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Two-photon Laser Scanning Microscopy (two-photon + laser_scanning_microscopy)
Selected AbstractsReduction in DNA Synthesis During Two-photon Microscopy of Intrinsic Reduced Nicotinamide Adenine Dinucleotide Fluorescence,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2005Michael G. Nichols ABSTRACT Two-photon laser scanning microscopy (TPLSM) of endogenous reduced nicotinamide adenine dinucleotide (NAD(P)H) provides important information regarding the cellular metabolic state. When imaging the punctate mitochondrial fluorescence originating from NAD(P)H in a rat basophilic leukemia (RBL) cell at low laser powers, no morphological changes are evident, and photobleaching is not observed when many images are taken. At higher powers, mitochondrial NAD(P)H fluorescence bleaches rapidly. To assess the limitations of this technique and to quantify the extent of photodamage, we have measured the effect of TPLSM on DNA synthesis. Although previous reports have indicated a threshold power for "safe" two-photon imaging, we find the laser power to be an insufficient indicator of photodamage. A more meaningful metric is a two-photon-absorbed dose that is proportional to the number of absorbed photon paris. A temporary reduction of DNA synthesis in RBL cells occurs whenever a threshold dose of approximately 2 × 1053 photon2 cm,4 s,1 is exceeded. This threshold is independent of laser intensity when imaging with average powers ranging from 5 to 17 mW at 740 nm. Beyond this threshold, the extent of the reduction is intensity dependent. DNA synthesis returns to control levels after a recovery period of several hours. [source] Bioimaging with Two-Photon-Induced Luminescence from Triangular Nanoplates and Nanoparticle Aggregates of GoldADVANCED MATERIALS, Issue 22 2009Yuqiang Jiang Cell imaging with two-photon-induced photoluminescence is demonstrated using gold nanoparticles (either triangular nanoplates or nanosphere aggregates) as imaging agents. On being conjugated to yeast cells, gold nanoplates exhibit visible two-photon excited luminescence that can be detected by two-photon laser scanning microscopy. The cells can be either dried (in the air) or alive (in water). [source] Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopyJOURNAL OF CUTANEOUS PATHOLOGY, Issue 8 2002Janine C. Malone Background: Various structural components of human skin biopsy specimens are difficult to visualize using conventional histologic approaches. Methods: We used two-photon microscopy and advanced imaging software to render three-dimensional (3D) images of in situ nerves, blood vessels, and hair follicles labeled with various fluorescent markers. Archived frozen human skin biopsy specimens were cryosectioned up to 150 µm in thickness and fluorescently stained with rhodamine- or fluorescein-labeled antibodies or lectins. Optical sections were collected by two-photon microscopy and the resulting data sets were analyzed in three dimensions using Voxx software. Results: Reconstructed image volumes demonstrated the complex 3D morphology of nerves, blood vessels and adnexal structures in normal mucocutaneous tissue. Conclusion: Two-photon microscopy and Voxx rendering software allow for detailed 3D visualization of structures within human mucocutaneous biopsy specimens, as they appear in situ, and facilitate objective interpretation of variations in their morphology. These techniques may be used to investigate disorders involving cutaneous structures that are difficult to visualize by means of traditional microscopy. [source] Complementary roles of platelets and coagulation in thrombus formation on plaques acutely ruptured by targeted ultrasound treatment: a novel intravital modelJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2009M. J. E. KUIJPERS Summary.,Background:,Atherothrombosis is a major cause of cardiovascular events. However, animal models to study this process are scarce. Objectives:,We describe the first murine model of acute thrombus formation upon plaque rupture to study atherothrombosis by intravital fluorescence microscopy. Methods:,Localized rupture of an atherosclerotic plaque in a carotid artery from Apoe,/, mice was induced in vivo using ultrasound. Rupture of the plaque and formation of localized thrombi were verified by two-photon laser scanning microscopy (TPLSM) in isolated arteries, and by immunohistochemistry. The thrombotic reaction was quantified by intravital fluorescence microscopy. Results:,Inspection of the ultrasound-treated plaques by histochemistry and TPLSM demonstrated local damage, collagen exposure, luminal thrombus formation as well as intra-plaque intrusion of erythrocytes and fibrin. Ultrasound treatment of healthy carotid arteries resulted in endothelial damage and limited platelet adhesion. Real-time intravital fluorescence microscopy demonstrated rapid platelet deposition on plaques and formation of a single thrombus that remained subocclusive. The thrombotic process was antagonized by thrombin inhibition, or by blocking of collagen or adenosine diphosphate receptor pathways. Multiple thrombi were formed in 70% of mice lacking CD40L. Conclusions:,Targeted rupture of murine plaques results in collagen exposure and non-occlusive thrombus formation. The thrombotic process relies on platelet activation as well as on thrombin generation and coagulation, and is sensitive to established and novel antithrombotic medication. This model provides new possibilities to study atherothrombosis in vivo. [source] | ||||||||||