STARSTEM Publications

STARSTEM Journal Articles

2022

  • A. Arangath, K. Neuhaus, S. Alexandrov and M. Leahy, “Evaluation of Signal Degradation Due to Birefringence in a Multiple Reference Optical Coherence Tomography System With Polarization-Based Balanced Detection,” in IEEE Photonics Journal, vol. 14, no. 4, pp. 1-12 (2022) Art no. 3944712: DOI: https://doi.org/10.1109/JPHOT.2022.3189809

    Download the pdf here.

Although time-domain optical coherence tomography (TD-OCT) systems are straightforward to realize, the imaging speed, sensitivity, and imaging depth limit their range of applications. Multiple reference optical coherence tomography (MR-OCT) based on TD-OCT increases imaging range by about tenfold while providing sensitivity to image highly scattering biological samples. The multiple path-delays and free-space construction make MR-OCT also interesting for hybrid and compact systems, filling the gap between fibre-based and wafer-level integrated optical systems. We describe an optical configuration using a balanced detection scheme and the resulting signal properties due to the required use of polarizing optical components. We numerically simulate the signal properties using Jones calculus and compare the results with measurements. We discuss the origin of signal degradation due to birefringence of the sample in OCT and show that the quarter-wave plate in the sample arm of the Michelson interferometer can be adjusted to optimize the signal returning from a birefringent sample thereby improving the visibility of structures of interest. The theory discussed will be useful to understand and minimize signal degradation due to birefringence in Time-Domain and Fourier-Domain OCT systems.

  • Dimitri A. Kessler, James W. MacKay, Stephen M. McDonnell, Robert L. Janiczek, Martin J. Graves, Joshua D. Kaggie, and Fiona J. Gilbert. “Segmentation of Knee MRI Data with Convolutional Neural Networks for Semi-Automated Three-Dimensional Surface-Based Analysis of Cartilage Morphology and Composition.” Osteoarthritis Imaging 1000010 (2022): In Press. Journal Pre-proof doi: 1016/j.ostima.2022.100010.

Objective

To assess automatic segmentations for surface-based analysis of cartilage morphology and composition on knee magnetic resonance (MR) images.

Methods

2D and 3D U-Nets were trained on double echo steady state (DESS) images from the publicly available Osteoarthritis Initiative (OAI) dataset with femoral and tibial bone and cartilage segmentations provided by the Zuse Institute Berlin (ZIB). The U-Nets were used to perform automatic segmentation of femoral and tibial bone-cartilage structures (bone and cartilage segmentations combined into one structure) from the DESS images. T2-weighted images from the OAI dataset were registered to the DESS images and used for T2 map calculation. Using the 3D cartilage surface mapping (3D-CaSM) method, surface-based analysis of cartilage morphology (thickness) and composition (T2) was performed using both manual and network-generated segmentations from OAI ZIB testing images. Bland-Altman analyses were performed to evaluate the accuracy of the extracted cartilage thickness and T2 measurements from both U-Nets compared to manual segmentations.

Results

Bland-Altman analysis showed a mean bias [95% limits of agreement] for femoral and tibial cartilage thickness measurements ranging between -0.12 to 0.33 [-0.28, 0.96] mm with 2D U-Net and 0.07 to 0.14 [-0.14, 0.39] mm with 3D U-Net. For T2, the mean bias [95% limits of agreement] ranged between -0.16 to 1.32 [-4.71, 4.83] ms with 2D U-Net and -0.05 to 0.46 [-2.47, 3.39] ms with 3D U-Net.

Conclusions

While both 2D and 3D U-Nets exemplified the time-efficiency benefit of using deep learning methods for generating the required segmentations, segmentations from 3D U-Nets demonstrated higher accuracy in the extracted thickness and T2 features using 3D-CaSM compared to the segmentations from 2D U-Nets.

  • Rajib Dey, Yi Zhou, Kai Neuhaus, Sergey Alexandrov, Andrew Nolan, Ting-Chiao Chang, and Martin Leahy. “Simple Characterisation Scheme for Optical Coherence Tomography Systems With Application to a Commercial and a Near-Isometric Resolution Fibre-Based System.” IEEE Photonics Journal 14, no. 1 (2022): 1–11. doi:1109/jphot.2021.3135058.

    Download the pdf here.

Optical Coherence Tomography (OCT) is a rapidly growing imaging modality in biomedical optics. OCT can perform high-resolution, cross-sectional imaging of the microstructure of biological tissues by measuring the coherent spectrum from the backscattered light. OCT systems with broad spectral bandwidths are often constructed using free-space optics to avoid dispersion by fibre optic components. This paper presents a fibre-based OCT system at a centre wavelength of 1300 nm with an axial resolution of 3.8 µm in air, surpassing any previously reported values to the best of our knowledge. Despite the challenges in transporting a broadband spectrum using fibre-optics, the system investigation was motivated by the ever-increasing demand for commercialization of high-resolution OCT systems and simplification of construction. We also evaluate and demonstrate the direct measurement method for axial resolution using an air wedge. Imaging of biomedical and other samples is demonstrated using a high numerical aperture sample lens and compared with images from a commercial OCT system. We discuss the effect of the improved structural visibility by achieving image voxels closer to an isometric shape with a high NA sample lens.

Why is this important?

For stem cells study a novel technique, nano-sensitive OCT (nsOCT), is used. To improve the dynamic range and depth resolution of the nsOCT, the imaging system with broadband light source, which provides extended spectral bandwidth, should be used. Development and characterization of such system is not a trivial task. In this paper we present fibre-based OCT system with a broadband laser from NKT corporation with central wavelength 1300nm and axial resolution of 3.8 μm in air, surpassing any previously reported values to the best of our knowledge.

Methods

The general physical principles were used to design, build and characterize the broadband fiber-based SDOCT system. To test the depth resolution we used the optical wedge with known parameters. Images of phantom (Scotch tape) and biological samples (cucumber and fingertip in vivo) were compared with one of the best commercial SDOCT system TELESTO III from Thorlabs Inc. Just fingertip of one human was imaged as an example.

How could this benefit citizens/ patients?

The nsOCT approach showed potential for study of different biomedical objects, including stem cells and humans in vivo. The developed SDOCT system for realization of the nsOCT approach improves dynamic range and resolution of the nsOCT. Advantages of the developed SDOCT system in comparison with one of the best commercial SDOCT system TELESTO III from Thorlabs Inc., including improved resolution, have been demonstrated. We expect that realization of the nsOCT approach using developed SDOCT system will improve the potential possibility of the nsOCT to study pathological changes in different biomedical objects.

Collaborators 

The research on application of the nsOCT for stem cells imaging and study was done with Regenerative Medicine Institute, School of Medicine, National University of Ireland, Galway, Ireland. It is expected that they also should have benefit from improved SDOCT system for nsOCT. The nsOCT is a complementary technique to other imaging modalities which are used in STARSTEM project, for example, photo-acoustic imaging. Thus additional information obtained using nsOCT will be useful for other STARSTEM participants.

Next steps

The nsOCT approach will be realized using this broadband fiber-based SDOCT system and applied for detection and imaging of different pathologies within biotissues.

2021

  • James, S., Neuhaus, K., Murphy, M. et al.Contrast agents for photoacoustic imaging: a review of stem cell tracking”Stem Cell Res Ther 12, 511 (2021). https://doi.org/10.1186/s13287-021-02576-3

With the advent of stem cell therapy for spinal cord injuries, stroke, burns, macular degeneration, heart diseases, diabetes, rheumatoid arthritis and osteoarthritis; the need to track the survival, migration pathways, spatial destination and differentiation of transplanted stem cells in a clinical setting has gained increased relevance. Indeed, getting regulatory approval to use these therapies in the clinic depends on biodistribution studies. Although optoacoustic imaging (OAI) or photoacoustic imaging can detect functional information of cell activities in real-time, the selection and application of suitable contrast agents is essential to achieve optimal sensitivity and contrast for sensing at clinically relevant depths and can even provide information about molecular activity. This review explores OAI methodologies in conjunction with the specific application of exogenous contrast agents in comparison to other imaging modalities and describes the properties of exogenous contrast agents for quantitative and qualitative monitoring of stem cells. Specific characteristics such as biocompatibility, the absorption coefficient, and surface functionalization are compared and how the labelling efficiency translates to both short and long-term visualization of mesenchymal stem cells is explored. An overview of novel properties of recently developed optoacoustic contrast agents and their capability to detect disease and recovery progression in clinical settings is provided which includes newly developed exogenous contrast agents to monitor stem cells in real-time for multimodal sensing.

Significance: Assessment of disease using optical coherence tomography (OCT) is an actively investigated problem, owing to many unresolved challenges in early disease detection, diagnosis, and treatment response monitoring. The early manifestation of disease or precancer is typically associated with subtle alterations in the tissue dielectric and ultrastructural morphology. In addition, biological tissue is known to have ultrastructural multifractality.

Aim: Detection and characterization of nano-sensitive structural morphology and multifractality in the tissue submicron structure. Quantification of nano-sensitive multifractality and its alteration in progression of tumor.

Approach: We have developed a novel label free nano-sensitive multifractal detrended fluctuation analysis (nsMFDFA) technique in combination with multifractal analysis and nano-sensitive optical coherence tomography (nsOCT). The proposed method deployed for extraction and quantification of nano-sensitive multifractal parameters in mammary fat pad (MFP).

Results: Initially, the nsOCT approach is numerically validated on synthetic submicron axial structures. The nsOCT technique was applied to pathologically characterized MFP of murine breast tissue to extract depth resolved nano-sensitive submicron structures. Subsequently, two dimensional MFDFA were deployed on submicron structural enface images to extract nano-sensitive tissue multifractality. In this study, we found that nano-sensitive multifractality increases in transition from healthy to tumor.

Conclusions: This novel method for extraction of nano-sensitive tissue multifractality promises to provide a non-invasive diagnostic tool for early disease detection and monitoring treatment response. The novel ability to delineate the dominant submicron scale nano-sensitive multifractal properties may also prove useful for characterizing a wide variety of complex scattering media of non-biological origin.

  •  Sergey Alexandrov, Anand Arangath, Yi Zhou, Mary Murphy, Niamh Duffy, Kai Neuhaus, Georgina Shaw, Ryan McAuley, Martin Leahy (2021), Accessing depth‑resolved high spatial frequency content from the optical coherence tomography signal.” Scientific Reports.

Abstract:

Optical coherence tomography (OCT) is a rapidly evolving technology with a broad range of applications, including biomedical imaging and diagnosis. Conventional intensity‐based OCT provides depth‐resolved imaging with a typical resolution and sensitivity to structural alterations of about 5–10 microns. It would be desirable for functional biological imaging to detect smaller features in tissues due to the nature of pathological processes. In this article, we perform the analysis of the spatial frequency content of the OCT signal based on scattering theory. We demonstrate that the OCT signal, even at limited spectral bandwidth, contains information about high spatial frequencies present in
the object which relates to the small, sub‐wavelength size structures. Experimental single frame imaging of phantoms with well‐known sub‐micron internal structures confirms the theory. Examples of visualization of the nanoscale structural changes within mesenchymal stem cells (MSC), which
are invisible using conventional OCT, are also shown. Presented results provide a theoretical and experimental basis for the extraction of high spatial frequency information to substantially improve the sensitivity of OCT to structural alterations at clinically relevant depths.

Why is this important?

Optical coherence tomography (OCT), the optical analogue of ultrasound imaging, has started to revolutionize medical diagnostics. OCT provides unique depth-resolved morphologic and functional information, which helps with the diagnosis and monitoring of different diseases. But spatial resolution and sensitivity to morphological changes are limited to microscale.

Detection of structural changes in biological samples at nanoscale for early diagnosis poses a significant challenge to both researchers and healthcare professionals. The existing clinical gold standards – magnetic resonance imaging and ultrasound are only sensitive to large scale changes (0.1 – 1 mm).

Methods

In this paper, we demonstrated theoretically and experimentally that information about the sub-micron structure of the sample is present in the OCT signal, and that this information can be visualized with nano-sensitivity to structural changes. Results of stem cells nsOCT imaging are presented in the paper.

How could this benefit citizens/ patients?

We presented an imaging technique that extends the abilities of the conventional OCT and can be used for any areas where the OCT is applied.

Collaborators 

The work was done in collaboration between TOMI and Regenerative Medicine Institute, School of Medicine.

Links

Optical coherence tomography (OCT) is a rapidly evolving technology with a broad range of applications, including biomedical imaging and diagnosis. Conventional intensity-based OCT provides depth-resolved imaging with a typical resolution and sensitivity to structural alterations of about 5–10 microns. It would be desirable for functional biological imaging to detect smaller features in tissues due to the nature of pathological processes. In this article, we perform the analysis of the spatial frequency content of the OCT signal based on scattering theory. We demonstrate that the OCT signal, even at limited spectral bandwidth, contains information about high spatial frequencies present in the object which relates to the small, sub-wavelength size structures. Experimental single frame imaging of phantoms with well-known sub-micron internal structures confirms the theory. Examples of visualization of the nanoscale structural changes within mesenchymal stem cells (MSC), which are invisible using conventional OCT, are also shown. Presented results provide a theoretical and experimental basis for the extraction of high spatial frequency information to substantially improve the sensitivity of OCT to structural alterations at clinically relevant depths.

Next steps

  • Further development of the nsOCT approach to improve the spatial resolution in both directions, in-depth and lateral.
  • Continue collaboration with Regenerative Medicine Institute, School of Medicine, to study fundamental properties of the stem cells and biological processes.

2020

  • Nguyen VT, Nardini M, Ruggiu A, Cancedda R, Descalzi F, Mastrogiacomo M.Platelet Lysate Induces in Human Osteoblasts Resumption of Cell Proliferation and Activation of Pathways Relevant for Revascularization and Regeneration of Damaged Bone”International Journal of Molecular Sciences. 2020; 21(14):5123. DOI: https://doi.org/10.3390/ijms21145123
    Download the pdf here.

To understand the regenerative effect of platelet-released molecules in bone repair one should investigate the cascade of events involving the resident osteoblast population during the reconstructive process. Here the in vitro response of human osteoblasts to a platelet lysate (PL) stimulus is reported. Quiescent or very slow dividing osteoblasts showed a burst of proliferation after PL stimulation and returned to a none or very slow dividing condition when the PL was removed. PL stimulated osteoblasts maintained a differentiation capability in vitro and in vivo when tested in absence of PL. Since angiogenesis plays a crucial role in the bone healing process, we investigated in PL stimulated osteoblasts the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) pathways, involved in both angiogenesis and bone regeneration. We observed phosphorylation of STAT3 and a strong induction, nuclear translocation and DNA binding of HIF-1α. In agreement with the induction of HIF-1α an enhanced secretion of vascular endothelial growth factor (VEGF) occurred. The double effect of the PL on quiescent osteoblasts, i.e., resumption of proliferation and activation of pathways promoting both angiogenesis and bone formation, provides a rationale to the application of PL as therapeutic agent in post-traumatic bone repair.

Magnetic resonance imaging of the pancreas is increasingly used as an important diagnostic modality for characterisation of pancreatic lesions. Pancreatic MRI protocols are mostly qualitative due to time constraints and motion sensitivity. MR Fingerprinting is an innovative acquisition technique that provides qualitative data and quantitative parameter maps from a single free‐breathing acquisition with the potential to reduce exam times. This work investigates the feasibility of MRF parameter mapping for pancreatic imaging in the presence of free-breathing exam. Sixteen healthy participants were prospectively imaged using MRF framework. Regions-of-interest were drawn in multiple solid organs including the pancreas and T1 and T2 values determined. MRF T1 and T2 mapping was performed successfully in all participants (acquisition time:2.4–3.6 min). Mean pancreatic T1 values were 37–43% lower than those of the muscle, spleen, and kidney at both 1.5 and 3.0 T. For these organs, the mean pancreatic T2 values were nearly 40% at 1.5 T and < 12% at 3.0 T. The feasibility of MRF at 1.5 T and 3 T was demonstrated in the pancreas. By enabling fast and free-breathing quantitation, MRF has the potential to add value during the clinical characterisation and grading of pathological conditions, such as pancreatitis or cancer.

What were you aiming to find out in this publication?

We would like to acquire MRI data faster and with higher quantitative accuracy. This work uses ‘MR Fingerprinting’, which combines MRI physics simulations with pseudorandom acquisitions to accelerate MRI.

Why is this important?

When MRI is performed, it is normally done in a non-quantitative manner using methods that have been relatively unchanged over the past few decades. MRI is used to diagnose an increasing number of diseases, which requires faster methods that are also precise. MR fingerprinting is a method that helps address this challenge by accelerating MRI throughput, while at the same time providing quantitative data.

In terms of iron-oxide imaging, this method will help improve the quantitation.

Describe the methods chosen.

In this work, we image the pancreas with MR fingerprinting and a challenging area to image due to its central location in the body – deep and far from the receivers placed outside the body.

We imaged 16 normal volunteers with MR fingerprinting. MR fingerprinting uses pseudorandom acquisitions that are combined with MRI physics simulations to accelerate the imaging. The images were acquired at two MRI field strengths common in hospitals, 1.5 T and 3.0 T, in order to demonstrate the feasibility on typical systems. The physics simulations required several hours, after which they could be applied to the data from each subject. The images were acquired in 2-4 minutes, which is a rapid time for quantitative parameter mapping.

How could this work benefit patients?

The intended impact is to improve patient throughput for routine MRI scans, while at the same time acquiring higher quality data. When successful, this will enable faster routine imaging, which will translate to cost savings, as well as a better diagnosis by improving the images (or maps) that are acquired with MRI. This also feeds into machine learning techniques, which benefit from higher repeatability between centres.

The main output of this work is demonstrating that we can reliably obtain MRF maps within the abdomen, which is a challenging area to image. This area is subject to breathing motion, but also small variations in magnetic fields that have large effects on images – including air in the lungs and bowels.

In addition, this work showed that these images could be obtained while a patient is free-breathing. Breathing artefacts are common to MRI in the abdomen, and having a technique that can ignore breathing is powerful, particularly when we want to image animals that can’t hold their breath on command or patients who have difficulties holding their breath for long periods.

Who were your collaborators?

This work was done in (external) collaboration with GE Healthcare and the University of Pisa.

What are the next steps?

The next step is to apply MR fingerprinting in sheep stifles (=knees) to measure the changes induced with iron labelled stem cells. The additional measurements with this are the presence of iron in abdominal sheep organs.

  • Kaggie, J.D., Markides, H., Graves, M.J. et al. “Ultra Short Echo Time MRI of Iron-Labelled Mesenchymal Stem Cells in an Ovine Osteochondral Defect Model.” Sci Rep 10, 8451 (2020). DOI: https://doi.org/10.1038/s41598-020-64423-4
    Download the pdf here.
Multipotent Mesenchymal Stem/Stromal Cells (MSCs) are widely used in cellular therapy for joint repair. However, the use of MSC therapies is complicated by a lack of understanding of the behaviour of cells and repair within the joint. Current methods of MSC tracking include labelling the cells with Super Paramagnetic Iron Oxide nanoparticles (SPIOs). However, standard acquisition sequences (T2 and T2*) give poor anatomical definition in the presence of SPIOs. To avoid anatomical compromise in the presence of SPIOs, we have investigated the use of Ultra-short Echo Time (UTE) MRI, using a 3D cones acquisition trajectory. This method was used to track SPIO labelled MSC injected into joints containing osteochondral defects in experimental sheep. This study demonstrates that multiple echo times from UTE with 3 T MRI can provide excellent anatomical detail of osteochondral defects and demonstrate similar features to histology. This work also monitors the location of SPIO-labelled cells for regenerative medicine of the knee with MRI, histology, and Prussian blue staining. With these methods, we show that the SPIOs do not hone to the site of defect but instead aggregate in the location of injection, which suggests that any repair mechanism with this disease model must trigger a secondary process.

What were you aiming to find out in this publication?

We would like to be able to image knee joints better, especially in the presence of stem cells labelled with iron oxides. Iron oxides cause rapid drop off in MRI signals, which can be compensated for by using a method called ‘ultra short echo time’ imaging to capture these signals.

Why is this important?

The use of stem cells is becoming increasingly studied, although we do not completely understand where these cells go and their effects on the joint. In order to ensure safe usage of the cells and push the technology, we need to understand how they interact inside live tissues, and thus, track them. To track these cells, they were labelled with iron-oxide particles or SPIONs – ‘super paramagnetic iron oxides’ – affect the magnetism in magnetic resonance imaging and cause rapid signal decays wherever the cells are concentrated. This imaging/tracking method allows us to confirm where the cells are located.

Interestingly, many models of osteoarthritis repair that work in mice do not work so well in larger animals, including sheep. Verification of osteoarthritic stem cell technologies in sheep is important in order to demonstrate safety prior to human trials.

Describe the methods chosen.

In this work, we use an MRI method, ‘ultra-short echo time imaging’ or UTE-MRI, captures the rapidly decaying MRI signals caused by these super-paramagnetic iron oxides and in hard-tissues like bone, to enable better measurements of

One of the fascinating aspects of UTE-MRI is that it uses a non-standard sampling pattern. In MRI, you can imagine that you sample the data like listening to a piano. You hit a key on the piano keyboard and listen. MRI is very similar to this, except this occurs with radiofrequencies rather than audible tones, but the principle is the same. You can sample the keyboard pattern one key at a time, up the musical scales one key at a time. This work alters the sampling pattern by collecting the signals earlier and uses a non-grid-like approach. It then also relies on advanced mathematics for image reconstruction beyond the typical MRI, called ‘non-linear Fourier transformations’.

This work imaged seven sheep stifles (or knees, except in sheep) with an osteoarthritis model, which was holes in their bone at the cartilage surface. We imaged their stifles with UTE-MRI and performed histology to confirm the presence SPIONs and to visualise how closely the histology matched the UTE-MRI data.

These two photos show cartilage and bone in a sheep that had an ‘osteochondral defect’, or hole in its bone. These images are an MRI (A) and histological sample imaged with a microscope (B). This MRI technique captures rapidly decaying MRI signals, thus called ‘ultra-short echo time imaging’.

How could this work benefit patients?

We were surprised in that the cells did not end up in the intended location, as we confirmed with histology and MRI. It was previously believed that stem cells honed to the site of the defect (or hole in the bone) in order to repair the tissue. This did not happen, which allows us to consider more interesting models, such as whether the cells recruit other cells to perform the repair.

If we can understand the mechanisms of repair, then we can target these mechanisms more accurately. By using more advanced imaging techniques, it allows us to visualise more clearly where the cells go into a large body system with a lot of different tissues.

We also hope that improvements on UTE-MRI can help in human imaging and diagnosis of bone/cartilage defects.

Who were your collaborators?

This work was done in collaboration with GE Healthcare.

What are the next steps?

This study leads into the follow-on study of using both MRI and photoacoustic imaging, and labelling the stem cells with MRI and photoacoustic agents, to confirm where the cells become located after injection and their effects on the repair of the tissue.

Our current work is on combining the use of iron-oxides and nanostars for imaging stem cells with two methods – magnetic resonance and photoacoustic imaging.

  • Dimitri A.Kessler, James W.MacKay, Victoria Crowe, Frances Henson, Martin J. Graves, Fiona J. Gilbert, Joshua D. Kaggie (2020), The Optimisation of Deep Neural Networks for Segmenting Multiple Knee Joint Tissues from MRIs.” Computerized Medical Imaging and Graphics. DOI: https://doi.org/10.1016/j.compmedimag.2020.101793
    Download the pdf here.

Automated semantic segmentation of multiple knee joint tissues is desirable to allow faster and more reliable analysis of large datasets and to enable further downstream processing e.g. automated diagnosis.

In this work, we evaluate the use of conditional Generative Adversarial Networks (cGANs) as a robust and potentially improved method for semantic segmentation compared to other extensively used convolutional neural network, such as the U-Net. As cGANs have not yet been widely explored for semantic medical image segmentation, we analysed the effect of training with different objective functions and discriminator receptive field sizes on the segmentation performance of the cGAN. Additionally, we evaluated the possibility of using transfer learning to improve the segmentation accuracy. The networks were trained on i) the SKI10 dataset which comes from the MICCAI grand challenge “Segmentation of Knee Images 2010”, ii) the OAI ZIB dataset containing femoral and tibial bone and cartilage segmentations of the Osteoarthritis Initiative cohort and iii) a small locally acquired dataset (Advanced MRI of Osteoarthritis (AMROA) study) consisting of 3D fat-saturated spoiled gradient recalled-echo knee MRIs with manual segmentations of the femoral, tibial and patellar bone and cartilage, as well as the cruciate ligaments and selected peri-articular muscles. The Sørensen–Dice Similarity Coefficient (DSC), volumetric overlap error (VOE) and average surface distance (ASD) were calculated for segmentation performance evaluation.

DSC ≥ 0.95 were achieved for all segmented bone structures, DSC ≥ 0.83 for cartilage and muscle tissues and DSC of ≈0.66 were achieved for cruciate ligament segmentations with both cGAN and U-Net on the in-house AMROA dataset. Reducing the receptive field size of the cGAN discriminator network improved the networks segmentation performance and resulted in segmentation accuracies equivalent to those of the U-Net. Pretraining not only increased segmentation accuracy of a few knee joint tissues of the fine-tuned dataset, but also increased the network’s capacity to preserve segmentation capabilities for the pretrained dataset.

cGAN machine learning can generate automated semantic maps of multiple tissues within the knee joint which could increase the accuracy and efficiency for evaluating joint health.

What are you aiming to find out?

We used machine learning to automatically segment multiple knee tissues, including bones, cartilage, muscles, and cruciate ligaments. This will help us enable better quantitative analysis of these tissues in future studies, and use the best machine learning network to do this.

Why does this research need to be done?

Tissue segmentation is a time-consuming process, but very useful for obtaining quantitative metrics – such as shape or signal intensity. Machine learning can accelerate this.

While machine learning is increasingly performed for the segmentation of tissues, this work shows that it can be used for many (10) different tissues, with highly varying characteristics. We expect this type of work to be constantly iterated on, although we were the first to show all of these tissues together and tissues like the ACL/PCL.

Describe the methods chosen.

We used several deep learning methods to automatically segment knee tissues – called the U-Net and the GAN, and compared these with different parameters. We wanted to know the best process to perform this measurement, as it can speed up analysis in future sets and patients.

We used publicly available knee datasets (SKI10, ZIB) and local data (AMROA).

The AMROA participants were questioned on their patient experience as they left the MRI system.

What are the next steps?

We would like to apply this to looking at how signal intensities change with the introduction of stem cells or nanoparticles.

  • Kessler, D.A., MacKay, J.W., McDonald, S., McDonnell, S., Grainger, A.J., Roberts, A.R., Janiczek, R.L., Graves, M.J., Kaggie, J.D. and Gilbert, F.J. (2020), Effectively Measuring Exerciserelated Variations in T1ρ and T2 Relaxation Times of Healthy Articular Cartilage.” Journal of Magnetic Resonance Imaging. DOI: 10.1002/jmri.27278
    Download the pdf here.

Background: Determining the compositional response of articular cartilage to dynamic joint loading using magnetic resonance imaging may be a more sensitive assessment of cartilage status than conventional static imaging. However, distinguishing the effects of joint loading versus inherent measurement variability remains difficult as the repeatability of these quantitative methods is often not assessed or reported. Purpose: To assess exercise-induced changes in femoral, tibial and patellar articular cartilage composition and compare these against measurement repeatability. Study Type: Prospective observational study. Population: Phantom and 19 healthy participants. Field Strength/Sequence: 3T; 3D fat-saturated spoiled gradient recalled-echo; T1ρ- and T2-prepared pseudo-steady-state 3D fast spin echo. Assessment: The intra-sessional repeatability of T1ρ and T2 relaxation mapping, with and without knee repositioning between two successive measurements, was determined in 10 knees. T1ρ and T2 relaxation mapping of nine knees was performed before and at multiple time points after a 5-minute repeated, joint-loading stepping activity. Three-dimensional surface models were created from patellar, femoral and tibial articular cartilage. Statistical Tests: Repeatability was assessed using root-mean-squared-CV (RMS-CV). Using Bland-Altman analysis, thresholds defined as the smallest detectable difference (SDD) were determined from the repeatability data with knee repositioning. Results: Without knee repositioning, both surface-averaged T1ρ and T2 were very repeatable on all cartilage surfaces with RMS-CV<1.1%. Repositioning of the knee had the greatest effect on T1ρ of patellar cartilage with the surface-averaged RMS-CV=4.8%. While T1ρ showed the greatest response to exercise at the patellofemoral cartilage region, the largest changes in T2 were determined in the lateral femorotibial region. Following thresholding, significant (> SDD) average exercise-induced in T1ρ and T2 of femoral (-8.0% and -5.3%), lateral tibial (-6.9% and -5.9%), medial tibial (+5.8% and +2.9%) and patellar (-7.9% and +2.8%) cartilage were observed. Data Conclusion: Joint loading with a stepping activity resulted in T1ρ and T2 changes above background measurement error.

Key Words: Articular Cartilage; MRI; Quantitative Imaging; Repeatability; Exercise; Relaxation Time

  • Nandan Das, Sergey Alexandrov, Yi Zhou, Katie Gilligan, Róisín M Dwyer, Martin J. Leahy  Nanoscale structure detection and monitoring of tumour growth with optical coherence tomography.” Nanoscale Advances 2020 DOI: doi.org/10.1039/D0NA00371A
    Download the pdf here.
Approximately 90% of cancers have their origins in epithelial tissues and this leads to epithelial thickening, but the ultrastructural changes and underlying architecture is less well known. Depth resolved label free visualization of nanoscale tissue morphology is required to reveal the extent and distribution of ultrastructural changes in underlying tissue, but is difficult to achieve with existing imaging modalities. We developed a nanosensitive optical coherence tomography (nsOCT) approach to provide such imaging based on dominant axial structure with a few nanometre detection accuracy. nsOCT maps the distribution of axial structural sizes an order of magnitude smaller than the axial resolution of the system. We validated nsOCT methodology by detecting synthetic axial structure via numerical simulations. Subsequently, we validated the nsOCT technique experimentally by detecting known structures from a commercially fabricated sample. nsOCT reveals scaling with different depth of dominant submicron structural changes associated with carcinoma which may inform the origins of the disease, its progression and improve diagnosis.

What are you aiming to find out?

Proposed research demonstrated detection of depth resolved submicron structure with few nanometre accuracies and quantified its alteration as tumour growth in mammary fat pad (MFP).

Why does this research need to be done?

Early detection of cancer can save millions of lives. It is known that tissue goes through submicron structural changes as cancer development initiates. However, developed technique rely on labelling tissue sample and information available from superficial region only.

Describe the methods chosen.

Here we have applied nano sensitive optical coherence tomography (nsOCT) which can detect depth resolved submicron structure and its alteration. Proposed method validated numerically and tested on mammary fat pad (MFP) in mice.

How will this benefit citizens/patients?

Clinical system can be develop based on proposed method to detect cancer in early stage. In initial study, we found a consistent change of submicron structure over tissue depth as tumour developed. Early detection of cancer can provide opportunity for better treatment and can improve quality of life.

What are the next steps?

We are presently developing nsOCT which expected to detect smaller submicron structure and can provide more insight about nano-sensitive changes as precancer progress.

  • Cerine Lal, Sergey Alexandrov, Sweta Rani, Yi Zhou, Thomas Ritter, and Martin Leahy Nanosensitive optical coherence tomography to assess wound healing within the cornea.” Biomedical Optics Express 2020 DOI: 10.1364/BOE.389342
    Download the pdf here.
Optical coherence tomography (OCT) is a non-invasive depth resolved optical imaging modality, that enables high resolution, cross-sectional imaging in biological tissues and materials at clinically relevant depths. Though OCT offers high resolution imaging, the best ultra-high-resolution OCT systems are limited to imaging structural changes with a resolution of one micron on a single B-scan within very limited depth. Nanosensitive OCT (nsOCT) is a recently developed technique that is capable of providing enhanced sensitivity of OCT to structural changes. Improving the sensitivity of OCT to detect structural changes at the nanoscale level, to a depth typical for conventional OCT, could potentially improve the diagnostic capability of OCT in medical applications. In this paper, we demonstrate the capability of nsOCT to detect structural changes deep in the rat cornea following superficial corneal injury.
What was the aim of this study?

The aim of the study is to demonstrate the capability of nsOCT to detect structural changes deep in the cornea following superficial corneal injury and subsequent healing.

Why is this important?

Studying nanoscale structural and dynamic changes in vivo is fundamental to understanding changes occurring at cellular level before the changes manifest at the tissue level. Detecting these submicron structural changes can help scientists and clinicians to diagnose the onset of a disease, its progression and in determining treatment effectiveness of drugs.

The cornea is the transparent, avascular layer of the eye that controls the entry of light into the eye and helps to refract the light onto the retina. Corneal transparency is vital to preserve its structure and function. Corneal injuries generally arise from thermal and chemical burns. Of these, 11.5–22% of all ocular injuries occur from chemical burns, from both acids and alkali. Among chemical induced corneal burns, alkali burn causes more damage to the corneal stroma and anterior chamber compared to acid injury. Alkali ions being lipophilic, penetrate into the corneal stroma disrupting the cells and denaturing the collagen matrix, which promotes further penetration into the anterior chamber.

Hence, it is imperative to understand the nanoscale structural changes occurring during ocular injury and subsequent wound healing process in vivo for assessment of wound repair and monitoring treatment efficacy.

What methods did you use?

In this paper, we have elucidated the capability of the nanosensitive OCT (nsOCT) technique to detect structural changes within the cornea to assess the impact of alkali injury and also to study the wound healing process. nsOCT offers much higher sensitivity to structural changes within the cornea compared to conventional OCT processing. The study reveals that nsOCT is able to detect structural changes with nanoscale sensitivity between healthy cornea, injured cornea and also during the reparative phase of the injury at all depths within the cornea with high statistical significance (p < 10−10).

How could this benefit citizens/patients?

The method presented offers potential for in vivo imaging applications especially in clinical imaging where sensitivity to changes in structure is of significance either to detect the onset of a disease or to evaluate the efficacy of treatment which cannot be obtained from conventional OCT processing.

It can be used for real time monitoring of corneal health following ocular trauma or ocular diseases.

What are the next steps?

Future steps will be the application of nsOCT technique to spatial and temporal structural changes within cornea following injury, treatment and healing and changes occurring due to elevated intra ocular pressure. The technique can also be used to detect pathophysiological changes in retina following diabetic retinopathy, glaucoma and other retinal diseases.

Further applications of the technique can be used to study morphological changes in biomedical samples, for example, to image progression of cancerous cells and tumours as they are known to undergo nanoscale structural changes within their vicinity long before the manifestation of the disease.

  • Yi Zhou, Sergey Alexandrov, Andrew Nolan, Nandan Das, Rajib Dey, Martin Leahy Noninvasive detection of nanoscale structural changes in cornea associated with cross‐linking treatment.” Journal of Biophotonics 2020 DOI: 10.1002/jbio.201960234
    Download the pdf here.
Corneal cross‐linking (CXL) using ultraviolet‐A (UVA) irradiation with a riboflavin photosensitizer has grown from an interesting concept to a practical clinical treatment for corneal ectatic diseases globally, such as keratoconus. To characterize the corneal structural changes, existing methods such as X‐ray microscopy, transmission electron microscopy, histology and optical coherence tomography (OCT) have been used. However, these methods have various drawbacks such as invasive detection, the impossibility for in vivo measurement, or limited resolution and sensitivity to structural alterations. Here, we report the application of oversampling nanosensitive OCT for probing the corneal structural alterations. The results indicate that the spatial period increases slightly after 30 minutes riboflavin instillation but decreases significantly after 30 minutes UVA irradiation following the Dresden protocol. The proposed noninvasive method can be implemented using existing OCT systems, without any additional components, for detecting nanoscale changes with the potential to assist diagnostic assessment during CXL treatment, and possibly to be a real‐time monitoring tool in clinics.

What are you aiming to find out?

The proposed method aims to detect nanoscale changes in cornea with the potential to assist diagnostic assessment during cross-linking treatment

Why does this research need to be done?

Corneal ectasia can severely impair vision, especially in the progressive form caused by the inherent structural weakness of the cornea. Keratoconus, the most common form of corneal ectasia affecting nearly 1 in 375 individuals globally, is an ocular disorder. Corneal cross-linking (CXL), was proved to be an effective way in halting the progression of keratoconus, meaning patients can avoid a corneal transplant.

What methods did you use and why?

In this study, we have presented the application of over-sampling nano-sensitive optical coherence tomography (nsOCT), which is proposed to retain the high spatial frequency information in the interference spectra, to probe the structural alterations inside ex vivo bovine cornea during CXL treatment with nanoscale sensitivity. The results suggest that the over-sampling nsOCT can be used to detect nano-sized structural changes valuable for corneal treatment methods.

How could this benefit citizens/ patients?

Due to its fast, non-invasive detecting method and nanoscale sensitivity, this unique technology is potential to be an indicator in diagnostic assessment associated with CXL treatment, and possibly to be a real-time monitoring tool in clinics as a fast way to receive feedback from patient’s tissue.

What are the next steps?

Future work will aim to implement this method for in vivo corneal detections associated with CXL treatment, including monitoring the nanoscale structural variations at different treating steps and also the postoperative assessment.

  • S. Carluccio, et. al  Progenitor Cells Activated by Platelet Lysate in Human Articular Cartilage as a Tool for Future Cartilage Engineering and Reparative Strategies.” Cells 2020 9(4) DOI: https://doi.org/10.3390/cells9041052
    Download the pdf here.
Regenerative strategies for human articular cartilage are still challenging despite the presence of resident progenitor cell population. Today, many efforts in the field of regenerative medicine focus on the use of platelet derivatives due to their ability to reactivate endogenous mechanisms supporting tissue repair. While their use in orthopedics continues, mechanisms of action and efficacy need further characterization. We describe that the platelet lysate (PL) is able to activate chondro-progenitor cells in a terminally differentiated cartilage tissue. Primary cultures of human articular chondrocytes (ACs) and cartilage explants were set up from donor hip joint biopsies and were treated in vitro with PL. PL recruited a chondro-progenitors (CPCs)-enriched population from ex vivo cartilage culture, that showed high proliferation rate, clonogenicity and nestin expression. CPCs were positive for in vitro tri-lineage differentiation and formed hyaline cartilage-like tissue in vivo without hypertrophic fate. Moreover, the secretory profile of CPCs was analyzed, together with their migratory capabilities. Some CPC-features were also induced in PL-treated ACs compared to fetal bovine serum (FBS)-control ACs. PL treatment of human articular cartilage activates a stem cell niche responsive to injury. These facts can improve the PL therapeutic efficacy in cartilage applications.
  • W. Hotham, and F.M.D. Henson “The use of large animals to facilitate the process of MSC going from laboratory to patient-‘bench to bedside’.” Cell Biol Toxicol 2020 DOI: 10.1007/s10565-020-09521-9
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Large animal models have been widely used to facilitate the translation of mesenchymal stem cells (MSC) from the laboratory to patient. MSC, with their multi-potent capacity, have been proposed to have therapeutic benefits in a number of pathological conditions. Laboratory studies allow the investigation of cellular and molecular interactions, while small animal models allow initial ‘proof of concept’ experiments. Large animals (dogs, pigs, sheep, goats and horses) are more similar physiologically and structurally to man. These models have allowed clinically relevant assessments of safety, efficacy and dosing of different MSC sources prior to clinical trials. In this review, we recapitulate the use of large animal models to facilitate the use of MSC to treat myocardial infarction-an example of one large animal model being considered the ‘gold standard’ for research and osteoarthritis-an example of the complexities of using different large animal models in a multifactorial disease. These examples show how large animals can provide a research platform that can be used to evaluate the value of cell-based therapies and facilitate the process of ‘bench to bedside’.

2019

  • Ivan Olefir, Ara Ghazaryan, Hong Yang, Jaber Malekzadeh-Najafabadi, Sarah Glasl, Panagiotis Symvoulidis, Valerie B. O’Leary, George Sergiadis, Vasilis Ntziachristos, and Saak V. Ovsepian. “Spatial and Spectral Mapping and Decomposition of Neural Dynamics and Organization of the Mouse Brain with Multispectral Optoacoustic Tomography.” Cell Reports 26, no. 10 (2019): 2833-2846.e3. doi:1016/j.celrep.2019.02.020.

    Download the pdf here.

In traditional optical imaging, limited light penetration constrains high-resolution interrogation to tissue surfaces. Optoacoustic imaging combines the superb contrast of optical imaging with deep penetration of ultrasound, enabling a range of new applications. We used multispectral optoacoustic tomography (MSOT) for functional and structural neuroimaging in mice at resolution, depth, and specificity unattainable by other neuroimaging modalities. Based on multispectral readouts, we computed hemoglobin gradient and oxygen saturation changes related to processing of somatosensory signals in different structures along the entire subcortical-cortical axis. Using temporal correlation analysis and seed-based maps, we reveal the connectivity between cortical, thalamic, and sub-thalamic formations. With the same modality, high-resolution structural tomography of intact mouse brain was achieved based on endogenous contrasts, demonstrating near-perfect matches with anatomical features revealed by histology. These results extend the limits of noninvasive observations beyond the reach of standard high-resolution neuroimaging, verifying the suitability of MSOT for small-animal studies.

  • Sergey Alexandrov, Paul M. McNamara, Nandan Das, Yi Zhou, Gillian Lynch, Josh Hogan, and Martin Leahy. “Spatial frequency domain correlation mapping optical coherence tomography for nanoscale structural characterization” Applied Physics Letters 115(2) 2019 DOI: https://doi.org/10.1063/1.5110459
    Download the pdf here.

Most of the fundamental pathological processes in living tissues exhibit changes at the nanoscale. Noninvasive, label-free detection of structural changes in biological samples pose a significant challenge to both researchers and healthcare professionals. It is highly desirable to be able to resolve these structural changes, during physiological processes, both spatially and temporally. Modern nanoscopy largely requires labeling, is limited to superficial 2D imaging, and is generally not suitable for in vivo applications. Furthermore, it is becoming increasingly evident that 2D biology often does not translate into the real 3D situation. Here, we present a method, spatial frequency domain correlation mapping optical coherence tomography (sf-cmOCT), for detection of depth resolved nanoscale structural changes noninvasively. Our approach is based on detection and correlation of the depth resolved spectra of axial spatial frequencies of the object which are extremely sensitive to structural alterations. The presented work describes the principles of this approach and demonstrates its feasibility by monitoring internal structural changes within objects, including human skin in vivo. Structural changes can be visualized at each point in the sample in space from a single image or over time using two or more images. These experimental results demonstrate possibilities for the study of nanoscale structural changes, without the need for biomarkers or labels. Thus, sf-cmOCT offers exciting and far-reaching opportunities for early disease diagnosis and treatment response monitoring, as well as a myriad of applications for researchers.

What are you aiming to find out?

Development of the new technologies for visualization of the sub-micron structure with nanoscale sensitivity to structural changes.

Why does this research need to be done?

For both the fundamental study of biological processes and early diagnosis of pathological processes, information about nanoscale tissue structure is crucial. Furthermore, it is becoming increasingly evident that 2D biology often does not translate into the real 3D situation. The research is motivated by current needs of optical science and technology for biomedical and other applications and is devoted to an important fundamental problem: investigation of new possibilities to detect the structural changes within 3D objects without labels by providing nanoscale sensitivity to structural alterations. One of the most appreciated and fast developed techniques for 3D biomedical imaging is optical coherence tomography (OCT), but resolution and sensitivity to structural alterations is typically limited to microscale. Proposed approach permits to dramatically improve sensitivity to structural changes, up to nanoscale, using just single frame.

Describe the methods chosen.

Label free non-contact optical imaging technologies have been developed. The ability to detect nano-scale structural changes has been demonstrated using different phantoms and human skin in vivo. Healthy volunteer was involved in this study.

What is the intended impact and how can others use the research?

According to the STARSTEM project these techniques will be used to detect changes in cell morphologies and extracellular vesicles at the nanometer scale; to investigate the potential of imaging of extracellular vesicle-mediated disease responses; for nanosensitive detection of tissue responses to disease.

What are the next steps?

  • Experiments to perform nanosensitive detection of tissue responses to disease.
  • Further development of these optical technologies.

Related research.

  1. Alexandrov, P. M. McNamara, N. Das, Y. Zhou, G. Lynch, J. Hogan, and M. Leahy “Spatial frequency domain correlation mapping optical coherence tomography for nanoscale structural characterization”. Appl. Phys. Lett. 2019, v.115, N12, 121105. https://doi.org/10.1063/1.5110459.
  2. Alexandrov, N. Das, J. McGrath, P. Owens, C. J. R. Sheppard, F. Boccafoschi, C. Giannini, T. Sibillano, H. Subhash, and M. Leahy. “Label free ultra-sensitive imaging with sub-diffraction spatial resolution”. 21st International Conference on Transparent Optical Networks ICTON, July 9-13, 2019 Angers, France, Invited, IEEE-Xplore Proceedings 2019 Fr.A6.3, pp.1-4. https://ieeexplore.ieee.org/xpl/conhome/1000766/all-proceedings

STARSTEM Conference Publications

2020

  • Nandan Das, Sergey Alexandrov, Róisín M. Dwyer, Rolf Saager, Nirmalya Ghosh, Martin Leahy. “Characterization of nano sensitive sub-micron scale tissue-structural multifractality and its alteration in tumor progress” Proceedings Volume 11239, Dynamics and Fluctuations in Biomedical Photonics XVII; 1123912 (2020).  DOI: 10.1117/12.2555840

    Download the pdf here.
Assessment of disease using OCT is an actively investigated problem, owing to many unresolved challenges in early disease detection, diagnosis and treatment response monitoring. The spatial scale to which the information can be obtained from the scattered light is limited by the diffraction limit (~λ/2; λ = wavelength of light is typically in the micron level) and the axial resolution of OCT systems is limited by the inverse of spectral bandwidth. Yet, onset or progression of disease /precancer is typically associated with subtle alterations in the tissue dielectric and its ultra-structural morphology. On the other hand, biological tissue is known to have ultra-structural multifractality. For both the fundamental study of biological processes and early diagnosis of pathological processes, information on the nanoscale in the tissue sub-micron structural morphology is crucial. Therefore, we have developed a novel spectroscopic and label-free 3D OCT system with nanoscale sensitivity in combination of multifractal analysis for extraction and quantification of tissue ultra-structural multifractal parameters. This present approach demonstrated its capability to measure nano-sensitive tissue ultra-structural multifractality. In an initial study, we found that nano-sensitive sub-micron structural multifractality changes in transition from healthy to tumor in pathologically characterized fresh tissue samples. This novel method for extraction of nanosensitive tissue multifractality promises to develop a non-invasive diagnosis tool for early cancer detection.
  • Yi Zhou, Sergey Alexandrov, Andrew Nolan, Rajib Dey, Nandan Das, Kai Neuhaus, Martin Leahy. “Application of over-sampling nano-sensitive optical coherence tomography for monitoring corneal internal structural changes in corneal cross-linking“. Proceedings Volume 11228, Optical Coherence Tomography and Coherence Domain Optical Methods in Biomedicine XXIV, 112282M (2020).  DOI: 10.1117/12.2547317

    Download the pdf here.
Corneal cross-linking (CXL) has grown from an interesting concept to a practical clinical treatment for corneal ectatic disease globally in the past three decades. In both understanding the principle of how CXL proceeds and monitoring the clinical procedure, detection of structural changes during cornea CXL plays a significant role. This paper demonstrates a novel over-sampling nano-sensitive optical coherence tomography (osnsOCT) method, which is potential to detect nanoscale structural changes in various tissues, to simultaneously measure the structural variations during the corneal CXL treatment.

2019

  • Sergey Alexandrov, Nandan Das, James McGrath, Peter Owens, Colin J. R. Sheppard, Francesca Boccafoschi, Cinzia Giannini, Teresa Sibillano, Hrebesh Subhash, and Martin Leahy. “Label free ultra-sensitive imaging with sub-diffraction spatial resolution” Paper presented at the 21st International Conference on Transparent Optical Networks, ICTON’2019, Angers, France, 09-13 July.  DOI: https://dx.doi.org/10.1109/ICTON.2019.8840220
    Download the pdf here.

In this paper, we show a new way to break the resolution limit and dramatically improve sensitivity to structural changes. To realize it we developed a novel label free contrast mechanism, based on the spectral encoding of spatial frequency (SESF) approach. The super-resolution SESF (srSESF) microscopy is based on reconstruction of the axial spatial frequency (period) profiles for each image point and comparison of these profiles to form super-resolution image. As a result, the information content of images is dramatically improved in comparison with conventional microscopy. Numerical simulation and experiments demonstrate significant improvement in sensitivity and resolution.

  • Sergey A. Alexandrov, James McGrath, Colin Sheppard, Francesca Boccafoschi, Cinzia Giannini, Teresa Sibillano, Hrebesh Subhash, Josh Hogan, and Martin Leahy. “Ultra-sensitive label free imaging below the resolution limit” Paper presented at the SPIE BiOS 2019 meeting, San Francisco, California, USA, 4 March 2019.  DOI: https://doi.org/10.1117/12.2502479
    Download the pdf here.

Almost all known nanoscopy methods rely upon the contrast created by fluorescent labels attached to the object of interest. This causes limitations on their applicability to in vivo imaging. A new label-free spectral encoding of spatial frequency (SESF) approach to nanoscale probing of three-dimensional structures has been developed. It has been demonstrated that spatial frequencies, encoded with optical wavelengths, can be passed though the optical system independent of the resolution of the imaging system. As a result information about small size structures can be detected even using a low resolution imaging system. Different versions of the SESF imaging have been published [1-7], including a novel contrast mechanism for high resolution imaging [1], real time nano-sensitive imaging [2], reconstruction the axial (along depth) spatial frequency profiles for each point with nano-sensitivity to structural changes [3], and the adaptation of the SESF approach to depth resolving imaging [4,5]. Recently the SESF approach has been applied to break the diffraction limit and dramatically improve resolution [6,7]. Here we present further development of the SESF approach including correlation mapping SESF imaging. Both results of numerical simulation and preliminary experimental results, including biological objects, will be presented. [1] Alexandrov, et.al., Opt. Lett. 36 3323 (2011). [2] Alexandrov, et.al., Opt. Express 20 (8) 9203 (2012). [3] Alexandrov, et.al., Appl. Phys. Let., 101 033702 (2012). [4] Uttam, et.al., Opt. Express, 21, 7488 (2013). [5] Alexandrov, et.al., Nanoscale, 6, 3545 (2014). [6] Alexandrov, et.al., Sci. Rep., 5, doi: 10.1038/srep13274 (2015). [7] Alexandrov, et.al., J. Biophotonics, https://doi.org/10.1002/jbio.201700385 (2018).