Label-free Imaging Technique Research Articles

2,5-Dihydroxyterephthalic Acid: A Matrix for Improved Detection and Imaging of Amino Acids.

Amino acids (AAs), which are low-molecular-weight (low-MW) metabolites, serve as essential building blocks not only for protein synthesis but also for maintaining the nitrogen balance in living systems. In situ detection and imaging of AAs are crucial for understanding more complex biological processes. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a label-free mass spectrometric imaging technique that enables the simultaneous detection and imaging of the spatial distribution and relative abundance of different endogenous/exogenous compounds in biological samples. The excellent efficiency of MALDI-MSI is attributed to the choice of the MALDI matrix. However, to the best of our knowledge, no matrix has been specifically developed for AAs. Herein, we report a MALDI matrix, 2,5-dihydroxyterephthalic acid (DHT), which can improve the detection and imaging of AAs in biological samples by MALDI-MS. Our results indicated that DHT exhibited strong ultraviolet-visible (UV-vis) absorption, uniform matrix deposition, and high vacuum stability. Moreover, the matrix-related ion signals produced from DHT were reduced by 50 and 71.8% at m/z < 500 compared to the commonly used matrices of 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA), respectively, in their respective organic solvents. In terms of quantitative performance, arginine, glutamic acid, glutamine, and proline can be detected with limits of detection of 6, 4, 6, and 4 ng/mL, respectively, using the DHT as the matrix. Using DHT as the matrix, all 20 protein AAs were successfully detected in human serum by MALDI-MS, whereas only 7 and 10 AAs were detected when DHB and CHCA matrices were used, respectively. Furthermore, 20 protein AAs and taurine were successfully detected and imaged in a section of edible Crassostrea gigas (oyster) tissue for the first time. Our study demonstrates that using DHT as a matrix can improve the detection and imaging of AAs in biological samples by MALDI-MS.

Artificial intelligence-enabled quantitative phase imaging methods for life sciences.

Quantitative phase imaging, integrated with artificial intelligence, allows for the rapid and label-free investigation of the physiology and pathology of biological systems. This review presents the principles of various two-dimensional and three-dimensional label-free phase imaging techniques that exploit refractive index as an intrinsic optical imaging contrast. In particular, we discuss artificial intelligence-based analysis methodologies for biomedical studies including image enhancement, segmentation of cellular or subcellular structures, classification of types of biological samples and image translation to furnish subcellular and histochemical information from label-free phase images. We also discuss the advantages and challenges of artificial intelligence-enabled quantitative phase imaging analyses, summarize recent notable applications in the life sciences, and cover the potential of this field for basic and industrial research in the life sciences.

Two-photon imaging reveals histopathological changes in the gastric tumor microenvironment induced by neoadjuvant treatment.

There is a close association between tumor response and survival in gastric cancer patients after receiving neoadjuvant treatment. An accurate and rapid assessment of therapeutic efficacy would be helpful for subsequent treatments and individual prognosis. At present, pathological examination is the gold standard for evaluating treatment response, however, it requires additional staining and the process is tedious, labor-intensive, as well as time-consuming. Here, we introduce a label-free imaging technique, two-photon imaging, to evaluate histopathological changes induced by pre-operative therapy, with a focus on assessing tumor regression as well as stromal response. Imaging data show that two-photon imaging allows label-free, rapid visualization of various aspects of pathological alterations in tumor microenvironment such as fibrotic reaction, inflammatory cell infiltration, mucinous response, isolated residual tumor cells. Moreover, a semi-automatic image processing approach is developed to extract the collagen morphological features, and statistical results show that there are significant differences in collagen area, length, width, cross-link space between the gastric cancer tissues with and without treatment. With the advent of a portable, miniaturized two-photon imaging device, we have enough reason to believe that this technique will become as an important auxiliary diagnostic tool in assessing neoadjuvant treatment response and thereby tailoring the most appropriate therapy strategies for the patients.

Visualization of metabolites and microbes at high spatial resolution using MALDI mass spectrometry imaging and in situ fluorescence labeling.

Label-free molecular imaging techniques such as matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) enable the direct and simultaneous mapping of hundreds of different metabolites in thin sections of biological tissues. However, in host-microbe interactions it remains challenging to localize microbes and to assign metabolites to the host versus members of the microbiome. We therefore developed a correlative imaging approach combining MALDI-MSI with fluorescence in situ hybridization (FISH) on the same section to identify and localize microbial cells. Here, we detail metaFISH as a robust and easy method for assigning the spatial distribution of metabolites to microbiome members based on imaging of nucleic acid probes, down to single-cell resolution. We describe the steps required for tissue preparation, on-tissue hybridization, fluorescence microscopy, data integration into a correlative image dataset, matrix application and MSI data acquisition. Using metaFISH, we map hundreds of metabolites and several microbial species to the micrometer scale on a single tissue section. For example, intra- and extracellular bacteria, host cells and their associated metabolites can be localized in animal tissues, revealing their complex metabolic interactions. We explain how we identify low-abundance bacterial infection sites as regions of interest for high-resolution MSI analysis, guiding the user to a trade-off between metabolite signal intensities and fluorescence signals. MetaFISH is suitable for a broad range of users from environmental microbiologists to clinical scientists. The protocol requires ~2 work days.

Image denoising in acoustic microscopy using block-matching and 4D filter

Scanning acoustic microscopy (SAM) is a label-free imaging technique used in biomedical imaging, non-destructive testing, and material research to visualize surface and sub-surface structures. In ultrasonic imaging, noises in images can reduce contrast, edge and texture details, and resolution, negatively impacting post-processing algorithms. To reduce the noises in the scanned image, we have employed a 4D block-matching (BM4D) filter that can be used to denoise acoustic volumetric signals. BM4D filter utilizes the transform domain filtering technique with hard thresholding and Wiener filtering stages. The proposed algorithm produces the most suitable denoised output compared to other conventional filtering methods (Gaussian filter, median filter, and Wiener filter) when applied to noisy images. The output from the BM4D-filtered images was compared to the noise level with different conventional filters. Filtered images were qualitatively analyzed using metrics such as structural similarity index matrix (SSIM) and peak signal-to-noise ratio (PSNR). The combined qualitative and quantitative analysis demonstrates that the BM4D technique is the most suitable method for denoising acoustic imaging from the SAM. The proposed block matching filter opens a new avenue in the field of acoustic or photoacoustic image denoising, particularly in scenarios with poor signal-to-noise ratios.

Nonlinear Imaging Histopathology: A Pipeline to Correlate Gold-Standard Hematoxylin and Eosin Staining With Modern Nonlinear Microscopy.

Hematoxylin and eosin (H&E) staining, the century-old technique, has been the gold standard tool for pathologists to detect anomalies in tissues and diseases such as cancer. H&E staining is a cumbersome, time-consuming process that delays and wastes precious minutes during an intraoperative diagnosis. However, even in the modern era, real-time label-free imaging techniques such as simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy have delivered several more layers of information to characterize a tissue with high precision. Still, they have yet to translate to the clinic. The slow translation rate can be attributed to the lack of direct comparisons between the old and new techniques. Our approach to solving this problem is to: 1) reduce dimensions by pre-sectioning the tissue in 500 μm slices, and 2) produce fiducial laser markings which appear in both SLAM and histological imaging. High peak-power femtosecond laser pulses enable ablation in a controlled and contained manner. We perform laser marking on a grid of points encompassing the SLAM region of interest. We optimize laser power, numerical aperture, and timing to produce axially extended marking, hence multilayered fiducial markers, with minimal damage to the surrounding tissues. We performed this co-registration over an area of 3 × 3 mm2 of freshly excised mouse kidney and intestine, followed by standard H&E staining. Reduced dimensionality and the use of laser markings provided a comparison of the old and new techniques, giving a wealth of correlative information and elevating the potential of translating nonlinear microscopy to the clinic for rapid pathological assessment.

Microsphere-assisted enhanced photothermal lens detection integrated with digital holographic microscopy for 3D particle sensing and thermal diffusivity measurement

In this paper, we show that by extending the concept of “microsphere-assisted imaging” into thermal lens (TL) detection, enhancement of photothermal detection is achieved. Furthermore, by integration of TL detection and digital holographic microscopy (DHM), we provide simultaneous measurement of photothermal properties as well as complementary imaging of the sample. Simultaneous acquisition of photothermal properties and imaging is essential for characterization of a variety of samples in biochemistry, material science, and process technologies. TL is a sensitive methodology to detect low concentrations of analytes in low-loss samples, and DHM in the transmission mode is an effective technique for label-free, non-contact, and real-time imaging and measurement of phase objects, such as biomaterials. We demonstrate the usefulness of the TL-DHM integrated system by applying it for acquisition of photothermal maps of samples as well as obtaining their morphometrical information. Additionally, we measure the thermal diffusivity of polydimethylsiloxane because of the focusing effect of polystyrene microspheres. The system has the potential to be applied for a variety of objects including bio-samples and may be proposed as a bench-top characterization device.

Label-Free Imaging Techniques to Evaluate Metabolic Changes Caused by Toxic Liver Injury in PCLS.

Abuse with hepatotoxic agents is a major cause of acute liver failure. The search for new criteria indicating the acute or chronic pathological processes is still a challenging issue that requires the selection of effective tools and research models. Multiphoton microscopy with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) are modern label-free methods of optical biomedical imaging for assessing the metabolic state of hepatocytes, therefore reflecting the functional state of the liver tissue. The aim of this work was to identify characteristic changes in the metabolic state of hepatocytes in precision-cut liver slices (PCLSs) under toxic damage by some of the most common toxins: ethanol, carbon tetrachloride (CCl4) and acetaminophen (APAP), commonly known as paracetamol. We have determined characteristic optical criteria for toxic liver damage, and these turn out to be specific for each toxic agent, reflecting the underlying pathological mechanisms of toxicity. The results obtained are consistent with standard methods of molecular and morphological analysis. Thus, our approach, based on optical biomedical imaging, is effective for intravital monitoring of the state of liver tissue in the case of toxic damage or even in cases of acute liver injury.

Expanding hyperspectral imaging applications to the clinical scene: non-invasive, label-free approaches for early diagnostics and precision medicine

Non-invasive, label-free hyperspectral imaging technologies are powerful tools that can provide critical insights into the spatiotemporal dynamics of evolving systems. They are already integrated into autonomous adaptive surveillance workflows for precision agriculture, remote sensing, environmental monitoring, and food analysis. However, these technologies are not yet routinely used in medicine. With these technologies applied to the biomedical sciences, we anticipate landmark advances in the physicochemical behavioral refinement of high signal-to-noise probes and in the rapid screening of patient exosome populations. To implement label-free spatiochemical imaging techniques while meeting the practical needs of clinicians, technology, workflow design and development must be tailored to the specific medical field while considering existing visualization modalities. This perspective merges basic, translational, and clinical views of label-free, non-invasive hyperspectral imaging. We explore its potential role in positively impacting cancer-related precision medicine and early diagnostics to support clinically implemented point-of-care technologies.

Single-filament imaging mass spectrometry lipidomics in Arthrospira platensis.

Elucidating intra-organismal biochemical and lipid organization in photosynthetic biological cell factories of filamentous cyanobacteria, such as Arthrospira platensis (Spirulina), is important for tracking physiological response mechanisms during growth. Little is known about the filaments' biochemical organization and cellular structure and no label-free imaging techniques exist that provide molecular mapping. We applied ultrahigh-resolution mass spectrometry (MS) with matrix-assisted laser desorption ionization (MALDI) imaging to immobilized Spirulina filaments to investigate the localization of lipids across distinct physiological regions. We optimized matrix selection and deposition methods with the goal of facilitating high spatial, and intra-filament, resolution using untargeted multivariate statistical spectral deconvolution across MS pixels. Our results demonstrate an improved two-step matrix application with an optimized procedure for intra-organismal lipid profiling to improve analyte sensitivity and achieve higher spatial resolution. We evaluate several conventional matrices, namely 2,5-dihydroxybenzoic acid (DHB), superDHB (sDHB), 1,5-diaminonaphthalene (DAN), and a 50:50 mix of DHB and sDHB, and compare delineation and pixel-based elucidation of intra-filament lipidomics. We identified a total of 1626 features that could be putatively assigned a lipid-like formula based on database query and 46 unique features, with associated lipid assignments that were significantly distinct in their intra-filament location. MALDI imaging MS with untargeted statistical spectral deconvolution was used to visualize intra-filament lipidomics organization in Spirulina filaments. Improvements in matrix deposition, including sequential sublimation and pneumatic spraying, increased signal abundance at high spatial resolution and allowed for identification of distinct lipid composition regions. This work outlines a methodology that may be used for micro-ecological untargeted molecular phenotyping.

An assessment of microvascular hemodynamics in human macula

An adequate blood supply to meet the energy demands is essential for any tissue, particularly for high energy demand tissues such as the retina. A critical question is: How is the dynamic match between neuronal demands and blood supply achieved? We present a quantitative assessment of temporal and spatial variations in perfusion in the macular capillary network in 10 healthy human subjects using a non-invasive and label-free imaging technique. The assessment is based on the calculation of the coefficient of variation (CoV) of the perfusion signal from arterioles, venules and capillaries from a sequence of optical coherence tomography angiography images centred on the fovea. Significant heterogeneity of the spatial and temporal variation was found within arterioles, venules and capillary networks. The CoV values of the capillaries and smallest vessels were significantly higher than that in the larger vessels. Our results demonstrate the presence of significant heterogeneity of spatial and temporal variation within each element of the macular microvasculature, particularly in the capillaries and finer vessels. Our findings suggest that the dynamic match between neuronal demands and blood supply is achieved by frequent alteration of local blood flow evidenced by capillary perfusion variations both spatially and temporally in the macular region.

Abstract 6054: Weight specific effects of hyaluronic acid on glioblastoma's energy metabolism and bio-molecular content

Abstract Glioblastoma (GBM) is one of the deadliest forms of brain cancer with inter and intra tumor heterogeneity and altered cell metabolism pathways. Developing multi-dimensional in vitro platforms can help explore different tumor micro-environments and can thus offer therapeutic opportunities. For a molecular-level understanding, non-destructive and label-free imaging techniques combined with in vitro tumor models offer discovery and automation potential for targeted studies. Here, we use the Fourier Transform Infrared (FTIR) spectroscopic imaging technique to profile biomolecular behavior of U87-MG GBM cell lines in a 2D culture in varied metabolic and tumor micro-environment conditions. As a micro-environmental cue, we chose to vary different molecular weights of hyaluronic acid (HA), a key component of brain’s extra-cellular matrix (ECM) and known to significantly influence cell-behavior. U87-MG cell lines were cultured for 72 hours in a DMEM supplemented with combinations of (i) glucose concentration: absent (0 mM), normal (5.5 mM) and elevated (25 mM) and (ii) molecular weights of 2 μg/ml HA: low (10 kDa)-LMW, medium (60 kDa)-MMW and high (500 kDa)-HMW. Fixed cells at different timepoints were imaged with FTIR and stained for HA-specific CD44 and RHAMM markers. Supernatant media was analyzed for metabolite expressions with LC/MS. FTIR study revealed that the Phosphate I/Amide II and Amide I/Amide II IR absorbance ratios were significantly influenced in the presence of 500 kDa HA. Visible differences in the phosphate (~1080 cm−1) and lipid (~2936 cm−1) intensities indicated signs of DNA breakages and changes in oxidative stress levels. Areas under the peaks representing α-helix (decrease) and β-sheet (increase) proteins revealed signs of protein denaturation in HMW HA cultures. These cultures also revealed elevated RHAMM expressions while the CD44 expression remained relatively unchanged. Metabolic profiling with LC/MS revealed an increase in metabolites like choline, histidine, and creatine, for cells cultured in the 0 mM glucose and HMW HA. Ornithine and Methionine remained relatively unaffected by the HA weight but showed some dependence on changing glucose levels. Proline and Tyrosine majorly showed HA weight-specific variations. Overall, this preliminary study has shown the impact of glucose and HA weight-specific effects on the biomolecular content and metabolic profiles of cancer cells. Most of the altered metabolite expressions seen are known to be involved in the Arginine-biosynthesis pathway which suggests a possible alteration in the same in response to environmental glucose and weight-specific HA levels. Further studies would involve the use of multi-dimensional hydrogel-cell models to assess variations in a more bio-mimetic surrounding. Highlighting altered metabolic pathways could help present novel therapeutic targets for future. Citation Format: Ashwin A. Bale, Brendan Harley, Rohit Bhargava. Weight specific effects of hyaluronic acid on glioblastoma's energy metabolism and bio-molecular content [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 6054.

Studying Oral Tissue via Real-Time High-Resolution Terahertz Spectroscopic Imaging

Terahertz (THz) waves are extremely sensitive to water content and biological composition (proteins, genes, and metabolites), which are the key regulator of cancer development. Hence, the application of THz spectroscopy and imaging is promising in the field of biomedicine, especially in cancer diagnosis. Currently, the diagnosis of cancer patients (including tumor borders, tumor stage, etc.) mainly rely on optical microscope observation of pathological slices. The preparation of the slices is complicated and time consuming, and the microscopic field of view is small, which cannot effectively guide the precise removal of the tumor during the operation in time. To solve this problem, a label-free real-time high-resolution near-field THz spectroscopy imaging technique was developed. In this study, we successfully apply this technique to the oral tissue. Compared with conventional point-by-point scanning terahertz time-domain spectroscopy imaging, this technique can exhibit a real-time imaging rate (as high as 20 frames/s at 512 \ifmmode\times\else\texttimes\fi{} 1024 pixels) and a field of view approximately 5 mm in diameter with a spatial resolution up to \ensuremath{\lambda}/10 at 1 THz in near-field condition. The experimental results show that the terahertz spectroscopic imaging in this paper can clearly detect and identify not only biological tissues with different components, but also normal and cancerous tissues, as well as tissues with the same composition but different slice directions. This work lays an experimental foundation for the application of THz spectroscopy imaging to rapid label-free intraoperative diagnosis, and it provides a basis for promoting the wide application of THz technology in the biomedical field.

A line-scanning hyperspectral Raman microscope based on Bessel beam illumination.

Carbohydrate metabolism within the photosynthetic processes of plants plays a major role in plant performance. Current techniques for live imaging the structural characteristics of plants are often destructive or lack spatially explicit information. Raman spectroscopy is a nondestructive, label-free imaging technique which, in combination with Bessel beam light sheet microscopy, offers a way to rapidly measure the abundance and flux of molecules as they travel through the three dimensions of living plants. Here we describe the development of a Hyperspectral Raman Light Sheet Microscope as an alternative to traditional confocal point-scanning imaging systems. We cover our methods for creating a self-reconstructing Bessel beam to excite scattering in the sample, as well as modifications made to the conventional slit-spectrometer setup for capturing low intensity Raman scattered infrared light. We demonstrate the system with various test samples including Fluorinated Ethylene Propylene and Acetaminophen, and then discuss future applications in biological materials.

Imaging of interactions of biomolecules with nanomaterials with interferometric scattering microscopy.

Interferometric scattering microscopy (iSCAT) is a label-free imaging technique that achieves single-molecule spatial and milli-second temporal resolution. In this technique, we use the combination of reflected and scattered light of the nanoparticle itself as our signal obviating the need of a label or tags as commonly used in fluorescence microscopy. With iSCAT, photobleaching is not an issue as the signal donor (the particle of interest) provides an infinite budget of scattered photons and hence indefinite imaging times. We apply this method to image and track biomolecules, such as DNA and proteins, without any fluorescent tags in their native state for upwards of seconds. We characterize and tracked the motion of DNA from the initial attachment, diffusion along the surface and finally detachment with single-molecule resolution. This allows us to image the nanoscopic interactions of native DNA with different 2D nanomaterials such as graphene and hBN. Together with other surface characterization techniques such as AFM, we identify sites of increased trapping potential and elucidate the nanoscopic mechanism behind the interaction between biomolecules and the 2D material surface. We envision that our studies allow us to inform on future biosensor design with engineered surface interactions.

DESI-MSI-based technique to unravel spatial distribution of COMT inhibitor Tolcapone

Ascertaining compound exposure and its spatial distribution are essential steps in the drug development process. Desorption electrospray ionization mass spectrometry (DESI-MSI) is a label-free imaging technique capable of simultaneously identify and visualize the distribution of a diverse range of biomolecules.In this study, DESI-MSI was employed to investigate spatial distribution of tolcapone in rat liver and brain coronal – frontal and striatal –sections after a single oral administration of 100 mg/Kg of tolcapone, brain-penetrant compound.Tolcapone was evenly distributed in liver tissue sections whereas in the brain it showed differential distribution across brain regions analyzed, being mainly located in the olfactory bulb, basal forebrain region, striatum, and pre-frontal cortex (PFC; cingulate, prelimbic and infralimbic area). Tolcapone concentration in tissues was compared using DESI-MSI and liquid-chromatography mass spectrometry (LC-MS/MS). DESI-MSI technique showed a higher specificity on detecting tolcapone in liver sections while in the brain samples DESI-MSI did not allow a feasible quantification. Indeed, DESI-MSI is a qualitative technique that allows to observe heterogeneity on distribution but more challenging regarding accurate measurements.Overall, tolcapone was successfully localized in liver and brain tissue sections using DESI-MSI, highlighting the added value that this technique could provide in assisting tissue-specific drug distribution studies.

Standardizing image assessment in optical diffraction tomography.

Optical diffraction tomography (ODT) has gradually become a popular label-free imaging technique that offers diffraction-limited resolution by mapping an object's three-dimensional (3D) refractive index (RI) distribution. However, there is a lack of comprehensive quantitative image assessment metrics in ODT for studying how various experimental conditions influence image quality, and subsequently optimizing the experimental conditions. In this Letter, we propose to standardize the image assessment in ODT by proposing a set of metrics, including signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and structural distinguishability (SD). To test the feasibility of the metrics, we performed experiments on angle-scanning ODT by varying the number of illumination angles, RI contrast of samples, sample feature sizes, and sample types (e.g., standard polystyrene beads and 3D printed structures) and evaluated the RI tomograms with SNR, CNR, and SD. We further quantitatively studied how image quality can be improved, and tested the image assessment metrics on subcellular structures of living cells. We envision the proposed image assessment metrics may greatly benefit end-users for assessing the RI tomograms, as well as experimentalists for optimizing ODT instruments.

Multi-exposure Speckle Imaging for Quantitative Evaluation of Cortical Blood Flow.

Laser speckle contrast imaging (LSCI) is a label-free optical imaging technique that can quantify flow dynamics across an entire image. Multi-exposure speckle imaging (MESI) is an extension of LSCI that allows for reproducible and quantifiable measurements of flow. MESI has the potential to provide quantitative cerebral blood flow information in both preclinical and clinical applications; in fact, MESI can be extended to resolve the flow dynamics in any exposed tissue. A MESI system can be divided into three primary components: (i) the illumination optics, consisting of the optical source and a method of modulating and gating the illumination intensity; (ii) the collection optics, consisting of a high-speed camera that can be triggered and gated to match the pulsed illumination; and finally (iii) post-processing hardware and software to extract the flow information from the recorded raw intensity images. In the following protocol, we offer a guide to design, operate, and test a MESI system.

Optical Coherence Tomography Is a Promising Tool for Zebrafish-Based Research-A Review.

The zebrafish is an established vertebrae model in the field of biomedical research. With its small size, rapid maturation time and semi-transparency at early development stages, it has proven to be an important animal model, especially for high-throughput studies. Three-dimensional, high-resolution, non-destructive and label-free imaging techniques are perfectly suited to investigate these animals over various development stages. Optical coherence tomography (OCT) is an interferometric-based optical imaging technique that has revolutionized the diagnostic possibilities in the field of ophthalmology and has proven to be a powerful tool for many microscopic applications. Recently, OCT found its way into state-of-the-art zebrafish-based research. This review article gives an overview and a discussion of the relevant literature and an outlook for this emerging field.

Characterization of two-photon photopolymerization fabrication using high-speed optical diffraction tomography

Two-photon photopolymerization (TPP) has recently become a popular method for the fabrication of three-dimensional (3D) micro- and nanostructures. The reproduction fidelity of the designed micro- and nanostructures is influenced by experimental writing conditions, including laser power, exposure time, etc. To determine the appropriate writing parameters, characterization of morphological features and surface roughness during the experiment is needed. Traditional characterization methods for TPP, e.g., scanning electron microscopy and atomic force microscopy, have limited speed and cannot study internal structures without invasive approaches. Optical diffraction tomography (ODT) is an emerging label-free 3D imaging technique based on reconstructing the object’s 3D refractive index (RI) distribution with diffraction-limited resolution. Here, we propose a non-invasive solution to fully characterize the TPP-fabricated structures using a high-speed ODT technique, which can eliminate the need for complex sample preparation, such as fluorescence labelling or metal-coating, and achieve a full 3D measurement time of 6 ms. By visualizing and studying different TPP-fabricated structures, including embedded spirals and cubes, via the ODT system, the fabrication quality, including 3D morphological features, exposure levels, and surface roughness, can be examined quantitatively. The results suggest our method can effectively improve the fabrication quality and reproducibility of TPP, generating impacts on the nanofabrication community.