Scopus Publications
- On the quantitative analysis of lamellar collagen arrangement with second-harmonic generation imaging
Pedro Guimarães, Miguel Morgado, Ana Batista
Biomedical Optics Express, 2024
Second harmonic generation (SHG) allows for the examination of collagen structure in collagenous tissues. Collagen is a fibrous protein found in abundance in the human body, present in bones, cartilage, the skin, and the cornea, among other areas, providing structure, support, and strength. Its structural arrangement is deeply intertwined with its function. For instance, in the cornea, alterations in collagen organization can result in severe visual impairments. Using SHG imaging, various metrics have demonstrated the potential to study collagen organization. The discrimination between healthy, keratoconus, and crosslinked corneas, assessment of injured tendons, or the characterization of breast and ovarian tumorous tissue have been demonstrated. Nevertheless, these metrics have not yet been objectively evaluated or compared. A total of five metrics were identified and implemented from the literature, and an additional approach adapted from texture analysis was proposed. In this study, we analyzed their effectiveness on a ground-truth set of artificially generated fibrous images. Our investigation provides the first comprehensive assessment of the performance of multiple metrics, identifying both the strengths and weaknesses of each approach and providing valuable insights for future applications of SHG imaging in medical diagnostics and research. - Corrigendum: Normative mice retinal thickness: 16-month longitudinal characterization of wild-type mice and changes in a model of Alzheimer's disease (Frontiers in Aging Neuroscience, (2023), 15, (1161847), 10.3389/fnagi.2023.1161847)
Ana Batista, Pedro Guimarães, João Martins, Paula I. Moreira, António Francisco Ambrósio, et al.
Frontiers in Aging Neuroscience, 2024 - Phase-Resolved Optical Coherence Elastography: An Insight into Tissue Displacement Estimation
Ana Batista, Pedro Serranho, Mário J. Santos, Carlos Correia, José P. Domingues, et al.
Sensors, 2023
Robust methods to compute tissue displacements in optical coherence elastography (OCE) data are paramount, as they play a significant role in the accuracy of tissue elastic properties estimation. In this study, the accuracy of different phase estimators was evaluated on simulated OCE data, where the displacements can be accurately set, and on real data. Displacement (∆d) estimates were computed from (i) the original interferogram data (Δφori) and two phase-invariant mathematical manipulations of the interferogram: (ii) its first-order derivative (Δφd) and (iii) its integral (Δφint). We observed a dependence of the phase difference estimation accuracy on the initial depth location of the scatterer and the magnitude of the tissue displacement. However, by combining the three phase-difference estimates (Δdav), the error in phase difference estimation could be minimized. By using Δdav, the median root-mean-square error associated with displacement prediction in simulated OCE data was reduced by 85% and 70% in data with and without noise, respectively, in relation to the traditional estimate. Furthermore, a modest improvement in the minimum detectable displacement in real OCE data was also observed, particularly in data with low signal-to-noise ratios. The feasibility of using Δdav to estimate agarose phantoms’ Young’s modulus is illustrated. - Evaluation of skin care products using Optical Coherence Elastography
Rita Bernardo, José Paulo Domingues, Rui Bernardes, António Miguel Morgado, Ana Batista
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2023
The biomechanical properties of the human skin are intrinsically correlated with changes associated with pathological conditions, aging, and hydration. Quantitative measurements can improve diagnostic tools, treatments, and cosmetic product evaluation. Using optical coherence elastography (OCE), an emerging imaging modality combining optical coherence tomography (OCT) with a localized excitation source to induce mechanical disturbances, a quantitative evaluation of tissue biomechanics can be achieved. OCE complements the structural information with elasticity data to attain a complete overview of skin status. In this study, we employed a home-built OCE system, combining a swept-source OCT system with a piezoelectric actuator for tissue displacement, to evaluate changes to the skin biomechanical properties due to the application of an anti-aging cream. Skin elasticity was monitored for a total of five weeks. Anti-aging cream was applied daily for four weeks. OCE measurements continued for one additional week to assess the effect of cream application interruption. Three female volunteers were included in this proof-of-principle investigation. Their counter-arm was used as control. Although no statistical significance was reached, a decrease in skin Young’s modulus was observed with the cream application, indicating an increase in skin elasticity. - Normative mice retinal thickness: 16-month longitudinal characterization of wild-type mice and changes in a model of Alzheimer's disease
Ana Batista, Pedro Guimarães, João Martins, Paula I. Moreira, António Francisco Ambrósio, et al.
Frontiers in Aging Neuroscience, 2023
Animal models of disease are paramount to understand retinal development, the pathophysiology of eye diseases, and to study neurodegeneration using optical coherence tomography (OCT) data. In this study, we present a comprehensive normative database of retinal thickness in C57BL6/129S mice using spectral-domain OCT data. The database covers a longitudinal period of 16 months, from 1 to 16 months of age, and provides valuable insights into retinal development and changes over time. Our findings reveal that total retinal thickness decreases with age, while the thickness of individual retinal layers and layer aggregates changes in different ways. For example, the outer plexiform layer (OPL), photoreceptor inner segments (ILS), and retinal pigment epithelium (RPE) thickened over time, whereas other retinal layers and layer aggregates became thinner. Additionally, we compare the retinal thickness of wild-type (WT) mice with an animal model of Alzheimer's disease (3 × Tg-AD) and show that the transgenic mice exhibit a decrease in total retinal thickness compared to age-matched WT mice, with statistically significant differences observed at all evaluated ages. This normative database of retinal thickness in mice will serve as a reference for future studies on retinal changes in neurodegenerative and eye diseases and will further our understanding of the pathophysiology of these conditions. - Retinal imaging in animal models: Searching for biomarkers of neurodegeneration
Ana Batista, Pedro Guimarães, Pedro Serranho, Ana Nunes, João Martins, et al.
Frontiers in Ophthalmology, 2023
There is a pressing need for novel diagnostic and progression biomarkers of neurodegeneration. However, the inability to determine disease duration and stage in patients with Alzheimer’s disease (AD) hinders their discovery. Because animal models of disease allow us to circumvent some of these limitations, they have proven to be of paramount importance in clinical research. Due to the clear optics of the eye, the retina combined with optical coherence tomography (OCT) offers the perfect opportunity to image neurodegeneration in the retina in vivo, non-invasively, directly, quickly, and inexpensively. Based on these premises, our group has worked towards uncovering neurodegeneration-associated changes in the retina of the triple-transgenic mouse model of familial AD (3×Tg-AD). In this work, we present an overview of our work on this topic. We report on thickness variations of the retina and retinal layers/layer aggregates caused by healthy aging and AD-like conditions and discuss the implications of focusing research efforts solely on retinal thickness. We explore what other information is embedded in the OCT data, extracted based on texture analysis and deep-learning approaches, to further identify biomarkers that could be used for early detection and diagnosis. We were able to detect changes in the retina of the animal model of AD as early as 1 month of age. We also discuss our work to develop an optical coherence elastography system to measure retinal elasticity, which can be used in conjunction with conventional OCT. Finally, we discuss the potential application of these technologies in human patients and the steps needed to make OCT a helpful screening tool for the detection of neurodegeneration. - Time-dependent elastic numerical model for Optical Coherence Elastography of the murine retina
Carlos Correia, Ana Batista, Sílvia Barbeiro, João Cardoso, José P. Domingues, et al.
2023 IEEE 7th Portuguese Meeting on Bioengineering Enbeng 2023, 2023
We present the initial stages of development of a Finite Element Method-based time-dependent elastic numerical model which seeks to support the employment of our Optical Coherence Elastography system for assessing murine retinal elasticity. The current model is able to reconstruct displacement maps in both homogeneous and heterogeneous domains with errors up to a few hundredths relatively to a known exact displacement map, within 1 millisecond. The results demonstrate the robustness of the numerical algorithm under different elastic domains, and model parametrization with real Optical Coherence Elastography data is already in progress. - Two-Photon Imaging for Non-Invasive Corneal Examination
Ana Batista, Pedro Guimarães, José Paulo Domingues, Maria João Quadrado, António Miguel Morgado
Sensors, 2022
Two-photon imaging (TPI) microscopy, namely, two-photon excited fluorescence (TPEF), fluorescence lifetime imaging (FLIM), and second-harmonic generation (SHG) modalities, has emerged in the past years as a powerful tool for the examination of biological tissues. These modalities rely on different contrast mechanisms and are often used simultaneously to provide complementary information on morphology, metabolism, and structural properties of the imaged tissue. The cornea, being a transparent tissue, rich in collagen and with several cellular layers, is well-suited to be imaged by TPI microscopy. In this review, we discuss the physical principles behind TPI as well as its instrumentation. We also provide an overview of the current advances in TPI instrumentation and image analysis. We describe how TPI can be leveraged to retrieve unique information on the cornea and to complement the information provided by current clinical devices. The present state of corneal TPI is outlined. Finally, we discuss the obstacles that must be overcome and offer perspectives and outlooks to make clinical TPI of the human cornea a reality. - Artificial Intelligence in Multiphoton Tomography: Atopic Dermatitis Diagnosis
Pedro Guimarães, Ana Batista, Michael Zieger, Martin Kaatz, Karsten Koenig
Scientific Reports, 2020
The diagnostic possibilities of multiphoton tomography (MPT) in dermatology have already been demonstrated. Nevertheless, the analysis of MPT data is still time-consuming and operator dependent. We propose a fully automatic approach based on convolutional neural networks (CNNs) to fully realize the potential of MPT. In total, 3,663 MPT images combining both morphological and metabolic information were acquired from atopic dermatitis (AD) patients and healthy volunteers. These were used to train and tune CNNs to detect the presence of living cells, and if so, to diagnose AD, independently of imaged layer or position. The proposed algorithm correctly diagnosed AD in 97.0 ± 0.2% of all images presenting living cells. The diagnosis was obtained with a sensitivity of 0.966 ± 0.003, specificity of 0.977 ± 0.003 and F-score of 0.964 ± 0.002. Relevance propagation by deep Taylor decomposition was used to enhance the algorithm’s interpretability. Obtained heatmaps show what aspects of the images are important for a given classification. We showed that MPT imaging can be combined with artificial intelligence to successfully diagnose AD. The proposed approach serves as a framework for the automatic diagnosis of skin disorders using MPT. - High-speed imaging of gas-bubble formation during femtosecond-laser cell optoporation
Ana Maria Gonçalves Batista, Hans Georg Breunig, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2020
Femtosecond-laser pulses can create transient holes in the membrane of a cell, making it briefly permeable to genetic macromolecules. This is a highly effective method for cell transfection and reprogramming. For sufficiently high irradiance values, the laser radiation leads to plasma formation through multiphoton ionization and a subsequent formation of gas-filled bubbles which are shortly visible after the irradiation. While this bubble formation is well known, the underlying microscopic processes, the optimal bubble size and duration which indicate the transient hole formation are less clear. The correspondence between bubble formation and successful optoporation is further complicated by the fact that the formation greatly depends on the irradiated cell position and laser irradiation parameters (power, exposure time). We have investigated the formation of bubbles resulting from short pulse irradiation with two commercial Ti:sapphire lasers using a high-speed camera. Higher laser powers and longer exposure times yielded bigger bubbles which took longer to collapse. Additionally, a correlation between the bubble characteristics and the cell’s metabolism and post-optoporation viability was found. These results can help to optimize the laser parameters for efficient optoporation and high post-treatment cell viability as well as to shine light on the microscopic excitation processes behind the bubble formation. - Towards Improving Human Corneal Care Using Two-Photon Imaging
Ana Batista, Hans Georg Breunig, Berthold Seitz, Karsten König
Ifmbe Proceedings, 2020 - Translation of two-photon microscopy to the clinic: Multimodal multiphoton CARS tomography of in vivo human skin
Karsten König, Hans Georg Breunig, Ana Batista, Andreas Schindele, Michael Zieger, et al.
Journal of Biomedical Optics, 2020 - Clinical multimodal multiphoton tomography of pigmented skin lesions with an ultracompact femtosecond fiber laser
Karsten König, Ana Maria Gonçalves Batista, Michael Zieger, Martin Kaatz, Holger Hänßle, et al.
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2020 - Early evaluation of corneal collagen crosslinking in ex-vivo human corneas using two-photon imaging
Ana Batista, Hans Georg Breunig, Tobias Hager, Berthold Seitz, Karsten König
Scientific Reports, 2019 - Riboflavin concentrations at the endothelium during corneal cross-linking in humans
Theo G. Seiler, Ana Batista, Beatrice E. Frueh, Karsten Koenig
Investigative Ophthalmology and Visual Science, 2019 - Multimodal multiphoton tomograph using a compact femtosecond fiber laser
Ana Batista, Karsten König, Aisada König, Hans Georg Breunig
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2019 - Towards laser-assisted microfluidic-cell transfection
Hans Georg Breunig, Ana Batista, Aisada König, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2019 - Non-invasive and label-free follow-up of accelerated-crosslinking using multiphoton tomography
Ana Batista, Hans Georg Breunig, Tobias Hager, Berthold Seitz, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2019 - Assessment of the metabolism and morphology of the porcine cornea, lens and retina by 2-photon imaging
Ana Batista, Hans G. Breunig, Aisada König, António M. Morgado, Karsten König
Journal of Biophotonics, 2018 - High-resolution, label-free two-photon imaging of diseased human corneas
Ana Batista, Hans Georg Breunig, Aisada König, Andreas Schindele, Tobias Hager, et al.
Journal of Biomedical Optics, 2018 - Rapid in vivo vertical tissue sectioning by multiphoton tomography
Ana Batista, Hans Georg Breunig, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2018 - Rapid vertical tissue imaging with clinical multiphoton tomography
Karsten Koenig, Hans G. Breunig, Benjamin Sauer, Ana Batista
Proceedings of SPIE the International Society for Optical Engineering, 2018 - Two-photon microscopy and fluorescence lifetime imaging of the cornea
Ana Batista, Hans Georg Breunig, Christoph Donitzky, Karsten König
Multiphoton Microscopy and Fluorescence Lifetime Imaging Applications in Biology and Medicine, 2018 - Femtosecond-laser setups for cell-membrane poration
Karsten König, Hans G. Breunig, Ana Batista, Benjamin Sauer, Aisada König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2018 - Assessment of human corneas prior to transplantation using high-resolution two-photon imaging
Ana Batista, Hans Georg Breunig, Aisada König, Andreas Schindele, Tobias Hager, et al.
Investigative Ophthalmology and Visual Science, 2018 - Multiphoton tomography of the human eye
Karsten König, Ana Batista, Tobias Hager, Berthold Seitz
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2017 - Towards in vivo breast skin characterization using multiphoton tomography
Ana Batista, Aisada Uchugonova, Hans Georg Breunig, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2017 - Femtosecond-laser assisted cell reprogramming
Hans Georg Breunig, Aisada Uchugonova, Ana Batista, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2017 - In vivo multiphoton imaging of the eyelid skin
Ana Batista, Hans Georg Breunig, Aisada Uchugonova, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2017 - Optical reprogramming of human cells in an ultrashort femtosecond laser microfluidic transfection platform
Aisada Uchugonova, Hans Georg Breunig, Ana Batista, Karsten König
Journal of Biophotonics, 2016 - Cell optoporation with a sub-15 fs and a 250-fs laser
Hans Georg Breunig, Ana Batista, Aisada Uchugonova, Karsten König
Journal of Biomedical Optics, 2016 - Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope
Ana Batista, Hans Georg Breunig, Aisada Uchugonova, António Miguel Morgado, Karsten König
Journal of Biomedical Optics, 2016 - Time-gated FLIM microscope for corneal metabolic imaging
Susana F. Silva, Ana Batista, José Paulo Domingues, Maria João Quadrado, António Miguel Morgado
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2016 - Optical reprogramming of human somatic cells using ultrashort Bessel-shaped near-infrared femtosecond laser pulses
Aisada Uchugonova, Hans Georg Breunig, Ana Batista, Karsten König
Journal of Biomedical Optics, 2015 - Software-aided automatic laser optoporation and transfection of cells
Hans Georg Breunig, Aisada Uchugonova, Ana Batista, Karsten König
Scientific Reports, 2015 - Fluorescence lifetime microscope for corneal metabolic imaging
Susana F. Silva, Ana Batista, Jose Paulo Domingues, Maria Joao Quadrado, Miguel Morgado
Proceedings 2015 IEEE 4th Portuguese Meeting on Bioengineering Enbeng 2015, 2015 - Optical cell cleaning with NIR femtosecond laser pulses
Aisada Uchugonova, Hans Georg Breunig, Ana Batista, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2015 - Software aided automatic cell optoporation system
Hans Georg Breunig, Ana Batista, Aisada Uchugonova, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2015 - Optical reprogramming with ultrashort femtosecond laser pulses
Aisada Uchugonova, Hans G. Breunig, Ana Batista, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2015 - Motionless polarization-resolved second harmonic generation imaging of corneal collagen
Hans G. Breunig, Ana Batista, Aisada Uchugonova, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2015 - Characterization of porcine eyes based on autofluorescence lifetime imaging
Ana Batista, Hans Georg Breunig, Aisada Uchugonova, António Miguel Morgado, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2015 - Development of a time-gated fluorescence lifetime microscope for in vivo corneal metabolic imaging
Susana F. Silva, Ana Batista, Olga C. Castejón, Maria João Quadrado, José Paulo Domingues, et al.
Optics Infobase Conference Papers, 2015 - Development of a time-gated fluorescence lifetime microscope for in vivo corneal metabolic imaging
Susana F. Silva, Ana Batista, Olga C. Castejón, Maria João Quadrado, José Paulo Domingues, et al.
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2015 - High-throughput continuous flow femtosecond laser-assisted cell optoporation and transfection
Hans Georg Breunig, Aisada Uchugonova, Ana Batista, Karsten König
Microscopy Research and Technique, 2014 - Detection of back-reflected SHG from corneal histological sections
Ana Batista, António Miguel Morgado, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2014 - Fluorescence lifetime imaging of induced pluripotent stem cells
Aisada Uchugonova, Ana Batista, Karsten König
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2014 - Label-free SHG imaging and spectral FLIM of corneas using a sub-15 fs laser microscope
Ana Batista, Hans Georg Breunig, Aisada Uchugonova, Berthold Seitz, António Miguel Morgado, et al.
Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2014 - Corneal metabolic imaging by FAD autofluorescence lifetime
A. Batista, S. F. Silva, J. P. Domingues, A. M. Morgado
3rd Portuguese Bioengineering Meeting Enbeng 2013 Book of Proceedings, 2013 - Exploring the impact of ecstasy on retinal physiology: A pioneer study
Ana Batista
2012 IEEE 2nd Portuguese Meeting in Bioengineering Enbeng 2012, 2012 - FLIM as a tool for metabolic imaging of the cornea
A. Batista, C. Loureiro, J. P. Domingues, J. S. Silva, M. Morgado
2012 IEEE 2nd Portuguese Meeting in Bioengineering Enbeng 2012, 2012 - Corneal Cells Metabolic Imaging using FAD Fluorescence Lifetime
Optics Infobase Conference Papers, 2012 - Effects of 3,4-methylenedioxymethamphetamine administration on retinal physiology in the rat
João Martins, Miguel Castelo-Branco, Ana Batista, Bárbara Oliveiros, Ana Raquel Santiago, et al.
Plos One, 2011
