| 1. |
- Borglin, Johan, 1986, et al.
(author)
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Improving multiphoton microscopy using annular beam shaping, focusing on imaging of human skin
- 2014
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In: Multiphoton Microscopy in the Biomedical Sciences XIV: 2-4 February 2014, San Francisco, California, United States. Progress in Biomedical Optics and Imaging - Proceedings of SPIE. - : SPIE. - 1605-7422. ; 8948
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Conference paper (peer-reviewed)abstract
- Multiphoton fluorescence microscopy (MPM) is a method for high resolution, non-invasive investigations of biological tissue. The aim of introducing an annular shaped laser beam is to reduce the ouf-of-focus generated background signal improving imaging of light scattering tissue such as human skin. Simulations show that 50% of the beam radius can be blocked, while preserving the shape of the point spread function. Initial experiments performed on a phantom consisting of fluorescein and fluorescent beads embedded in agar by using a custom built MPM-set up show that by introducing a simple beam blocker to create an annular beam, the background signal is reduced with approximately 5%. Future work will include optimizing the set up, and creating phantoms with more light scattering properties. © 2014 SPIE.
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| 2. |
- Borglin, Johan, 1986, et al.
(author)
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Insights on proximity effect and multiphoton induced luminescence from gold nanospheres in far field optical microscopy
- 2015
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In: Applied Physics Letters. - : AIP Publishing. - 0003-6951 .- 1077-3118. ; 107:23
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Journal article (peer-reviewed)abstract
- Goldnanoparticles can be visualized in far-field multiphoton laser-scanning microscopy (MPM) based on the phenomena of multiphoton induced luminescence (MIL). This is of interest for biomedical applications, e.g., for cancer diagnostics, as MPM allows for working in the near-infrared(NIR) optical window of tissue. It is well known that the aggregation of particles causes a redshift of the plasmon resonance, but its implications for MIL applying far-field MPM should be further exploited. Here, we explore MIL from 10nm goldnanospheres that are chemically deposited on glass substrates in controlled coverage gradients using MPM operating in NIR range. The substrates enable studies of MIL as a function of inter-particle distance and clustering. It was shown that MIL was only detected from areas on the substrates where the particle spacing was less than one particle diameter, or where the particles have aggregated. The results are interpreted in the context that the underlying physical phenomenon of MIL is a sequential two-photon absorption process, where the first event is driven by the plasmon resonance. It is evident that goldnanospheres in this size range have to be closely spaced or clustered to exhibit detectable MIL using far-field MPM operating in the NIR region.
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| 3. |
- Ericson, Marica B, 1974, et al.
(author)
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Two-photon laser-scanning fluorescence microscopy applied for studies of human skin
- 2008
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In: Journal of Biophotonics. - : Wiley. - 1864-0648 .- 1864-063X. ; 1:4, s. 320-330
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Journal article (peer-reviewed)abstract
- Two-photon laser scanning fluorescence microscopy (TPM) has been shown to be advantageous for imaging optically turbid media such as human skin. The ability of performing three-dimensional imaging without presectioning of the samples makes the technique not only suitable for noninvasive diagnostics but also for studies of topical delivery of xenobiotics. Here, TPM is used as a method to visualize both autofluorescent and exogenous fluorophores in skin. Samples exposed to sulforhodamine B have been scanned from two directions to investigate attenuation effects. It is shown that optical effects play a major role. Thus, TPM is excellent for visualizing the localization and distribution of fluorophores in human skin, although quantification might be difficult. Furthermore, an image-analysis algorithm has been implemented to facilitate interpretation of TPM images of autofluorescent features of nonmelanoma skin cancer obtained ex vivo. The algorithm was designed to detect cell nuclei and currently has a sensitivity and specificity of 82% and 78% to single cell nuclei. However, in order to detect multinucleated cells, the algorithm needs further development. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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| 4. |
- Guldbrand, Stina, 1970, et al.
(author)
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Measuring the diffusion of fluorophores in human skin by two-photon fluorescence correlation spectroscopy combined with measurements of point spread function
- 2011
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In: MULTIPHOTON MICROSCOPY scigloo.IN THE BIOMEDICAL SCIENCES XI Book Series: Proceedings of SPIE. ; 7903, s. 7903291-7903296
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Journal article (peer-reviewed)abstract
- Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been used in combination with measurements of the point spread function (PSF), for quantitative analysis of fluorophores in excised human skin. Measurements have been performed at depths between 0 and 40 μm. The PSF, measured as full width at half maximum, was found not to depend on the depth. Measurements revealed difference in diffusion coefficient depending on extra- or intracellular location of fluorophore. The number of molecules was accumulating close to the surface and then decreased by the depth. The results from our study show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.
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| 5. |
- Guldbrand, Stina, 1970
(author)
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Non-linear Optical Microscopy and Spectroscopy for Biomedical Studies
- 2012
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Doctoral thesis (other academic/artistic)abstract
- This thesis is based on the application of non-linear optical microscopy and spectroscopy techniques within biomedical research. Non-linear optical microscopy gives the possibility of exciting fluorophores using near infrared light. This is an advantage when working with biological tissue, which has low absorption in this wavelength area, making up an ”open window”for non-invasive three dimensional imaging. Of particular interest has been the study of fluorescent xenobiotics in human skin using two-photon fluorescence laser scanning microscopy. The background is the desire to develop new non-invasive tools to study topical drug delivery and improve the understanding of mechanisms involved in contact allergy. In addition, two-photon fluorescence microscopy is a potential tool for non-invasive skin cancer diagnostics, which also is a topic of this thesis. In order to acquire quantitative data, two-photon fluorescence microscopy has been combined with fluorescence correlation spectroscopy (TPFCS). This is to the best of my knowledge the first time TPFCS has been applied to study the diffusion and distribution of fluorescent molecules in human skin.By the use of this method a reactive compound, acting as a contact allergen, has been demonstrated to bind to proteins in the top epidermal layers of the skin, resulting in a significantly slower diffusion. It has been proposed that endogenously formed protoporphyrin IX (PpIX) can be applied to improve contrast when performing two-photon fluorescence microscopy for diagnostics of non-melanoma skin cancer. In this thesis, it is demonstrated that detection of two-photon excited fluorescence of endogenous PpIX in human skin is not possible. Instead, it is preferable to use a slightly shorter wavelength, i.e. 710 nm, to induce one-photon anti-Stokes fluorescence. This finding is of great importance for continued work in the field, bringing non-linear optical microscopy into the clinics. Plasmonic noble metal nanoparticles, e.g. gold nanoparticles, have been proposed as contrast enhancers for several biomedical applications. In this thesis, gold nanoparticles have been explored with respect to their multiphoton induced luminescence when combined with non-linear optical microscopy. By investigating 10 nm gold nanoparticles deposited on glass plates, it is here demonstrated that aggregation and short inter-particle distances are prerequisites in order to detect multiphoton induced luminescence. Thus detection of single particles in a biological environment is unlikely, and future work should be undertaken to explore how the clustering can be controlled in a biological environment to, e.g, be used as a contrast mechanism.
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| 6. |
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| 7. |
- Guldbrand, Stina, 1970, et al.
(author)
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Two-photon fluorescence correlation microscopy combined with measurements of point spread function; investigations made in human skin
- 2010
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In: Optics Express. - 1094-4087. ; 18:15, s. 15289-15302
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Journal article (peer-reviewed)abstract
- Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been applied in connection to measurements of the point spread function (PSF) for quantitative analysis of sulphorhodamine B (SRB) in excised human skin. The PSF was measured using subresolution fluorescent beads embedded in the skin specimen. The PSF, measured as full width at half maximum (FWHM) was found to be 0.41 ± 0.05 μm in the lateral direction, and 1.2 ± 0.4 μm in the axial direction. The molecular diffusion of SRB inside the skin ranged between 0.5 and 15.0 × 10 −8 cm2/s. The diffusion coefficient is not dependent on depths down to 40 μm. The fluorophores were found to accumulate on the upper layers of the skin. This work is the first TPFCS study in human skin. The results show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.
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| 8. |
- Guldbrand, Stina, 1970, et al.
(author)
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Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular diffusion within human skin
- 2013
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In: European Journal of Pharmaceutics and Biopharmaceutics. - : Elsevier BV. - 0939-6411. ; 84:2, s. 430-436
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Journal article (peer-reviewed)abstract
- There is a need for tools enabling quantitative imaging of biological tissue for pharmaceutical applications. In this study, two-photon fluorescence microscopy (TPM) has been combined with fluorescence correlation spectroscopy (FCS), demonstrating proof-of-principle providing quantitative data of fluorophore concentration and diffusion in human skin. Measurements were performed on excised skin exposed to either rhodamine B (RB) or rhodamine B isothiocyanate (RBITC), chosen based on their similarity in fluorescence yield and molecular weight, but difference in chemical reactivity. The measurements were performed at tissue depths in the range 0 and 20 pm, and the diffusion coefficients at skin depths 5 and 10 mu m were found to be significantly different (P < 0.05). Overall median values for the diffusion coefficients were found to be 4.0 x 10(-13) m(2)/s and 2.0 x 10(-13) m(2)/s for RB and RBITC, respectively. These values correspond to the diffusion of a hard sphere with a volume eight times larger for RBITC compared to RB. This indicates that the RBITC have bound to biomolecules in the skin, and the measured signal is obtained from the RBITC-biomolecule complexes, demonstrating the potential of the TPM-FCS method to track molecular interactions in an intricate biological matrix such as human skin.
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| 9. |
- Kantere, Despoina, et al.
(author)
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Anti-Stokes fluorescence from endogenously formed protoporphyrin IX - Implications for clinical multiphoton diagnostics.
- 2013
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In: Journal of biophotonics. - : Wiley. - 1864-0648 .- 1864-063X. ; 6:5, s. 409-415
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Journal article (peer-reviewed)abstract
- Multiphoton imaging based on two-photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce. No report shows conclusive evidence that endogenously formed PpIX increases tumor contrast when performing multiphoton imaging in the clinical situation. We here demonstrate by spectral analysis that two-photon excitation of endogenously formed PpIX does not provide additional contrast in superficial basal cell carcinomas. In fact, the PpIX signal is overshadowed by the autofluorescent background. The results show that PpIX should be excited at a wavelength giving rise to one-photon anti-Stokes fluorescence, to overcome the autofluorescent background. Thus, this study reports on a plausible method, which can be implemented for clinical investigations on endogenously formed PpIX using multiphoton microscopy (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
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| 10. |
- Kirejev, Vladimir, 1984, et al.
(author)
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Multiphoton microscopy – a powerful tool in skin research and topical drug delivery science
- 2012
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In: Journal of Drug Delivery Science and Technology. - 1773-2247. ; 22:3, s. 250-259
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Journal article (peer-reviewed)abstract
- Multiphoton microscopy (MPM) has become a powerful complementary tool in biomedical research, enabling non-invasive three-dimensional imaging of tissue with high resolution. The major advantage is that investigations and visualization can be performed without mechanical destruction of the sample through tissue sectioning. This review will give a brief introduction to the technology, accompanied by examples of how the technique can be implemented within the field of skin research. Specifically, MPM has already made it possible to visualize cellular morphology and the cutaneous distribution of topically applied compounds applied to intact skin. MPM provides information that can be used to assess the bioavailability of drugs and to visualize drug penetration pathways into skin. MPM has also been implemented as a tool for obtaining non-invasive tissue biopsy based on skin autofluorescence in connection to diagnostics of skin cancer. We will also briefly present some recent results where MPM has been used to track cyclodextrin based drugs applied topically. Finally, we will discuss some future challenges of the technology, including label-free imaging, multimodal techniques, and quantitative imaging.
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| 11. |
- Kirejev, Vladimir, 1984, et al.
(author)
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Novel nanocarriers for topical drug delivery: investigating delivery efficiency and distribution in skin using two-photon microscopy
- 2011
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In: Proc. SPIE, Multiphoton Microscopy in the Biomedical Sciences XI, editors: Ammasi Periasamy, Karsten König, Peter T. C. So, 23 January 2011. - : SPIE. ; 7903:1
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Conference paper (peer-reviewed)abstract
- The complex structure of skin represents an effective barrier against external environmental factors, as for example, different chemical and biochemical compounds, yeast, bacterial and viral infections. However, this impermeability prevents efficient transdermal drug delivery which limits the number of drugs that are able to penetrate the skin efficiently. Current trends in drug application through skin focus on the design and use of nanocarriers for transport of active compounds. The transport systems applied so far have several drawbacks, as they often have low payload, high toxicity, a limited variability of inclusion molecules, or long degradation times. The aim of these current studies is to investigate novel topical drug delivery systems, e.g. nanocarriers based on cyclic oligosaccharides - cyclodextrins (CD) or iron (III)-based metal-organic frameworks (MOF). Earlier studies on cell cultures imply that these drug nanocarriers show promising characteristics compared to other drug delivery systems. In our studies, we use two-photon microscopy to investigate the ability of the nanocarriers to deliver compounds through ex-vivo skin samples. Using near infrared light for excitation in the so called optical window of skin allows deep-tissue visualization of drug distribution and localization. In addition, it is possible to employ two-photon based fluorescence correlation spectroscopy for quantitative analysis of drug distribution and concentrations in different cell layers.
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