3D Refractive Index Research Articles

DMD and microlens array as a switchable module for illumination angle scanning in optical diffraction tomography.

Optical diffraction tomography (ODT) enables label-free and morphological 3D imaging of biological samples using refractive-index (RI) contrast. To accomplish this, ODT systems typically capture multiple angular-specific scattering measurements, which are used to computationally reconstruct a sample's 3D RI. Standard ODT systems employ scanning mirrors to generate angular illuminations. However, scanning mirrors are limited to illuminating the sample from only one angle at a time. Furthermore, when operated at high speeds, these mirrors may exhibit mechanical instabilities that compromise image quality and measurement speed. Recently, newer ODT systems have been introduced that utilize digital-micromirror devices (DMD), spatial light modulators (SLMs), or LED arrays to achieve switchable angle-scanning with no physically-scanning components. However, these systems associate with power inefficiencies and/or spurious diffraction orders that can also limit imaging performance. In this work, we developed a novel non-interferometric ODT system that utilizes a fully switchable module for angle scanning composed of a DMD and microlens array (MLA). Compared to other switchable ODT systems, this module enables each illumination angle to be generated fully independently from every other illumination angle (i.e., no spurious diffraction orders) while also optimizing the power efficiency based on the required density of illumination angles. We validate the quantitative imaging capability of this system using calibration microspheres. We also demonstrate its capability for imaging multiple-scattering samples by imaging an early-stage zebrafish embryo.

Lens-free on-chip 3D microscopy based on wavelength-scanning Fourier ptychographic diffraction tomography

Lens-free on-chip microscopy is a powerful and promising high-throughput computational microscopy technique due to its unique advantage of creating high-resolution images across the full field-of-view (FOV) of the imaging sensor. Nevertheless, most current lens-free microscopy methods have been designed for imaging only two-dimensional thin samples. Lens-free on-chip tomography (LFOCT) with a uniform resolution across the entire FOV and at a subpixel level remains a critical challenge. In this paper, we demonstrated a new LFOCT technique and associated imaging platform based on wavelength scanning Fourier ptychographic diffraction tomography (wsFPDT). Instead of using angularly-variable illuminations, in wsFPDT, the sample is illuminated by on-axis wavelength-variable illuminations, ranging from 430 to 1200 nm. The corresponding under-sampled diffraction patterns are recorded, and then an iterative ptychographic reconstruction procedure is applied to fill the spectrum of the three-dimensional (3D) scattering potential to recover the sample’s 3D refractive index (RI) distribution. The wavelength-scanning scheme not only eliminates the need for mechanical motion during image acquisition and precise registration of the raw images but secures a quasi-uniform, pixel-super-resolved imaging resolution across the entire imaging FOV. With wsFPDT, we demonstrate the high-throughput, billion-voxel 3D tomographic imaging results with a half-pitch lateral resolution of 775 nm and an axial resolution of 5.43 μm across a large FOV of 29.85 mm2 and an imaging depth of >200 μm. The effectiveness of the proposed method was demonstrated by imaging various types of samples, including micro-polystyrene beads, diatoms, and mouse mononuclear macrophage cells. The unique capability to reveal quantitative morphological properties, such as area, volume, and sphericity index of single cell over large cell populations makes wsFPDT a powerful quantitative and label-free tool for high-throughput biological applications.

GAN-based quantitative oblique back-illumination microscopy enables computationally efficient epi-mode refractive index tomography.

Quantitative oblique back-illumination microscopy (qOBM) is a novel imaging technology that enables epi-mode 3D quantitative phase imaging and refractive index (RI) tomography of thick scattering samples. The technology uses four oblique back illumination images captured at the same focal plane and a fast 2D deconvolution reconstruction algorithm to reconstruct 2D phase cross-sections of thick samples. Alternatively, a through-focus z-stack of oblique back illumination images can be used to recover 3D RI tomograms with improved RI quantitative fidelity at the cost of a more computationally expensive reconstruction algorithm. Here, we report on a generative adversarial network (GAN) assisted approach to reconstruct 3D RI tomograms with qOBM that achieves high fidelity and greatly reduces processing time. The proposed approach achieves high-fidelity 3D RI tomography using differential phase contrast images from three adjacent z-planes. A ∼9-fold improvement in volumetric reconstruction time is achieved. We further show that this technique provides high SNR RI tomograms with high quantitative fidelity, reduces motion artifacts, and generalizes to different tissue types. This work can lead to real-time, high-fidelity RI tomographic imaging for in-vivo pre-clinical and clinical applications.

Single-exposure multi-wavelength optical diffraction tomography based on space-angle dual multiplexing holography.

We present a space-angle dual multiplexing holographic recording system for realizing single-exposure multi-wavelength optical diffraction tomographic (ODT) imaging. This system is achieved by combining the principle of single-exposure multi-wavelength holographic imaging technique based on angle-division multiplexing with the principle of single-exposure ODT imaging technique based on microlens array multi-angle illuminations and space-division multiplexing. Compared with the existing multi-wavelength ODT imaging methods, it enables the holographic recording of all the diffraction tomography information of a measured specimen at multiple illumination wavelengths in a single camera exposure without any scan mechanism. Using our proposed data processing method, the multi-wavelength three-dimensional (3D) refractive index tomograms of a specimen can be eventually reconstructed from single recorded multiplexing hologram. Experimental results of a static polystyrene bead and a living C. elegans worm demonstrate the feasibility of this system.

Label-Free Three-Dimensional Morphological Characterization of Cell Death Using Holographic Tomography.

This study presents a novel label-free approach for characterizing cell death states, eliminating the need for complex molecular labeling that may yield artificial or ambiguous results due to technical limitations in microscope resolution. The proposed holographic tomography technique offers a label-free avenue for capturing precise three-dimensional (3D) refractive index morphologies of cells and directly analyzing cellular parameters like area, height, volume, and nucleus/cytoplasm ratio within the 3D cellular model. We showcase holographic tomography results illustrating various cell death types and elucidate distinctive refractive index correlations with specific cell morphologies complemented by biochemical assays to verify cell death states. These findings hold promise for advancing in situ single cell state identification and diagnosis applications.

Deep-learning based 3D birefringence image generation using 2D multi-view holographic images

Refractive index stands as an inherent characteristic of a material, allowing non-invasive exploration of the three-dimensional (3D) interior of the material. Certain materials with different refractive indices produce a birefringence phenomenon in which incident light is split into two polarization components when it passes through the materials. Representative birefringent materials appear in calcite crystals, liquid crystals (LCs), biological tissues, silk fibers, polymer films, etc. If the internal 3D shape of these materials can be visually expressed through a non-invasive method, it can greatly contribute to the semiconductor, display industry, optical components and devices, and biomedical diagnosis. This paper introduces a novel approach employing deep learning to generate 3D birefringence images using multi-viewed holographic interference images. First, we acquired a set of multi-viewed holographic interference pattern images and a 3D volume image of birefringence directly from a polarizing DTT (dielectric tensor tomography)-based microscope system about each LC droplet sample. The proposed model was trained to generate the 3D volume images of birefringence using the two-dimensional (2D) interference pattern image set. Performance evaluations were conducted against the ground truth images obtained directly from the DTT microscopy. Visualization techniques were applied to describe the refractive index distribution in the generated 3D images of birefringence. The results show the proposed method’s efficiency in generating the 3D refractive index distribution from multi-viewed holographic interference images, presenting a novel data-driven alternative to traditional methods from the DTT devices.

Electrochemical Etching of Nitrogen Ion‐Implanted Gallium Nitride – A Route to 3D Nanoporous Patterning

Dopant‐selective electrochemical etching (ECE) of gallium nitride (GaN) results in well‐defined porous layers with tunable refractive index, which is extremely interesting for integrating photonic components into nitride technology. Herein, the impact of nitrogen implantation with and without subsequent rapid thermal annealing (RTA) on the porosification process of highly n‐doped GaN ([Si] 3 × 1019 cm−3) is investigated. Implantation is expected to compensate the donors of the n‐GaN layer to spatially suppress porosification during ECE. Optical transmission, electrochemical capacitance–voltage, and X‐Ray diffractometry of as‐grown and as‐implanted GaN suggest successful compensation of n‐dopants. Cross‐sectional scanning electron microscopy reveals the presence of mesopores (diameter 2–50 nm) after ECE of the as‐grown n‐GaN. In the case of implanted n‐GaN, it is found that ECE results in macropores (diameter > 50 nm), which can be suppressed by an intermediate RTA step. The implanted and annealed n‐GaN layers solely exhibit mesopores at the top and bottom, while the intermediate region remains unimpaired. Chronoamperometry and gravimetry provide additional insight and confirm the presence of macro‐ and mesopores in samples without and with RTA, respectively. The results demonstrate a successful implementation of etch‐resisting subsurface layers, which are required for 3D refractive index engineering in porous GaN.

Dynamic tracking of onion-like carbon nanoparticles in cancer cells using limited-angle holographic tomography with self-supervised learning

This research presents a novel approach for the dynamic monitoring of onion-like carbon nanoparticles inside colorectal cancer cells. Onion-like carbon nanoparticles are widely used in photothermal cancer therapy, and precise 3D tracking of their distribution is crucial. We proposed a limited-angle digital holographic tomography technique with unsupervised learning to achieve rapid and accurate monitoring. A key innovation is our internal learning neural network. This network addresses the information limitations of limited-angle measurements by directly mapping coordinates to measured data and reconstructing phase information at unmeasured angles without external training data. We validated the network using standard SiO2 microspheres. Subsequently, we reconstructed the 3D refractive index of onion-like carbon nanoparticles within cancer cells at various time points. Morphological parameters of the nanoparticles were quantitatively analyzed to understand their temporal evolution, offering initial insights into the underlying mechanisms. This methodology provides a new perspective for efficiently tracking nanoparticles within cancer cells.

Super-resolution based number-theoretical phase unwrapping method applicable to 2D spiral fringe patterns

Spiral interferometry is a technique that accurately measures changes in 3D structures or refractive indices by converting phase changes into spiral fringe patterns. The spiral fringe patterns consist of phase distributions, which are wrapped from 0 to 2π, requiring phase unwrapping techniques. Phase unwrapping transforms a discontinuous phase image from 0 to 2π into a continuous phase image, which can ultimately be reconstructed into 3D information. The 3D reconstructed information is affected by the unwrapped phase information with noise. Among the phase unwrapping methods, the number-theoretical phase unwrapping method used in 1D structured light is a unique approach that unwraps the phase into a step function. Due to its insensitivity to noise, this method is an effective solution for improving phase unwrapping in noisy environments. However, due to the same characteristics, the resolution inevitably decreases. In this paper, we propose a super-resolution based numerical unwrapping method applicable to two-dimensional spiral patterns. Based on multi-wavelength interpolation, this method not only retains the advantage of the number-theoretical phase unwrapping method, which is insensitive to noise, but also compensates for its disadvantage of resolution degradation. The proposed method uses the basic concept of generating sub-step information by multi-wavelength interpolation and performing super-resolution based on this concept. We perform simulations and experiments to verify the validity. As a result of the verification, it is confirmed that the super-resolution effect of the number-theoretical unwrapping in the 2D spiral fringe patterns can be achieved by the proposed method.

Abstract 2530: Advancing ICK assay: Real-time, label-free imaging of lymphocyte subsets

Abstract Background: Immune Cell Killing (ICK) Assay is pivotal in immunology and clinical research, uncovering the intricate connections between the immune system and diseases. Traditional ICK assays utilizing antibody staining for endpoint data generation pose challenges in capturing dynamic interactions. Moreover, antibody labeling for data quantification may compromise sensitivity, especially with low-affinity antibodies or a limited number of cells. The anticipation of label-free lymphocyte subset classification and real-time effector/target cell interaction analysis in one field of view is expected to provide more valuable insights than traditional ICK assays. Previous studies achieved 70-80% accuracy in image-based lymphocyte subset classification using digital holographic microscopy, light scattering, etc. but the cells’ condition during the cell sorting or sample preparation process may affect the lymphocyte subset status, resulting in real morphological discrepancies. Our approach focuses on directly sorting human PBMCs to enable label-free, dynamic interaction analysis of natural immune system responses in one field of view. Method: In this study, PBMC samples from 6 individuals were separated using standard-density gradient centrifugation and labeled with antibodies. Data were acquired using the Holotomographic Microscope HT-X1 (Tomocube Inc.). We employed Densenet 121 as the deep learning model architecture, considering the trade-off between memory consumption and performance. The cross-entropy loss function was utilized, and the AdamW optimizer was chosen for model parameter optimization. Input data consisted of 3D refractive index (RI) data for individual cells, each of sized (20, 54, 54). Individual cell labeling was performed using staining information. Results: An overall accuracy of 93.75% in Human PBMC subtype cell classification was achieved. In stage 1, an accuracy of 97.56% was demonstrated in classifying [CD14, CD15, Others]. Among the cells classified as "Others" in stage 2, [CD3, CD19, CD16&CD56] displayed an accuracy of 93.5%. In stage 3, classifying [CD4, CD8] within the CD3 subtype achieved an accuracy of 90.2%. This classification is presumably based on pattern recognition of the chromosomal landscape in holotomography images. Conclusion: Our research has successfully demonstrated the feasibility of real-time label-free lymphocyte subset classification. This approach enabled the classification of effector cells as live cells in ICK assay and the analysis of effector-target cell dynamic interactions. Further training in the classification of more detailed subtype cells and various cell death types will provide additional insights. The advancement of this technology is expected to play a pivotal role in disease prevention, treatment, vaccine development, medical research, and academic studies. Citation Format: Sanggeun Oh, Jaephil Do, Hyun-Seok Min, Dongmin Ryu, Wei sun Park. Advancing ICK assay: Real-time, label-free imaging of lymphocyte subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2530.

Alternating projection combined with fast gradient projection (FGP-AP) method for intensity-only measurement optical diffraction tomography in LED array microscopy.

Optical diffraction tomography (ODT) is a powerful label-free measurement tool that can quantitatively image the three-dimensional (3D) refractive index (RI) distribution of samples. However, the inherent "missing cone problem," limited illumination angles, and dependence on intensity-only measurements in a simplified imaging setup can all lead to insufficient information mapping in the Fourier domain, affecting 3D reconstruction results. In this paper, we propose the alternating projection combined with the fast gradient projection (FGP-AP) method to compensate for the above problem, which effectively reconstructs the 3D RI distribution of samples using intensity-only images captured from LED array microscopy. The FGP-AP method employs the alternating projection (AP) algorithm for gradient descent and the fast gradient projection (FGP) algorithm for regularization constraints. This approach is equivalent to incorporating prior knowledge of sample non-negativity and smoothness into the 3D reconstruction process. Simulations demonstrate that the FGP-AP method improves reconstruction quality compared to the original AP method, particularly in the presence of noise. Experimental results, obtained from mouse kidney cells and label-free blood cells, further affirm the superior 3D imaging efficacy of the FGP-AP method.

Optical diffraction tomography for assessing single cell models in angular light scattering.

Angularly resolved light scattering (ALS) has become a useful tool for assessing the size and refractive index of biological scatterers at cellular and organelle length scales. Sizing organelle populations with ALS relies on Mie scattering theory models, which require significant assumptions about the object, including spherical scatterers and a homogeneous medium. These assumptions may incur greater error at the single cell level, where there are fewer scatterers to be averaged over. We investigate the validity of these assumptions using 3D refractive index (RI) tomograms measured via optical diffraction tomography (ODT). We compute the angular scattering on digitally manipulated tomograms with increasingly strong model assumptions, including RI-matched immersion media, homogeneous cytosol, and spherical organelles. We also compare the tomogram-computed angular scattering to experimental measurements of angular scattering from the same cells to ensure that the ODT-based approach accurately models angular scattering. We show that enforced RI-matching with the immersion medium and a homogeneous cytosol significantly affects the angular scattering intensity shape, suggesting that these assumptions can reduce the accuracy of size distribution estimates.

3D Holo-tomographic Mapping of COVID-19 Microclots in Blood to Assess Disease Severity.

The coronavirus disease 2019 (COVID-19) has impacted health globally. Cumulative evidence points to long-term effects of COVID-19 such as cardiovascular and cognitive disorders, diagnosed in patients even after the recovery period. In particular, micrometer-sized blood clots and hyperactivated platelets have been identified as potential indicators of long COVID. Here, we resolve microclot structures in the plasma of patients with different subphenotypes of COVID-19 in a label-free manner, using 3D digital holo-tomographic microscopy (DHTM). Based on 3D refractive index (RI) tomograms, the size, dry mass, and prevalence of microclot composites were quantified and then parametrically differentiated from fibrin-rich microclots and platelet aggregates in the plasma of COVID-19 patients. Importantly, fewer microclots and platelet aggregates were detected in the plasma of healthy controls compared to COVID-19 patients. Our imaging and analysis workflow is built around a commercially available DHT microscope capable of operation in clinical settings with a 2 h time period from sample preparation and data acquisition to results.

Quantitative real-time phase microscopy for extended depth-of-field imaging based on the 3D single-shot differential phase contrast (ssDPC) imaging method.

Optical diffraction tomography (ODT) is a promising label-free imaging method capable of quantitatively measuring the three-dimensional (3D) refractive index distribution of transparent samples. In recent years, partially coherent ODT (PC-ODT) has attracted increasing attention due to its system simplicity and absence of laser speckle noise. Quantitative phase imaging (QPI) technologies represented by Fourier ptychographic microscopy (FPM), differential phase contrast (DPC) imaging and intensity diffraction tomography (IDT) need to collect several or hundreds of intensity images, which usually introduce motion artifacts when shooting fast-moving targets, leading to a decrease in image quality. Hence, a quantitative real-time phase microscopy (qRPM) for extended depth of field (DOF) imaging based on 3D single-shot differential phase contrast (ssDPC) imaging method is proposed in this research study. qRPM incorporates a microlens array (MLA) to simultaneously collect spatial information and angular information. In subsequent optical information processing, a deconvolution method is used to obtain intensity stacks under different illumination angles in a raw light field image. Importing the obtained intensity stack into the 3D DPC imaging model is able to finally obtain the 3D refractive index distribution. The captured four-dimensional light field information enables the reconstruction of 3D information in a single snapshot and extending the DOF of qRPM. The imaging capability of the proposed qRPM system is experimental verified on different samples, achieve single-exposure 3D label-free imaging with an extended DOF for 160 µm which is nearly 30 times higher than the traditional microscope system.

Imaging and Quantitative Analysis on the Etching of Diatom Frustules via Digital Holographic Microscopy.

Frustules, whose length spans from a few micrometers to more than a hundred micrometers, have been the subject of various modifications to improve their physical properties because of their complex porous silica structure. However, three-dimensional measurements of these changes can be challenging because of the complex 3D architecture and limitations of known methods. In this study, we present a new method that applies digital holographic microscopy (DHM) to analyze controlled etched frustules and observe real-time degradation of frustules at the single-cell level. Frustules obtained from Craspedostauros sp. diatoms were etched in 1 N NaOH for 5 min at 25 and 60 °C, respectively, and the frustule's valve was analyzed using DHM. DHM uses a combination of holography and tomography to reconstruct a 3D refractive index image of the frustule. Measurements of the width, volume, and surface area are achieved. Results showed that at 60 °C of etching, a significant difference with the unetched frustule was observed for all measurements but with high fluctuation values. Finally, real-time observation of the degradation of the frustule is observed when immersed in a high concentration of NaOH. This is the first time the real-time etching of the frustule is observed at the single-cell level. This research provides an easy estimation of the 3D measurements of frustules that may provide new fundamental information and applications.

Three-dimensional refractive index microscopy based on the multi-layer propagation model with obliquity factor correction

Multi-layer propagation model-based intensity diffraction tomography (IDT) is a three-dimensional (3D) microscopy imaging technique to reveal the structural information of optically thick or multiple scattering samples. However, the performance of existing multi-layer propagation models deteriorates with increasing illumination angle, which can prevent good 3D refractive index (RI) imaging. To overcome this limitation, we incorporate the obliquity factor (OF) into the multi-layer propagation model. Numerical simulation validates that the forward propagation and the RI reconstruction both can be considerably improved. Then, the 3D RI reconstruction performance is quantitatively and qualitatively validated by optical experiments on polystyrene microspheres and peony pollen grains, respectively. In addition, excellent optical sectioning and high spatial resolution are experimentally demonstrated on various unlabeled fixed and living samples, including an Arabidopsis root, HeLa and HCT-116 cells. Time-lapse 3D imaging of living samples is performed for the first time for multi-layer propagation model-based IDT, demonstrating its potential application to some biological and medical studies.

Multi-harmonic structured illumination-based optical diffraction tomography

Imaging speed and spatial resolution are key factors in optical diffraction tomography (ODT), while they are mutually exclusive in 3D refractive index imaging. This paper presents a multi-harmonic structured illumination-based optical diffraction tomography (MHSI-ODT) to acquire 3D refractive index (RI) maps of transparent samples. MHSI-ODT utilizes a digital micromirror device (DMD) to generate structured illumination containing multiple harmonics. For each structured illumination orientation, four spherical spectral crowns are solved from five phase-shifted holograms, meaning that the acquisition of each spectral crown costs 1.25 raw images. Compared to conventional SI-ODT, which retrieves two spectral crowns from three phase-shifted raw images, MHSI-ODT enhances the imaging speed by 16.7% in 3D RI imaging. Meanwhile, MHSI-ODT exploits both the 1st-order and the 2nd-order harmonics; therefore, it has a better intensity utilization of structured illumination. We demonstrated the performance of MHSI-ODT by rendering the 3D RI distributions of 5 µm polystyrene (PS) microspheres and biological samples.

#264 : 3D Time-Lapse Refractive-Index Imaging of Intact Mouse Embryos for Non-Invasive Evaluation of the Embryonic Development

Background and Aims: Time-lapse technology is an emerging assisted reproductive technology (ART) that enables simultaneous culture and monitoring of embryos in vitro during their development. It provides extra information on key events during embryo development, which can assist embryologists in selecting embryos alongside traditional morphology evaluations. Various imaging technologies have been utilised to visualise and evaluate embryos such as brightfield microscopy, darkfield microscopy, and Hoffman modulation contrast microscopy. Although these imaging technologies allow for non-invasive imaging of human embryos in a clinical setting, 3D imaging of a whole embryo with subcellular resolution remains a challenge. Method: We demonstrate 3D time-lapse label-free imaging of live mouse embryos using the 3D refractive index (RI) imaging technique. The proposed method is a quantitative phase imaging technique that measures multiple-intensity images of transmitted light through the embryos via axial scanning to reconstruct the 3D RI map of the embryos. Results: Using the proposed method, live mouse embryos were monitored at different developmental stages, ranging from 2-cell embryos to expanded blastocysts. The measured 3D RI maps of the developing embryos reveal 3D landscape of subcellular features inside the embryos (Fig. 1). In the quantitative assessment of embryonic development, various morphokinematic parameters were extracted from the measured 3D RI maps of the embryos. Conclusion: Based on the results, we envision that our method has broad applications where non-invasive and quantitative assessment of embryonic development is required.

3D refractive index reconstruction from phaseless coherent optical microscopy data using multiple scattering-based inverse solvers—a study

Reconstructing 3D refractive index profile of scatterers using optical microscopy measurements presents several challenges over the conventional microwave and RF domain measurement scenario. These include phaseless and polarization-insensitive measurements, small numerical aperture, as well as a Green’s function where spatial frequencies are integrated in a weighted manner such that far-field angular spectrum cannot be probed and high spatial frequencies that permit better resolution are weighed down. As a result of these factors, the non-linearity and the ill-posedness of the inverse problem are quite severe. These limitations have imposed that inverse scattering problems in the microscopy domain largely consider scalar wave approximations and neglect multiple scattering. Here, we present first inverse scattering results for optical microscopy setup where full-wave vectorial formulation and multiple scattering is incorporated. We present (a) how three popular inverse scattering solvers from microwave domain can be adapted for the present inverse problem, (b) the opportunities and challenges presented by each of these solvers, (c) a comparative insight into these solvers and contrast with the simpler Born approximation approach, and (d) potential routes to improve the performance of these solvers for the hard inverse problem of optical microscopy.

The intrinsic mode activation and evolution in fiber splicing based on 3D refractive index profile characterization

Fiber fusion splicing tremendously affects the modal stability, insertion loss and transmission quality of the optical fiber communication systems. Especially, in the few-mode fibers, a tiny dissatisfactory fiber splicing operation may lead to a big modal perturbation or unexpected mode coupling. In this paper, we propose a 3D refractive index profile visualization method to demonstrate the mode activation and evolution in fiber fusion splicing. A refractive index profile measuring instrument is established and applied to detect the 3D refractive index profile deformation caused by the fiber splicing. Then, based on the measured values of 3D refractive index profile, the mode activation is analyzed for fiber splicing between a single-mode fiber and a two-mode fiber with different core-offset distance. Results show that the mode begins to tremble with a core-offset of 4 μm and the high-order modes are effectively activated at the core-offset of 7 μm. The basic theoretical model and experiment results provide a profound support in almost all kinds of fiber splicing operation, mode interference construction and mode coupling modulation. Additionally, the method can be expanded to the research on fiber cleaving and bending.