Low-coherence Quantitative Phase Microscopy Research Articles

High space–time bandwidth product imaging in low coherence quantitative phase microscopy

Current low coherence quantitative phase microscopy (LC-QPM) systems suffer from either reduced field of view (FoV) or reduced temporal resolution due to the short temporal coherence (TC) length of the light source. Here, we propose a hybrid, experimental and numerical approach to address this core problem associated with LC-QPM. We demonstrate high spatial resolution and high phase sensitivity in LC-QPM at high temporal resolution. High space–time bandwidth product is achieved by employing incoherent light source for sample illumination in QPM to increase the spatial resolution and single-shot Hilbert spiral transform (HST) based phase recovery algorithm to enhance the temporal resolution without sacrificing spatial resolution during the reconstruction steps. The high spatial phase sensitivity comes by default due to the use of incoherent light source in QPM which has low temporal coherence length and does not generate speckle noise and coherent noise. The spatial resolution achieved by the HST is slightly inferior to the temporal phase-shifting (TPS) method when tested on a specimen but surpasses that of the single-shot Fourier transform (FT) based phase recovery method. Contrary to HST method, FT method requires high density fringes for lossless phase recovery, which is difficult to achieve in LC-QPM over entire FoV. Consequently, integration of HST algorithm with LC-QPM system makes an attractive route. Here, we demonstrate scalable FoV and resolution in single-shot LC-QPM and experimentally corroborate it on a test object and on both live and fixed biological specimen such as MEF, U2OS and human red blood cells (RBCs). LC-QPM system with HST reconstruction offer high-speed single-shot QPM imaging at high phase sensitivity and high spatial resolution enabling us to study sub-cellular dynamic inside U2OS for extended duration (3 h) and observe high-speed (50 fps) dynamics of human RBCs. The experimental results validate the effectiveness of the present approach and will open new avenues in the domain of biomedical imaging in the future.

Spatial-domain low-coherence quantitative phase microscopy to improve the cytological diagnosis of pancreatic cancer

Use of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytology to detect pancreatic cancer is limited, with a high false negative rate mainly due to the relatively fewer number of completely...

Quantitative Phase-Contrast Imaging for Distinction between Different States of Human Breast Cancer Cells

We present label-free phase imaging to differentiate human breast cancer cells for breast cancer diagnosis. Low-coherent quantitative phase microscopy (QPM) is exploited to provide speckle-free interference images of live cells adhered to a plate, from which the phase information of the individual cells can be extracted. The phase measurement performance of QPM is validated by the phase-derived optical thickness of a sub-wavelength featured sample, consistent with the results when using conventional atomic force microscopy (AFM). The phase sensitivity is recorded as ~44 mrad, corresponding to ~2.3 nm depth fluctuation during measurement. Phase-contrast imaging is performed with three breast epithelial cell lines: non-tumorous (MCF-10A), weakly malignant (MCF-7), and highly malignant (MDA-MB-231), which mimic an in vivo environments in development of human breast cancer. Our results indicate that MCF-10A and MDA-MB-231 exhibited significantly lower phase-shifts than those of MCF-7, but no difference in phase was observed between MCF-10A and MDA-MB-231. We expect that the use of the QPM approach would be beneficial for clinicians to quantitatively evaluate the breast cancer states from the unmodified specimen of breast biopsy.

Low coherence quantitative phase microscopy with machine learning model and Raman spectroscopy for the study of breast cancer cells and their classification.

Early-stage detection of breast cancer is the primary requirement in modern healthcare as it is the most common cancer among women worldwide. Histopathology is the most widely preferred method for the diagnosis of breast cancer, but it requires long processing time and involves qualitative assessment of cancer by a trained person/doctor. Here, we present an alternate technique based on white light interference microscopy (WLIM) and Raman spectroscopy, which has the capability to differentiate between cancerous and normal breast tissue. WLIM provides quantitative phase information about the biological tissues/cells, whereas Raman spectroscopy can detect changes in their molecular structure and chemical composition during cancer growth. Further, both the techniques can be implemented very quickly without staining the sample. The present technique is employed to perform ex vivo study on a total of 80 normal and cancerous tissue samples collected from 16 different patients. A generalized machine learning model is developed for the classification of normal and cancerous tissues, which is based on texture features obtained from phase maps with an accuracy of 90.6%. The correlation of outcomes from these two techniques can open a new avenue for fast and accurate detection of cancer without any trained personnel.

Dynamic changes in the cell membrane on three dimensional low coherent quantitative phase microscopy (3D LC-QPM) after treatment with the near infrared photoimmunotherapy.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer therapy that relies on the binding of a near-infrared antibody photoabsorber conjugate (APC) to a cancer cell. Subsequent exposure to NIR light selectively induces rapid necrotic cell death on target-expressing cells with minimal off-target effects. When treated with NIR-PIT, targeted cells become swollen, develop blebs and burst within minutes of light exposure. Detailed spatial and temporal morphological changes of the cellular membrane of targeted cells treated with NIR-PIT have not been fully explored with state-of-the-art microscopic methods. In this study, we investigated the morphologic and kinetic effects of PIT on two types of cells, a spindle-shaped 3T3/Her cell and a spheric-shaped MDA-MB468 cell, after NIR-PIT using three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM). Adhesive cells treated with NIR-PIT demonstrated region-specific cell membrane rupture occurring first on the distal free edge of the cell near the site of adhesion, in a process that was independent of cell shape. The results show that the peripheral portions of the cell membrane near the site of adhesion are particularly vulnerable to the effects of NIR-PIT, likely because these sites exhibit higher baseline surface tension.

Alterations of filopodia by near infrared photoimmunotherapy: evaluation with 3D low-coherent quantitative phase microscopy.

Filopodia are highly organized cellular membrane structures that facilitate intercellular communication. Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that causes necrotic cell death. Three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM) is based on a newly established low-coherent interference microscope designed to obtain serial topographic images of the cellular membrane. Herein, we report rapid involution of filopodia after NIR-PIT using 3D LC-QPM. For 3T3/HER2 cells, the number of filopodia decreased immediately after treatment with significant differences. Volume and relative height of 3T3/HER2 cells increased immediately after NIR light exposure, but significant differences were not observed. Thus, disappearance of filopodia, evaluated by 3D LC-QPM, is an early indicator of cell membrane damage after NIR-PIT.

Fourier phase in Fourier-domain optical coherence tomography

Phase of an electromagnetic wave propagating through a sample-of-interest is well understood in the context of quantitative phase imaging in transmission-mode microscopy. In the past decade, Fourier-domain optical coherence tomography has been used to extend quantitative phase imaging to the reflection-mode. Unlike transmission-mode electromagnetic phase, however, the origin and characteristics of reflection-mode Fourier phase are poorly understood, especially in samples with a slowly varying refractive index. In this paper, the general theory of Fourier phase from first principles is presented, and it is shown that Fourier phase is a joint estimate of subresolution offset and mean spatial frequency of the coherence-gated sample refractive index. It is also shown that both spectral-domain phase microscopy and depth-resolved spatial-domain low-coherence quantitative phase microscopy are special cases of this general theory. Analytical expressions are provided for both, and simulations are presented to explain and support the theoretical results. These results are further used to show how Fourier phase allows the estimation of an axial mean spatial frequency profile of the sample, along with depth-resolved characterization of localized optical density change and sample heterogeneity. Finally, a Fourier phase-based explanation of Doppler optical coherence tomography is also provided.

Identifying Colonic Dysplasia and Cancer From Rectal Nuclear Nano-morphology Markers in Ulcerative Colitis Patients

Introduction: Current guidelines recommend that ulcerative colitis (UC) pts undergo annual surveillance colonoscopy with random colonic biopsies for detection of colorectal dysplasia and cancer. This is a suboptimal invasive technique for detecting all colorectal dysplasia. Less invasive surveillance measures that would stratify pts based on risk of developing dysplasia/cancer are needed. We hypothesize, based on the premise of cancer field effect, that markers from rectal biopsies can predict the presence of dysplastic/neoplastic lesions in the colon. This pilot study aims to evaluate whether Spatial-domain Low-coherence Quantitative Phase Microscopy (SL-QPM) derived nuclear nano-morphology markers from flat normal appearing rectal mucosa can detect remote colonic dysplasia/neoplasia in UC pts. Methods: We prospectively recruited UC pts undergoing surveillance colonoscopy with random colon biopsies as per the American Gastroenterological Association surveillance guidelines. Two extra biopsies from flat rectal mucosa were obtained, fixed in formalin and processed per standard histology protocol. According to the final histologic diagnoses from all random biopsies, pts were categorized into a lowrisk group, if pathology showed no dysplasia/cancer, or a high-risk group, if there was any dysplasia, including adenomatous tissue or colon adenocarcinoma. For each pt, SL-QPM analysis was performed on nuclei from 40 to 60 rectal columnar epithelial cells.Figure 1Results: A total of 148 pts were included; 127 low risk and 21 high risk. The high risk group included pts with colorectal cancer (n=1), low-grade dysplasia (n=5), high-grade dysplasia (n=1), indefinite dysplasia (n=2) or sporadic tubular adenoma (n=12). A set of nuclear nano-morphology markers distinguished low risk from high risk UC pts with high statistical significance. Results were not confounded by presence of active inflammation anywhere in the colon (p-value>0.5). The performance characterized by area under receiver operating characteristics curve is 0.82 in distinguishing low from high risk UC pts (86% sensitivity at 61% specificity). Conclusion: Nuclear nano-morphology markers from non-dysplastic rectal tissue identify remote colonic dysplasia in UC pts. This technology is unaffected by the presence of inflammation and stratifies patients into low versus high risk for harboring dysplasia/cancer. This approach could enhance the efficiency of dysplasia surveillance in UC patients and an ongoing study is underway to validate these findings.Figure 2Figure 3

Investigation of nanoscale structural alterations of cell nucleus as an early sign of cancer

BackgroundThe cell and tissue structural properties assessed with a conventional bright-field light microscope play a key role in cancer diagnosis, but they sometimes have limited accuracy in detecting early-stage cancers or predicting future risk of cancer progression for individual patients (i.e., prognosis) if no frank cancer is found. The recent development in optical microscopy techniques now permit the nanoscale structural imaging and quantitative structural analysis of tissue and cells, which offers a new opportunity to investigate the structural properties of cell and tissue below 200 – 250 nm as an early sign of carcinogenesis, prior to the presence of microscale morphological abnormalities. Identification of nanoscale structural signatures is significant for earlier and more accurate cancer detection and prognosis.ResultsOur group has recently developed two simple spectral-domain optical microscopy techniques for assessing 3D nanoscale structural alterations – spectral-encoding of spatial frequency microscopy and spatial-domain low-coherence quantitative phase microscopy. These two techniques use the scattered light from biological cells and tissue and share a common experimental approach of assessing the Fourier space by various wavelengths to quantify the 3D structural information of the scattering object at the nanoscale sensitivity with a simple reflectance-mode light microscopy setup without the need for high-NA optics. This review paper discusses the physical principles and validation of these two techniques to interrogate nanoscale structural properties, as well as the use of these methods to probe nanoscale nuclear architectural alterations during carcinogenesis in cancer cell lines and well-annotated human tissue during carcinogenesis.ConclusionsThe analysis of nanoscale structural characteristics has shown promise in detecting cancer before the microscopically visible changes become evident and proof-of-concept studies have shown its feasibility as an earlier or more sensitive marker for cancer detection or diagnosis. Further biophysical investigation of specific 3D nanoscale structural characteristics in carcinogenesis, especially with well-annotated human cells and tissue, is much needed in cancer research.

Investigation of depth-resolved nanoscale structural changes in regulated cell proliferation and chromatin decondensation

We present depth-resolved spatial-domain low-coherence quantitative phase microscopy, a simple approach that utilizes coherence gating to construct a depth-resolved structural feature vector quantifying sub-resolution axial structural changes at different optical depths within the sample. We show that this feature vector is independent of sample thickness variation, and identifies nanoscale structural changes in clinically prepared samples. We present numerical simulations and experimental validation to demonstrate the feasibility of the approach. We also perform experiments using unstained cells to investigate the nanoscale structural changes in regulated cell proliferation through cell cycle and chromatin decondensation induced by histone acetylation.

Full-field Hilbert phase microscopy using nearly common-path low coherence off-axis interferometry for quantitative imaging of biological cells

We demonstrate single shot low coherence quantitative Hilbert phase microscopy (HPM) for the reconstruction of the two dimensional (2D) phase map of biological cells. The system is based on a compact and nearly common-path high magnification Mirau-interferometric objective lens. The spatial carrier frequency of the interference fringes was increased by means of introducing tilt in one of the arms of the interferometer, thus making the system off-axis. The system is user friendly as the interference fringes and imaging of objects with high lateral and axial resolution can be obtained quickly using a low cost commercially available microscope. Experimental results for the 2D phase map of polystyrene spheres and human red blood cells (RBCs) are presented. Hilbert transform fringe analysis was used for reconstructing the phase map and refractive index (RI) of the objects. For dynamic substances which change rapidly, single shot low coherence interferometric microscopy is an important method for obtaining the phase. Experimental results with increased field-of-view and large tilt angle are also presented. It is well known that on increasing the tilt angle for improved spatial phase sampling the object remains focused in only a small area even though the field-of-view is large. This limitation was overcome by means of vertical scanning low coherence interferometry. Due to the low coherence properties of the light source the interference occurs only at the desired location of the object, i.e., where the object is sharply focused. The object was vertically scanned and the single shot interferograms were recorded for every scan and analyzed by Hilbert transform. In this way a large area of the sample can be imaged quantitatively.

Nuclear nano-morphology markers of histologically normal cells detect the “field effect” of breast cancer

Accurate detection of breast malignancy from histologically normal cells ("field effect") has significant clinical implications in a broad base of breast cancer management, such as high-risk lesion management, personalized risk assessment, breast tumor recurrence, and tumor margin management. More accurate and clinically applicable tools to detect markers characteristic of breast cancer "field effect" that are able to guide the clinical management are urgently needed. We have recently developed a novel optical microscope, spatial-domain low-coherence quantitative phase microscopy, which extracts the nanoscale structural characteristics of cell nuclei (i.e., nuclear nano-morphology markers), using standard histology slides. In this proof-of-concept study, we present the use of these highly sensitive nuclear nano-morphology markers to identify breast malignancy from histologically normal cells. We investigated the nano-morphology markers from 154 patients with a broad spectrum of breast pathology entities, including normal breast tissue, non-proliferative benign lesions, proliferative lesions (without and with atypia), "malignant-adjacent" normal tissue, and invasive carcinoma. Our results show that the nuclear nano-morphology markers of "malignant-adjacent" normal tissue can detect the presence of invasive breast carcinoma with high accuracy and do not reflect normal aging. Further, we found that a progressive change in nuclear nano-morphology markers that parallel breast cancer risk, suggesting its potential use for risk stratification. These novel nano-morphology markers that detect breast cancerous changes from nanoscale structural characteristics of histologically normal cells could potentially benefit the diagnosis, risk assessment, prognosis, prevention, and treatment of breast cancer.

Investigation of nuclear nano-morphology marker as a biomarker for cancer risk assessment using a mouse model

The development of accurate and clinically applicable tools to assess cancer risk is essential to define candidates to undergo screening for early-stage cancers at a curable stage or provide a novel method to monitor chemoprevention treatments. With the use of our recently developed optical technology--spatial-domain low-coherence quantitative phase microscopy (SL-QPM), we have derived a novel optical biomarker characterized by structure-derived optical path length (OPL) properties from the cell nucleus on the standard histology and cytology specimens, which quantifies the nano-structural alterations within the cell nucleus at the nanoscale sensitivity, referred to as nano-morphology marker. The aim of this study is to evaluate the feasibility of the nuclear nano-morphology marker from histologically normal cells, extracted directly from the standard histology specimens, to detect early-stage carcinogenesis, assess cancer risk, and monitor the effect of chemopreventive treatment. We used a well-established mouse model of spontaneous carcinogenesis--Apc(Min) mice, which develop multiple intestinal adenomas (Min) due to a germline mutation in the adenomatous polyposis coli (Apc) gene. We found that the nuclear nano-morphology marker quantified by OPL detects the development of carcinogenesis from histologically normal intestinal epithelial cells, even at an early pre-adenomatous stage (six weeks). It also exhibits a good temporal correlation with the small intestine that parallels the development of carcinogenesis and cancer risk. To further assess its ability to monitor the efficacy of chemopreventive agents, we used an established chemopreventive agent, sulindac. The nuclear nano-morphology marker is reversed toward normal after a prolonged treatment. Therefore, our proof-of-concept study establishes the feasibility of the SL-QPM derived nuclear nano-morphology marker OPL as a promising, simple and clinically applicable biomarker for cancer risk assessment and evaluation of chemopreventive treatment.

Use of optical biomarkers from nondysplastic metaplastic cells on the detection of high-grade dysplasia and adenocarcinoma from Barrett’s esophagus.

14 Background: Patients with Barrett’s esophagus (BE), defined as presence of intestinal metaplasia (IM) in the esophagus, require surveillance due to an increased risk of developing esophageal adenocarcinoma (EAC). The biggest challenge in the current surveillance methodology (i.e., Seattle protocol) is random sampling with the inability to identify those patients with non-dysplastic BE on surveillance who have occult high grade dysplasia (HGD) or will eventually progress to HGD or EAC. We propose a novel approach based on the concept of field effect to detect HGD or EAC through the analysis of non-dysplastic IM, thus identifying a high risk BE population. Our group uses a unique microscope – spatial-domain low-coherence quantitative phase microscopy (SL-QPM) to detect changes in nuclear structure as small as 0.9 nm, a scale 1000 times smaller than what conventional microscopy detects. We hypothesize that the SL-QPM-derived optical biomarkers of non-dysplastic IM would distinguish BE patients with EAC/HGD from those without neoplasia. Methods: We performed a retrospective study of 60 BE patients who underwent Seattle protocol biopsies: 33 BE patients with IM only and 27 BE patients with HGD or EAC (21 HGD, 6 EAC). H&E stained slides with non-dysplastic IM on review by an expert pathologist were used. The distance between the selected IM biopsy and HGD/EAC was 1 to 4 cm. Forty to 60 columnar cells from each case were analyzed. Results: We identified three optical biomarkers (nuclear optical path length, intra-nuclear uniformity, entropy) that can distinguish non-dysplastic BE from patients with HGD and EAC with statistical significance (P < 0.01). A prediction model combining all three optical biomarkers can distinguish BE patients with HGD/EAC from those with IM only at 89% sensitivity and 76% specificity (accuracy = 0.87). Conclusions: The accurate assessment of nanoscale optical biomarkers by SL-QPM is a promising approach for detecting dysplastic/neoplastic Barrett’s epithelium from non-dysplastic IM. This approach could potentially simplify BE surveillance by identifying a subset of high-risk BE patients that warrant intensive surveillance.

Spatial-Domain Low-Coherence Quantitative Phase Microscopy to Improve the Cytological Diagnosis of Pancreatic Cancer

Spatial-Domain Low-Coherence Quantitative Phase Microscopy to Improve the Cytological Diagnosis of Pancreatic Cancer

Label-free imaging of intracellular motility by low-coherent quantitative phase microscopy

The subject study demonstrates the imaging of cell activity by quantitatively assessing the motion of intracellular organelles and cell plasma membranes without any contrast agent. The low-coherent interferometric technique and phase-referenced phase shifting technique were integrated to reveal the depth-resolved distribution of intracellular motility. The transversal and vertical spatial resolutions were 0.56 μm and 0.93 μm, respectively, and the mechanical stability of the system was 1.2 nm. The motility of the cell was assessed by mean squared displacement (MSD) and we have compensated for the MSD by applying statistical noise analysis. Thus we show the significant change of intracellular motility after paraformaldehyde treatment in non-labeled cells.

Correction of stain variations in nuclear refractive index of clinical histology specimens

For any technique to be adopted into a clinical setting, it is imperative that it seamlessly integrates with well-established clinical diagnostic workflow. We recently developed an optical microscopy technique-spatial-domain low-coherence quantitative phase microscopy (SL-QPM) that can extract the refractive index of the cell nucleus from the standard histology specimens on glass slides prepared via standard clinical protocols. This technique has shown great potential in detecting cancer with a better sensitivity than conventional pathology. A major hurdle in the clinical translation of this technique is the intrinsic variation among staining agents used in histology specimens, which limits the accuracy of refractive index measurements of clinical samples. In this paper, we present a simple and easily generalizable method to remove the effect of variations in staining levels on nuclear refractive index obtained with SL-QPM. We illustrate the efficacy of our correction method by applying it to variously stained histology samples from animal model and clinical specimens.

Spatial-domain low-coherence quantitative phase microscopy for cancer diagnosis

A microscopy technique, spatial-domain low-coherence quantitative phase microscopy (SL-QPM) is proposed for speckle-free, quantitative phase imaging of subcellular structures with subnanometer sensitivity. We quantified, for the first time to our knowledge, the refractive index of the cell nuclei on original unmodified histology specimens. We demonstrate that the refractive index of cell nucleus is highly sensitive in detecting cancer, especially in histologically normal-appearing cells from cancer patients. Because this technique is sensitive and does not require special sample processing, it can be disseminated to all clinical settings.

Nanoscale nuclear architecture for cancer diagnosis beyond pathology via spatial-domain low-coherence quantitative phase microscopy

Definitive diagnosis of malignancy is often challenging due to limited availability of human cell or tissue samples and morphological similarity with certain benign conditions. Our recently developed novel technology-spatial-domain low-coherence quantitative phase microscopy (SL-QPM)-overcomes the technical difficulties and enables us to obtain quantitative information about cell nuclear architectural characteristics with nanoscale sensitivity. We explore its ability to improve the identification of malignancy, especially in cytopathologically non-cancerous-appearing cells. We perform proof-of-concept experiments with an animal model of colorectal carcinogenesis-APC(Min) mouse model and human cytology specimens of colorectal cancer. We show the ability of in situ nanoscale nuclear architectural characteristics in identifying cancerous cells, especially in those labeled as "indeterminate or normal" by expert cytopathologists. Our approach is based on the quantitative analysis of the cell nucleus on the original cytology slides without additional processing, which can be readily applied in a conventional clinical setting. Our simple and practical optical microscopy technique may lead to the development of novel methods for early detection of cancer.