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1.
Life (Basel) ; 14(2)2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38398740

RESUMO

Nonlinear microscopy (NM) enables us to investigate the morphology or monitor the physiological processes of the skin through the use of ultrafast lasers. Fiber (or fiber-coupled) lasers are of great interest because they can easily be combined with a handheld, scanning nonlinear microscope. This latter feature greatly increases the utility of NM for pre-clinical applications and in vivo tissue imaging. Here, we present a fiber-coupled, sub-ps Ti-sapphire laser system being optimized for in vivo, stain-free, 3D imaging of skin alterations with a low thermal load of the skin. The laser is pumped by a low-cost, 2.1 W, 532 nm pump laser and delivers 0.5-1 ps, high-peak-power pulses at a ~20 MHz repetition rate. The spectral bandwidth of the laser is below 2 nm, which results in a low sensitivity for dispersion during fiber delivery. The reduction in the peak intensity due to the increased pulse duration is compensated by the lower repetition rate of our laser. In our proof-of-concept imaging experiments, a ~1.8 m long, commercial hollow-core photonic bandgap fiber was used for fiber delivery. Fresh and frozen skin biopsies of different skin alterations (e.g., adult hemangioma, basal cell cancer) and an unaffected control were used for high-quality, two-photon excitation fluorescence microscopy (2PEF) and second-harmonic generation (SHG) z-stack (3D) imaging.

2.
Postepy Dermatol Alergol ; 37(4): 548-558, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32994778

RESUMO

INTRODUCTION: The anti-cancer properties of high-dose intravenous ascorbic acid have been demonstrated in various malignancies. In our recent study, we tested topically applied ascorbic acid to treat basal cell carcinoma (BCC), and achieved a good clinical response. AIM: Based on these results, we decided to examine the efficacy and tolerability of high-dose intravenous ascorbic acid (IVA) for locally advanced BCC. MATERIAL AND METHODS: In this pilot study, patients diagnosed with locally advanced BCC who were not amenable to radiation, surgical or local therapy (no other treatment option was available at the time) received intravenous ascorbic acid (1-1.8 g/kg), in an outpatient setting, 1-3 times per week for a mean duration of 42 ±23.6 weeks. This therapy was generally well tolerated. RESULTS: Among 4 patients who had a total of 165 (mean: 41 ±51, range: 1-114) skin lesions, 3 patients achieved stable disease and one had progressive disease. There was substantial variability in individual tumor response to therapy. With the aid of two-photon microscopy and second harmonic generation imaging techniques, alterations in collagen structure were observed between tumor nests during IVA therapy. CONCLUSIONS: Our results suggest that IVA is well tolerated in a small group of patients with extensive BCCs. However, in the era of smoothened (Smo) receptor inhibitors, it may only be considered as an adjuvant therapy in treatment-resistant cases.

3.
Lasers Med Sci ; 35(8): 1821-1830, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32372237

RESUMO

Pseudoxanthoma elasticum (PXE, OMIM 264800) is a rare autosomal recessive disorder with ectopic mineralization and fragmentation of elastin fibers. It is caused by mutations of the ABCC6 gene that leads to decreased serum levels of inorganic pyrophosphate (PPi) anti-mineralization factor. The occurrence of severe complications among PXE patients highlights the importance of early diagnosis so that prompt multidisciplinary care can be provided to patients. We aimed to examine dermal connective tissue with nonlinear optical (NLO) techniques, as collagen emits second-harmonic generation (SHG) signal, while elastin can be excited by two-photon excitation fluorescence (TPF). We performed molecular genetic analysis, ophthalmological and cardiovascular assessment, plasma PPi measurement, conventional histopathological examination, and ex vivo SHG and TPF imaging in five patients with PXE and five age- and gender-matched healthy controls. Pathological mutations including one new variant were found in the ABCC6 gene in all PXE patients and their plasma PPi level was significantly lower compared with controls. Degradation and mineralization of elastin fibers and extensive calcium deposition in the mid-dermis was visualized and quantified together with the alterations of the collagen structure in PXE. Our data suggests that NLO provides high-resolution imaging of the specific histopathological features of PXE-affected skin. In vivo NLO may be a promising tool in the assessment of PXE, promoting early diagnosis and follow-up.


Assuntos
Microscopia Óptica não Linear/métodos , Pseudoxantoma Elástico/diagnóstico por imagem , Pele/diagnóstico por imagem , Estudos de Casos e Controles , Colágeno/metabolismo , Tecido Conjuntivo/patologia , Elastina/metabolismo , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Pseudoxantoma Elástico/metabolismo , Pseudoxantoma Elástico/patologia , Pele/metabolismo , Pele/patologia
4.
Science ; 367(6477): 528-537, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31831638

RESUMO

Microglia are the main immune cells in the brain and have roles in brain homeostasis and neurological diseases. Mechanisms underlying microglia-neuron communication remain elusive. Here, we identified an interaction site between neuronal cell bodies and microglial processes in mouse and human brain. Somatic microglia-neuron junctions have a specialized nanoarchitecture optimized for purinergic signaling. Activity of neuronal mitochondria was linked with microglial junction formation, which was induced rapidly in response to neuronal activation and blocked by inhibition of P2Y12 receptors. Brain injury-induced changes at somatic junctions triggered P2Y12 receptor-dependent microglial neuroprotection, regulating neuronal calcium load and functional connectivity. Thus, microglial processes at these junctions could potentially monitor and protect neuronal functions.


Assuntos
Lesões Encefálicas/imunologia , Encéfalo/imunologia , Junções Intercelulares/imunologia , Microglia/imunologia , Neurônios/imunologia , Receptores Purinérgicos P2Y12/fisiologia , Animais , Encéfalo/ultraestrutura , Lesões Encefálicas/patologia , Cálcio , Comunicação Celular/imunologia , Células HEK293 , Humanos , Camundongos , Mitocôndrias/imunologia , Canais de Potássio Shab/genética , Canais de Potássio Shab/fisiologia , Transdução de Sinais
5.
Pathol Oncol Res ; 25(3): 1015-1021, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29981012

RESUMO

Basal cell carcinoma (BCC) is the most frequent malignant neoplasm in the Caucasian population. There are several therapeutic options for BCC, but surgical excision is considered gold standard treatment. As BCCs often have poorly defined borders, the clinical assessment of the tumor margins can be challenging. Therefore, there is an increasing demand for efficient in vivo imaging techniques for the evaluation of tumor borders prior to and during surgeries. In the near future, nonlinear microscopy techniques might meet this demand. We measured the two-photon excitation fluorescence (TPEF) signal of nicotinamide adenine dinucleotide hydride (NADH) and elastin and second harmonic generation (SHG) signal of collagen on 10 ex vivo healthy control and BCC skin samples and compared the images by different quantitative image analysis methods. These included integrated optical density (IOD) measurements on TPEF and SHG images and application of fast Fourier transform (FFT), CT-FIRE and CurveAlign algorithms on SHG images to evaluate the collagen structure. In the BCC samples, we found significantly lower IOD of both the TPEF and SHG signals and higher collagen orientation index utilizing FFT. CT-FIRE algorithm revealed increased collagen fiber length and decreased fiber angle while CurveAlign detected higher fiber alignment of collagen fibers in BCC. These results are in line with previous findings which describe pronounced changes in the collagen structure of BCC. In the future, these novel image analysis methods could be integrated in handheld nonlinear microscope systems, for sensitive and specific identification of BCC.


Assuntos
Carcinoma Basocelular/patologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neoplasias Cutâneas/patologia , Pele/patologia , Estudos de Casos e Controles , Colágeno/metabolismo , Análise de Fourier , Humanos , Prognóstico
6.
Arch Dermatol Res ; 310(5): 463-473, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29725758

RESUMO

Ehlers-Danlos syndrome (EDS) is the name for a heterogenous group of rare genetic connective tissue disorders with an overall incidence of 1 in 5000. The histological characteristics of EDS have been previously described in detail in the late 1970s and early 1980s. Since that time, the classification of EDS has undergone significant changes, yet the description of the histological features of collagen morphology in different EDS subtypes has endured the test of time. Nonlinear microscopy techniques can be utilized for non-invasive in vivo label-free imaging of the skin. Among these techniques, two-photon absorption fluorescence (TPF) microscopy can visualize endogenous fluorophores, such as elastin, while the morphology of collagen fibers can be assessed by second-harmonic generation (SHG) microscopy. In our present work, we performed TPF and SHG microscopy imaging on ex vivo skin samples of one patient with classical EDS and two patients with vascular EDS and two healthy controls. We detected irregular, loosely dispersed collagen fibers in a non-parallel arrangement in the dermis of the EDS patients, while as expected, there was no noticeable impairment in the elastin content. Based on further studies on a larger number of patients, in vivo nonlinear microscopic imaging could be utilized for the assessment of the skin status of EDS patients in the future.


Assuntos
Tecido Conjuntivo/diagnóstico por imagem , Tecido Conjuntivo/patologia , Síndrome de Ehlers-Danlos/diagnóstico por imagem , Microscopia Óptica não Linear/métodos , Pele/diagnóstico por imagem , Colágeno Tipo III/genética , Colágeno Tipo V/genética , Síndrome de Ehlers-Danlos/genética , Elastina/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Linhagem , Conformação Proteica , Pele/patologia
7.
Brain Res Bull ; 137: 277-284, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29325992

RESUMO

Degradation of myelin sheath is thought to be the cause of neurodegenerative diseases, such as multiple sclerosis (MS), but definitive agreement on the mechanism of how myelin is lost is currently lacking. Autoimmune initiation of MS has been recently questioned by proposing that the immune response is a consequence of oligodendrocyte degeneration. To study the process of myelin breakdown, we induced demyelination with cuprizone and applied coherent anti-Stokes Raman scattering (CARS) microscopy, a non-destructive label-free method to image lipid structures in living tissue. We confirmed earlier results showing a brain region dependent myelin destructive effect of cuprizone. In addition, high resolution in situ CARS imaging revealed myelin debris forming lipid droplets alongwith myelinated axon fibers. Quantification of lipid debris with custom-made software for segmentation and three dimensional reconstruction revealed brain region dependent accumulation of lipid drops inversely correlated with the thickness of myelin sheaths. Finally, we confirmed that in situ CARS imaging is applicable to living human brain tissue in brain slices derived from a patient. Thus, CARS microscopy is potent tool for quantitative monitoring of myelin degradation in unprecedented spatiotemporal resolution during oligodendrocyte damage. We think that the accumulation of lipid drops around degrading myelin might be instrumental in triggering subsequent inflammatory processes.


Assuntos
Encéfalo/patologia , Cuprizona/toxicidade , Doenças Desmielinizantes/patologia , Metabolismo dos Lipídeos , Bainha de Mielina/patologia , Idoso , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/cirurgia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Doenças Desmielinizantes/metabolismo , Feminino , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/patologia , Masculino , Camundongos Endogâmicos C57BL , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Técnicas de Cultura de Tecidos
8.
Pathol Oncol Res ; 24(4): 927-930, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29103202

RESUMO

Basal cell carcinoma (BCC) is the most common malignancy in Caucasians. Nonlinear microscopy has been previously utilized for the imaging of BCC, but the captured images do not correlate with H&E staining. Recently, Freudiger et al. introduced a novel method to visualize tissue morphology analogous to H&E staining, using coherent anti-Stokes Raman scattering (CARS) technique. In our present work, we introduce a novel algorithm to post-process images obtained from dual vibration resonance frequency (DVRF) CARS measurements to acquire high-quality pseudo H&E images of BCC samples. We adapted our CARS setup to utilize the distinct vibrational properties of CH3 (mainly in proteins) and CH2 bonds (primarily in lipids). In a narrowband setup, the central wavelength of the pump laser is set to 791 nm and 796 nm to obtain optimal excitation. Due to the partial overlap of the excitation spectra and the 5-10 nm FWHM spectral bandwidth of our lasers, we set the wavelengths to 790 nm (proteins) and 800 nm (lipids). Nonresonant background from water molecules also reduces the chemical selectivity which can be significantly improved if we subtract the DVRF images from each other. As a result, we acquired two images: one for "lipids" and one for" proteins" when we properly set a multiplication factor to minimize the non-specific background. By merging these images, we obtained high contrast H&E "stained" images of BBC's. Nonlinear microscope systems upgraded for real time DVRF CARS measurements, providing pseudo H&E images can be suitable for in vivo assessment of BCC in the future.


Assuntos
Carcinoma Basocelular/diagnóstico , Interpretação de Imagem Assistida por Computador/métodos , Microscopia Óptica não Linear/métodos , Neoplasias Cutâneas/diagnóstico , Algoritmos , Humanos , Vibração
9.
Arch Dermatol Res ; 309(3): 209-215, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28180933

RESUMO

Obesity is a risk factor for several cardiovascular and metabolic diseases. Its influence on the skin is less obvious, yet certain negative effects of adipose tissue inflammation on the dermis have been suggested. Excess weight is closely associated with sedentary behavior, so any increase in physical activity is considered beneficial against obesity. To investigate the effects of obesity and physical exercise on the skin, we established a mouse model in which mice were kept either on a high-fat diet or received standard chow. After the two groups achieved a significant weight difference, physical exercise was introduced to both. Animals were given the opportunity to perform voluntary exercise for 40 min daily in a hamster wheel for a period of 8 weeks. We evaluated the status of the dermis at the beginning and at the end of the exercise period by in vivo nonlinear microscopy. Obese mice kept on high-fat diet lost weight steadily after they started to exercise. In the high-fat diet group, we could detect significantly larger adipocytes and a thicker layer of subcutaneous tissue; both changes started to normalize after exercise. Nonlinear microscopy revealed an impaired collagen structure in obese mice that improved considerably after physical activity was introduced. With the ability to detect damage on collagen structure, we set out to address the question whether this process is reversible. With the use of a novel imaging method, we were able to show the reversibility of connective tissue deterioration as a benefit of physical exercise.


Assuntos
Tecido Adiposo/patologia , Tecido Conjuntivo/fisiologia , Derme/fisiologia , Dieta Hiperlipídica/efeitos adversos , Condicionamento Físico Animal/fisiologia , Adipócitos/patologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/patologia , Comportamento Sedentário
10.
Biomed Opt Express ; 7(11): 4480-4489, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27895989

RESUMO

Obesity related metabolic syndrome and type 2 diabetes have severe consequences on our skin. Latest developments in nonlinear microscopy allow the use of noninvasive, label free imaging methods, such as second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS), for early diagnosis of metabolic syndrome-related skin complications by 3D imaging of the skin and the connective tissue. Our aim was to study effects of various types of diet-induced obesity in mice using these methods. We examined mice on different diets for 32 weeks. The collagen morphology was evaluated four times in vivo by SHG microscopy, and adipocytes were examined once at the end of experiment by ex vivo CARS method. A strong correlation was found between the body weight and the adipocyte size, while we found that the SHG intensity of dermal collagen reduces considerably with increasing body weight. Obese mice on high-fat diet showed worse results than those on high-fat - high-fructose diet. Animals on high-fructose diet did not gain more weight than those on ordinary diet despite of the increased calorie intake, but their collagen damage was nonetheless significant. Obesity and high sugar intake damages the skin, mainly the dermal connective tissue and subcutaneous adipose tissue, which efficiently can be monitored by in vivo SHG and ex vivo CARS microscopy.

11.
Biomed Opt Express ; 7(9): 3531-3542, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27699118

RESUMO

A novel, Yb-fiber laser based, handheld 2PEF/SHG microscope imaging system is introduced. It is suitable for in vivo imaging of murine skin at an average power level as low as 5 mW at 200 kHz sampling rate. Amplified and compressed laser pulses having a spectral bandwidth of 8 to 12 nm at around 1030 nm excite the biological samples at a ~1.89 MHz repetition rate, which explains how the high quality two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG) images are obtained at the average power level of a laser pointer. The scanning, imaging and detection head, which comprises a conventional microscope objective for beam focusing, has a physical length of ~180 mm owing to the custom designed imaging telescope system between the laser scanner mirrors and the entrance aperture of the microscope objective. Operation of the all-fiber, all-normal dispersion Yb-fiber ring laser oscillator is electronically controlled by a two-channel polarization controller for Q-switching free mode-locked operation. The whole nonlinear microscope imaging system has the main advantages of the low price of the fs laser applied, fiber optics flexibility, a relatively small, light-weight scanning and detection head, and a very low risk of thermal or photochemical damage of the skin samples.

12.
Microsc Res Tech ; 78(9): 823-30, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26208320

RESUMO

Nonlinear microscopy techniques are being increasingly used to perform in vivo studies in dermatology. These methods enable us to investigate the morphology and monitor the physiological process in the skin by the use of femtosecond lasers operating in the red, near-infrared spectral range (680-1,300 nm). In this work we used two different techniques that require no labeling: second harmonic generation (SHG) for collagen detection and coherent anti-Stokes Raman scattering (CARS) to assess lipid distribution in genetically obese murine skin. Obesity is one of the most serious public health problems due to its high and increasing prevalence and the associated risk of type 2 diabetes and cardiovascular diseases. Other than these diseases, nearly half of patients with diabetes mellitus suffer from dermatological complications such as delayed wound healing, foot ulcers and several other skin changes. In our experiment we investigated and followed the effects of obesity on dermal collagen alterations and adipocyte enlargement using a technique not reported in the literature so far. Our results indicate that the in vivo SHG and ex vivo CARS imaging technique might be an important tool for diagnosis of diabetes-related skin disorders in the near future.


Assuntos
Colágeno/análise , Fibroblastos/fisiologia , Processamento de Imagem Assistida por Computador , Lipídeos/análise , Microscopia/métodos , Obesidade/patologia , Pele/patologia , Animais , Fibroblastos/química , Humanos , Lasers , Camundongos Obesos , Pele/química , Análise Espectral Raman
13.
Exp Dermatol ; 23(8): 596-605, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24903756

RESUMO

Epidermal Langerhans cells (LCs) function as professional antigen-presenting cells of the skin. We investigated the LC-targeting properties of a special mannose-moiety-coated pathogen-like synthetic nanomedicine DermaVir (DV), which is capable to express antigens to induce immune responses and kill HIV-infected cells. Our aim was to use multiphoton laser microscopy (MLM) in vivo in order to visualize the uptake of Alexa-labelled DV (AF546-DV) by LCs. Knock-in mice expressing enhanced green fluorescent protein (eGFP) under the control of the langerin gene (CD207) were used to visualize LCs. After 1 h, AF546-DV penetrated the epidermis and entered the eGFP-LCs. The AF546-DV signal was equally distributed inside the LCs. After 9 h, we observed AF546-DV signal accumulation that occurred mainly at the cell body. We demonstrated in live animals that LCs picked up and accumulated the nanoparticles in the cell body.


Assuntos
Vacinas contra a AIDS/farmacocinética , Células de Langerhans/metabolismo , Nanomedicina/métodos , Nanopartículas , Animais , Transporte Biológico , Proteínas de Fluorescência Verde/genética , Células de Langerhans/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Modelos Animais
14.
J Invest Dermatol ; 134(1): 105-111, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23884312

RESUMO

Recently, a transglutaminase 3 knockout (TGM3/KO) mouse was generated that showed impaired hair development, but no gross defects in the epidermal barrier, although increased fragility of isolated corneocytes was demonstrated. Here we investigated the functionality of skin barrier in vivo by percutaneous sensitization to FITC in TGM3/KO (n=64) and C57BL/6 wild-type (WT) mice (n=36). Cutaneous inflammation was evaluated by mouse ear swelling test (MEST), histology, serum IgE levels, and by flow cytometry from draining lymph nodes. Inflammation-induced significant MEST difference (P<0.0001) was detected between KO and WT mice and was supported also by histopathology. A significant increase of CD4+ CD25+-activated T cells (P<0.01) and elevated serum IgE levels (P<0.05) in KO mice indicated more the development of FITC sensitization than an irritative reaction. Propionibacter acnes-induced intracutaneous inflammation showed no difference (P=0.2254) between the reactivity of WT and KO immune system. As in vivo tracer, FITC penetration from skin surface followed by two-photon microscopy demonstrated a more invasive percutaneous penetration in KO mice. The clinically uninvolved skin in TGM3/KO mice showed impaired barrier function and higher susceptibility to FITC sensitization indicating that TGM3 has a significant contribution to the functionally intact cutaneous barrier.


Assuntos
Dermatite de Contato/imunologia , Dermatite de Contato/microbiologia , Infecções por Bactérias Gram-Positivas/imunologia , Propionibacterium acnes/imunologia , Transglutaminases/imunologia , Animais , Dermatite de Contato/etiologia , Edema/imunologia , Edema/metabolismo , Feminino , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/toxicidade , Infecções por Bactérias Gram-Positivas/metabolismo , Imunoglobulina E/imunologia , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Propionibacterium acnes/metabolismo , Pele/imunologia , Pele/metabolismo , Pele/microbiologia , Transglutaminases/genética
15.
Eur J Neurosci ; 27(2): 364-77, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18215234

RESUMO

Stratum radiatum interneurons, unlike pyramidal cells, are rich in nicotinic acetylcholine receptors (nAChRs); however, the role of these receptors in plasticity has remained elusive. As opposed to previous physiological studies, we found that functional alpha7-subunit-containing nAChRs (alpha7-nAChRs) are abundant on interneuron dendrites of rats. Moreover, dendritic Ca2+ transients induced by activation of alpha7-nAChRs increase as a function of distance from soma. The activation of these extrasynaptic alpha7-nAChRs by cholinergic agonists either facilitated or depressed backpropagating action potentials, depending on the timing of alpha7-nAChR activation. We have previously shown that dendritic alpha7-nAChRs are involved in the regulation of synaptic transmission, suggesting that alpha7-nAChRs may play an important role in the regulation of the spike timing-dependent plasticity. Here we provide evidence that long-term potentiation is indeed boosted by stimulation of dendritic alpha7-nAChRs. Our results suggest a new mechanism for a cholinergic switch in memory encoding and retrieval.


Assuntos
Potenciais de Ação/fisiologia , Dendritos/fisiologia , Interneurônios/fisiologia , Potenciação de Longa Duração/fisiologia , Receptores Nicotínicos/fisiologia , Animais , Interneurônios/citologia , Masculino , Plasticidade Neuronal/fisiologia , Ratos , Ratos Wistar , Receptor Nicotínico de Acetilcolina alfa7
16.
Appl Opt ; 46(10): 1860-5, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17356631

RESUMO

We propose a two-photon microscope scheme capable of real-time, three-dimensional investigation of the electric activity pattern of neural networks or signal summation rules of individual neurons in a 0.6 mm x 0.6 mm x 0.2 mm volume of the sample. The points of measurement are chosen according to a conventional scanning two-photon image, and they are addressed by separately adjustable optical fibers. This allows scanning at kilohertz repetition rates of as many as 100 data points. Submicrometer spatial resolution is maintained during the measurement similarly to conventional two-photon microscopy.


Assuntos
Potenciais de Ação/fisiologia , Tecnologia de Fibra Óptica/instrumentação , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Rede Nervosa/fisiologia , Neurônios/fisiologia , Células Cultivadas , Humanos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Neurônios/citologia , Fibras Ópticas
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