42 frames per second (fps) with around 1 µm lateral resolution. The quantitative phase images have revealed membrane height fluctuations of red blood cells with nanometer-scale sensitivity, while the intensity images have resolved subcellular features in stained cancer tissue slices.

Polarization grating based on diffraction phase microscopy for quantitative phase imaging of paramecia.

This study presents a polarization grating based diffraction phase microscopy (PG-DPM) and its application in bio-imaging. Compared with traditional diffraction phase microscopy (DPM) of which the fringe contrast is sample-dependent, the fringe contrast of PG-DPM is adjustable by changing the polarization of the illumination beam. Moreover, PG-DPM has been applied to real-time phase imaging of live paramecia for the first time. The study reveals that paramecium has self-helical forward motion characteristics, or more specifically, 77% clockwise and 23% anti-clockwise rotation when moving forward. We can envisage that PG-DPM will be applied to many different fields.

Single-shot two-channel Fresnel bimirror interferometric microscopy for quantitative phase imaging of biological cell

A simple slight-off-axis, single-shot, and two-channel interferometric microscopy is demonstrated for the quantitative phase imaging of a biological sample, only using a cube beamsplitter and a Fresnel bimirror interferometer. The incident beam is replicated into two parallel beams using a tilted cube beamsplitter, while the transmission beam is the replica and the reflection beam is the mirror-reverted replica. The two replicas encounter different mirrors of the Fresnel bimirror interferometer, and are reflected and brought to interfere with each other at the image plane of a digital camera. Subsequently, two interference channels with a relative π (rad) phase shift in one interferogram are acquired simultaneously using only one digital camera. The proposed method is capable of obtaining two-channel, slight-off-axis interference in a single shot. The use of small amount of ready-made and low-cost optical components renders the system compact, stable, and easy to implement. In addition, two identical mirrors of the Fresnel bimirror interferometer are symmetrically arranged with a very small angle. Their reflected beams vary in direction over a wide space range with the rotation of the Fresnel bimirror interferometer and the interference always occurs. Based on this characteristic, we are able to capture the interferograms over a large space range and obtain the quantitative phase information. This is a suitable method for the quantitative phase imaging of biological samples.

Color-coded LED microscopy for quantitative phase imaging: Implementation and application to sperm motility analysis.

Color-coded light-emitting diode (LED) microscopy (cLEDscope) is a novel computational microscopy technique capable of multi-contrast and quantitative phase imaging of biological specimens using color-multiplexed illumination. Using specially designed LED patterns, it is capable of recording multiple differential phase contrast (DPC) images in a single exposure and employs a computational algorithm to retrieve the phase distribution of the specimens. Herein, we describe the detailed procedures in the cLEDscope implementation for quantitative phase imaging. Several notable features and caveats in the cLEDscope setup and image processing are also outlined. The imaging model is derived for our specific configuration, and the associated phase-retrieval algorithms are presented on the basis of a weak-object transfer function. As an illustrative application of the quantitative cLEDscope, we demonstrate its utility as a sperm-motility analyzer by exploiting its real-time quantitative imaging capability.

Dual-wavelength common-path digital holographic microscopy for quantitative phase imaging of biological cells

Biological cells are usually transparent with a small refractive index gradient. Digital holographic interferometry can be used in the measurement of biological cells. We propose a dual-wavelength common-path digital holographic microscopy for the quantitative phase imaging of biological cells. In the proposed configuration, a parallel glass plate is inserted in the light path to create the lateral shearing, and two lasers with different wavelengths are used as the light source to form the dual-wavelength composite digital hologram. The information of biological cells for different wavelengths is separated and extracted in the Fourier domain of the hologram, and then combined to a shorter wavelength in the measurement process. This method could improve the system’s temporal stability and reduce speckle noises simultaneously. Mouse osteoblastic cells and peony pollens are measured to show the feasibility of this method.

Quadriwave lateral shearing interferometric microscopy with wideband sensitivity enhancement for quantitative phase imaging in real time

Real-time quantitative phase imaging has tremendous potential in investigating live biological specimens in vitro. Here we report on a wideband sensitivity-enhanced interferometric microscopy for quantitative phase imaging in real time by employing two quadriwave lateral shearing interferometers based on randomly encoded hybrid gratings with different lateral shears. Theoretical framework to analyze the measurement sensitivity is firstly proposed, from which the optimal lateral shear pair for sensitivity enhancement is also derived. To accelerate the phase retrieval algorithm for real-time visualization, we develop a fully vectorized path-independent differential leveling phase unwrapping algorithm ready for parallel computing, and the framerate for retrieving the phase from each pair of two 4 mega pixel interferograms is able to reach 47.85 frames per second. Experiment results demonstrate that the wideband sensitivity-enhanced interferometric microscopy is capable of eliminating all the periodical error caused by spectral leaking problem and reducing the temporal standard deviation to the half level compared with phase directly retrieved by the interferogram. Due to its high adaptability, the wideband sensitivity-enhanced interferometric microscopy is promising in retrofitting existing microscopes to quantitative phase microscopes with high measurement precision and real-time visualization.

Dual-wavelength common-path digital holographic microscopy for quantitative phase imaging based on lateral shearing interferometry.

A dual-wavelength common-path digital holographic microscopy based on a single parallel glass plate is presented to achieve quantitative phase imaging, which combines the dual-wavelength technique with lateral shearing interferometry. Two illumination laser beams with different wavelengths (λ1=532 nm and λ2=632.8 nm) are reflected by the front and back surfaces of the parallel glass plate to create the lateral shear and form the digital hologram, and then the hologram is reconstructed to obtain the phase distribution with a synthetic wavelength Λ=3339.8 nm. The experimental configuration is very compact, with the advantages of vibration resistance and measurement range extension. The experimental results of the laser-ablated pit, groove, and staircase specimens show the feasibility of the proposed configuration.

Multispectral quantitative phase imaging of human red blood cells using inexpensive narrowband multicolor LEDs.

We report multispectral phase-shifting interference microscopy for quantitative phase imaging of human red blood cells (RBCs). A wide range of wavelengths are covered by means of using multiple color light emitting diodes (LEDs) with narrow spectral bandwidth ranging from violet to deep red color. The multicolor LED light source was designed and operated sequentially, which works as a multispectral scanning light source. Corresponding to each color LED source, five phase-shifted interferograms were recorded sequentially for the measurement of phase maps, as well as the refractive index of RBCs within the entire visible region. The proposed technique provides information about the effect of wavelengths on the morphology and refractive index of human RBCs. The system does not require expensive multiple color filters or any wavelength scanning mechanism along with broadband light source.

Abstract 5186: Label-free imaging identification of white blood cells using quantitative phase microscopy for negative selection of circulating tumor cells

Abstract Background: Circulating tumor cells (CTCs) are cancer cells which are shed from primary tumors and circulating in the bloodstream, and thought to play a key role in metastasis. Existing CTC capture methods are largely based on surface markers such as cytokeratins. However, invasive tumor cells tend to lose their epithelial markers and change into mesenchymal phenotype by the epithelial mesenchymal transition (EMT). It seems that conventional CTC capture methods miss the mesenchymal CTCs which are supposed to have highly malignant potentials. Because the features of CTCs are not fully understood, it is difficult to retrieve all CTCs by positive selection using CTC-surface markers. Considering the fact that vast majority of nucleated cells circulating in the blood stream are WBCs, eliminating WBCs from circulation is an efficient way to purify CTCs. In this study, we attempted to develop a novel method for the identification of WBCs in the blood using Quantitative Phase Imaging Microscopy (QPM). Methods: We analyzed WBCs and cancer cell lines using QPM that can image optical path-length of living cells with a high resolution of 1nm without staining in non-cytotoxic way. Obtained images, quantitative phase images (QPIs), were analyzed by a computer vision application which automatically identifies an object in digital images based on features extracted from the dataset we had developed by machine learning. Results: We imaged 325 WBCs obtained from healthy donors and 325 cell-line cells (including 5 different cell lines) with QPM. Then, we extracted certain features from the QPIs and used them as training images for machine learning. We employed 5-fold cross validation to create an algorithm to detect WBCs circulating in the blood. The algorithm successfully recognized WBCs among QPIs mixed in with WBCs and cancer cell lines. The ROC AUC value was 0.98, indicating that the algorithm was not random selection. Conclusions: We successfully differentiated WBCs from cancer cell lines based on the features of QPIs. The object recognition method applying to QPIs of the cell is expected as a useful, non-cytotoxic, marker free isolation of CTCs. Citation Format: Yusuke Ozaki, Hidenao Iwai, Hirotoshi Kikuchi, Tomohiro Matsumoto, Shinichiro Miyazaki, Toshiki Kawabata, Yoshihiro Hiramatsu, Manabu Ohta, Kinji Kamiya, Megumi Baba, Kentaro Goto, Toyohiko Yamauchi, Yukio Ueda, Shigetoshi Okazaki, Hiroyuki Konno. Label-free imaging identification of white blood cells using quantitative phase microscopy for negative selection of circulating tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5186. doi:10.1158/1538-7445.AM2015-5186

Vertical focusing and cell ordering in a microchannel via viscoelasticity: Applications for cell monitoring using a digital holographic microscopy

We demonstrate sheath-free vertical concentration and positioning of cells induced by fluid viscoelasticity in a rectangular channel. The high uniformity in depth position and the reduction in cell overlap eliminate out-of-focus blurring and increase detection sensitivity. Additionally, orientational ordering of red blood cells improves the monitoring and counting performance of an optical imaging system. As a proof of concept of vertical focusing and cell ordering, we demonstrate counting and monitoring of red blood cells flowing in a microchannel using phase-contrast holographic microscopy for quantitative phase imaging.

High-speed synthetic aperture microscopy for live cell imaging

We present a high-speed synthetic aperture microscopy for quantitative phase imaging of live biological cells. We measure 361 complex amplitude images of an object with various directions of illumination covering an NA of 0.8 in less than one-thirteenth of a second and then combine the images with a phase-referencing method to create a synthesized phase image. Because of the increased depth selectivity, artifacts from diffraction that are typically present in coherent imaging are significantly suppressed, and lateral resolution of phase imaging is improved. We use the instrument to demonstrate high-quality phase imaging of live cells, both static and dynamic, and thickness measurements of a nanoscale cholesterol helical ribbon.

Transport-of-intensity approach to differential interference contrast (TI-DIC) microscopy for quantitative phase imaging

Differential interference contrast (DIC) microscopy is an inherently qualitative phase-imaging technique. What is obtained is an image with mixed phase-gradient and amplitude information rather than a true linear mapping of actual optical path length (OPL) differences. Here we investigate an approach that combines the transport-of-intensity equation (TIE) with DIC microscopy, thus improving direct visual observation. There is little hardware modification and the computation is noniterative. Numerically solving for the propagation of light in a series of through-focus DIC images allows linear phase information in a single slice to be completely determined and restored from DIC intensity values.