| 1. |
- Ericson, Marica B, 1974, et al.
(författare)
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Exploring plasmonic coupling as a stimuli responsive contrast mechanism in multiphoton microscopy
- 2018
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Ingår i: Proceedings Volume 10509, Plasmonics in Biology and Medicine XV. - : SPIE. - 1605-7422. - 9781510615045 - 9781510615038
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Konferensbidrag (refereegranskat)abstract
- A novel approach for optical biosensing can be obtained based multiphoton induced luminescence (MIL) and its dependence on plasmonic coupling. It has been shown that the proximity of spherical AuNPs determines the generation of MIL in far-field multiphoton laser scanning microscopy (MPM). A stimuli responsive contrast mediator with high sensitivity can be created by controlling the aggregated state of AuNP. In this study we explore a system based on spherical AuNPs functionalized with beta-cyclodextrin and multiple beta-D-lactose units (lacto-CD-AuNP). The aim of the beta-D- lactose units is to target cancer cells, based on overexpression of galectin3 (Gal-3) receptors. The results demonstrate that clustering of particles, and thereby MIL signal, was only acquired from tumor cell lines, i.e., SK-MEL-28 and A431, while not from normal keratinocytes (HEKn). Thus further studies should be undertaken to translate the concept to a preclinical setting.
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| 2. |
- Eskilson, Olof, 1992-, et al.
(författare)
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Self-Assembly of Mechanoplasmonic Bacterial Cellulose-Metal Nanoparticle Composites
- 2020
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Ingår i: Advanced Functional Materials. - : Wiley-VCH Verlagsgesellschaft. - 1616-301X .- 1616-3028. ; 30:40
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Tidskriftsartikel (refereegranskat)abstract
- Nanocomposites of metal nanoparticles (NPs) and bacterial nanocellulose (BC) enable fabrication of soft and biocompatible materials for optical, catalytic, electronic, and biomedical applications. Current BC-NP nanocomposites are typically prepared by in situ synthesis of the NPs or electrostatic adsorption of surface functionalized NPs, which limits possibilities to control and tune NP size, shape, concentration, and surface chemistry and influences the properties and performance of the materials. Here a self-assembly strategy is described for fabrication of complex and well-defined BC-NP composites using colloidal gold and silver NPs of different sizes, shapes, and concentrations. The self-assembly process results in nanocomposites with distinct biophysical and optical properties. In addition to antibacterial materials and materials with excellent senor performance, materials with unique mechanoplasmonic properties are developed. The homogenous incorporation of plasmonic gold NPs in the BC enables extensive modulation of the optical properties by mechanical stimuli. Compression gives rise to near-field coupling between adsorbed NPs, resulting in tunable spectral variations and enhanced broadband absorption that amplify both nonlinear optical and thermoplasmonic effects and enables novel biosensing strategies.
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| 3. |
- James, Jeemol, et al.
(författare)
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Fluorescence Correlation Spectroscopy Combined with Multiphoton Laser Scanning Microscopy-A Practical Guideline
- 2021
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Ingår i: Applied Sciences. - : MDPI AG. - 2076-3417. ; 11:5
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Tidskriftsartikel (refereegranskat)abstract
- Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue-being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research.
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| 4. |
- James, Jeemol
(författare)
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Laser Scanning Multiphoton Microscopy – Focusing on Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging for Biomedical Applications
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Laser scanning multiphoton microscopy (MPM) is considered as a non-invasive technology for three-dimensional imaging of complex biological tissues. The quantitative potential of the MPM is not investigated as much as qualitative imaging. To explore the quantitative aspects, MPM is here combined with fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging (FLIM) for different biomedical applications. The first part of the thesis (papers 1 and 2) emphasizes the importance of validation and optimization of an experimental MPM set up to develop a systematic methodology to combine MPM with FCS utilizing a single-photon counting method. A practical guideline featuring the theoretical and experimental boundaries to implement two-photon excited FCS in the MPM experimental setup is developed in paper 1. Concentration range, numerical aperture of the objective lens, and laser excitation power were found as prime factors to be optimized to study the diffusion time using MPM-FCS. To extend the applicability of MPM-FCS in biological samples, proof of principle was demonstrated by measuring the viscosity of collagen gel from the diffusion time measurements of Rhodamine B in different water glycerol mixtures (Paper 2). In the final part of the thesis (papers 3 and 4), MPM-FLIM was employed for different biomedical applications. An exploratory study was performed using MPM-FLIM for ex vivo investigations in positive and negative sentinel lymph nodes derived from melanoma patients (Paper 3). MPM-FLIM demonstrates the potential to differentiate atypical cells, healthy lymphocytes, and blood vessels in sentinel lymph nodes along with morphological features and fluorescence lifetime data. Two-photon spectral and FLIM characterization of the complex intrinsic cellular fluorophores such as nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and keratin in keratinocytes were performed to facilitate the non-invasive imaging of epidermal and dermal tissue cultures in vitro (paper 4). This study exposed the importance of keratin signal and should not be neglected when FLIM data is interpreted which needs to be done very carefully in complex biological samples. Taken together, this thesis demonstrates how to adopt MPM in combination with FCS and FLIM highlighting both the methodology development and biomedical applications.
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| 5. |
- James, Jeemol, et al.
(författare)
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Optimizing Ti:Sapphire laser for quantitative biomedical imaging
- 2018
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Ingår i: Progress in Biomedical Optics and Imaging - Proceedings of SPIE. 1049824. - : SPIE. - 1605-7422 .- 1996-756X. - 9781510614826
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Konferensbidrag (refereegranskat)abstract
- Ti:Sapphire lasers are powerful tools in the field of scientific research and industry for a wide range of applications such as spectroscopic studies and microscopic imaging where tunable near-infrared light is required. To push the limits of the applicability of Ti:Sapphire lasers, fundamental understanding of the construction and operation is required. This paper presents two projects, (i) dealing with the building and characterization of custom built tunable narrow linewidth Ti:Sapphire laser for fundamental spectroscopy studies; and the second project (ii) the implementation of a fs-pulsed commercial Ti:Sapphire laser in an experimental multiphoton microscopy platform. For the narrow linewidth laser, a gold-plated diffraction grating with a Littrow geometry was implemented for highresolution wavelength selection. We demonstrate that the laser is tunable between 700 to 950 nm, operating in a pulsed mode with a repetition rate of 1 kHz and maximum average output power around 350 mW. The output linewidth was reduced from 6 GHz to 1.5 GHz by inserting an additional 6 mm thick etalon. The bandwidth was measured by means of a scanning Fabry Perot interferometer. Future work will focus on using a fs-pulsed commercial Ti:Sapphire laser (Tsunami, Spectra physics), operating at 80 MHz and maximum average output power around 1 W, for implementation in an experimental multiphoton microscopy set up dedicated for biomedical applications. Special focus will be on controlling pulse duration and dispersion in the optical components and biological tissue using pulse compression. Furthermore, time correlated analysis of the biological samples will be performed with the help of time correlated single photon counting module (SPCM, Becker & Hickl) which will give a novel dimension in quantitative biomedical imaging.
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| 6. |
- James, Jeemol, et al.
(författare)
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Report on fluorescence lifetime imaging using multiphoton laser scanning microscopy targeting sentinel lymph node diagnostics.
- 2020
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Ingår i: Journal of biomedical optics. - 1560-2281. ; 25:7, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- Sentinel lymph node (SLN) biopsy is an important method for metastasis staging in, e.g., patients with malignant melanoma. Tools enabling prompt histopathological analysis are expected to facilitate diagnostics; optical technologies are explored for this purpose.The objective of this exploratory study was to investigate the potential of adopting multiphoton laser scanning microscopy (MPM) together with fluorescence lifetime analysis (FLIM) for the examination of lymph node (LN) tissue ex vivo.Five LN tissue samples (three metastasis positive and two negative) were acquired from a biobank comprising tissues from melanoma patients. Tissues were deparaffinized and subjected to MPM-FLIM using an experimental MPM set-up equipped with a time correlated single photon counting module enabling FLIM.The data confirm that morphological features similar to conventional histology were observed. In addition, FLIM analysis revealed elevated morphological contrast, particularly for discriminating between metastatic cells, lymphocytes, and erythrocytes.Taken together, the results from this investigation show promise for adopting MPM-FLIM in the context of SLN diagnostics and encourage further translational studies on fresh tissue samples.
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| 7. |
- Malak, Monika, 1993, et al.
(författare)
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Contribution of autofluorescence from intracellular proteins in multiphoton fluorescence lifetime imaging.
- 2022
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Multiphoton fluorescence lifetime imaging microscopy (MPM-FLIM) is extensively proposed as a non-invasive optical method to study tissue metabolism. The approach is based on recording changes in the fluorescence lifetime attributed to metabolic co-enzymes, of which nicotinamide adenine dinucleotide (NADH) is of major importance. However, intrinsic tissue fluorescence is complex. Particularly when utilizing two-photon excitation, as conventionally employed in MPM. This increases the possibility for spectral crosstalk and incorrect assignment of the origin of the FLIM signal. Here we demonstrate that in keratinocytes, proteins such as keratin may interfere with the signal usually assigned to NADH in MPM-FLIM by contributing to the lifetime component at 1.5ns. This is supported by a change in fluorescence lifetime distribution in KRT5- and KRT14-silenced cells. Altogether, our results suggest that the MPM-FLIM data originating from cellular autofluorescence is far more complex than previously suggested and that the contribution from other tissue constituents should not be neglected-changing the paradigm for data interpretation in this context.
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| 8. |
- Thomsen, Hanna, 1989, et al.
(författare)
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Spatially confined photoinactivation of bacteria: Towards novel tools for detailed mechanistic studies
- 2018
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Ingår i: Progress in Biomedical Optics and Imaging - Proceedings of SPIE. Multiphoton Microscopy in the Biomedical Sciences XVIII 2018; San Francisco; United States; 28 January 2018 through 30 January 2018. Vol 10498. - : SPIE. - 1605-7422 .- 1996-756X. - 9781510614826
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Konferensbidrag (refereegranskat)abstract
- © 2018 SPIE. Antimicrobial resistance is a serious global threat fueling an accelerated field of research aimed at developing novel antimicrobial therapies. A particular challenge is the treatment of microbial biofilms formed upon bacterial growth and often associated with chronic infections. Biofilms comprise bacteria that have adhered to a surface and formed 3D microcolonies, and demonstrate significantly increased antimicrobial resistance compared to the planktonic counterpart. A challenge in developing novel strategies for fighting these chronic infections is a lack of mechanistic understanding of what primarily contributes to enhanced drug resistance. Tools for noninvasive study of live biofilms are necessary to begin to understand these mechanisms on both a single cell and 3D level. Herein, a method by which multiphoton microscopy is implemented to study a biofilm model of Staphylococcus epidermidis to noninvasively visualize and measure penetration of compounds in 3D biofilm structure and two photon excitation was exploited for spatially confined photoinactivation and microscopy optimized for evaluation of microbiological viability at a microscopic level. Future studies are aimed at future development of the proposed techniques for detailed studies of, e.g., quorum sensing and mechanisms contributing to antimicrobial resistance.
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