Multicolor nonlinear microscopy of living tissue using two-and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer.M ultiphoton microscopy (MPM) (1, 2) is well suited for high-resolution imaging of intrinsic molecular signals in living specimens. It provides convenient excitation of the characteristic UV absorption bands of intrinsic fluorophores using IR illumination, leaving a broad uninterrupted spectral region for efficient multicolor fluorescence collection. The ability of MPM to produce images deep in optically thick preparations is crucial for intravital tissue microscopy. In addition, second harmonic generation (SHG) enables direct imaging (3) of anisotropic biological structures possessing large hyperpolarizabilities, such as collagen (4, 5). These imaging modalities are easy to implement simultaneously and differ only in optical filter selection and detector placement.To date, most biological MPM has depended on labeling with conventional fluorophores or fluorescent proteins such as the GFPs; however, a few studies have used two-photon excitation (2PE) of intrinsic molecules such as NAD(P)H (6-8) and flavins (9), three-photon excitation (3PE) of serotonin (10-12), and SHG of collagen, skeletal muscle, and microtubules (2, 13). The combination of intrinsic and extrinsic signals is particularly powerful. For example, the process of tumor cell migration along collagen fibers can be observed by using GFP-labeled tumor cells and intrinsic collagen SHG (14). 2PE fluorescence spectra currently exist for NAD(P)H and some flavins (9, 15), and 3PE spectra exist for serotonin, tryptophan, and dopamine (10). Here we report the SHG efficiency spectrum for various collagens and 2PE cross sections of a ''basis set'' of tissue 2PE fluorophores. We demonstrate m-resolution multiphoton imaging of normal tissue structure and of disease states such as Alzheimer's disease (AD) and cancer. Intrinsic emission MPM in living specimens yields detail that may ultimately prove useful to clinical diagnostics as well as to basic biological research. Materials and MethodsInstrumentation and the associated methodologies used in these investigations are described in detail in Supporting Materials and Methods, which is published as supporting information on the PNAS web site, www.pnas.org...

No abstract

We investigated the role of neuronal (type I) nitric oxide synthase (nNOS) in NMDA-mediated excitotoxicity in wild-type (SV129 and C57BL/6J) and type I NOS knock-out (nNOS Ϫ/Ϫ ) mice and examined its relationship to apoptosis. Excitotoxic lesions were produced by intrastriatal stereotactic NMDA microinjections (10-20 nmol). Lesion size was dose-and timedependent, completely blocked by MK-801 pretreatment, and smaller in nNOS knock-out mice compared with wild-type littermates (nNOS ϩ/ϩ , 11.7 Ϯ 1.7 mm 3 ; n ϭ 8; nNOS Ϫ/Ϫ , 6.4 Ϯ 1.8 mm 3 ; n ϭ 7). The density and distribution of striatal NMDA binding sites, determined by NMDA receptor autoradiography, did not differ between strains. Pharmacological inhibition of nNOS by 7-nitroindazole (50 mg/kg, i.p.) decreased NMDA lesion size by 32% in wild-type mice (n ϭ 7). Neurochemical and immunohistochemical measurements of brain nitrotyrosine, a product of peroxynitrite formation, were increased markedly in wild-type but not in the nNOS Ϫ/Ϫ mice. Moreover, elevations in 2,3-and 2,5-dihydroxybenzoic acid levels were significantly reduced in the mutant striatum, as a measure of hydroxyl radical production.The importance of apoptosis to NMDA receptor-mediated toxicity was evaluated by DNA laddering and by quantitative histochemistry [terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) staining]. DNA laddering was first detected within lesioned tissue after 12-24 hr. TUNEL-positive cells were first observed at 12 hr, increased in number at 48 hr and 7 d, and were located predominantly in proximity to the lesion border. The density was significantly lower in nNOS Ϫ/Ϫ mice. Hence, oligonucleosomal DNA breakdown suggesting apoptosis develops as a late consequence of NMDA microinjection and is reduced in nNOS mutants. The mechanism of protection in nNOS Ϫ/Ϫ mice may relate to decreased oxygen free radical production and related NO reaction products and, in part, involves mechanisms of neuronal death associated with the delayed appearance of apoptosis.

The deposition of amyloid A␤ peptide in the wall of cerebral vessels (cerebral amyloid angiopathy), can lead to weakness and rupture of the vessel wall, resulting in hemorrhagic stroke. The Tg2576 transgenic mouse line, overexpressing mutant amyloid precursor protein in an age-dependent manner, forms amyloid angiopathy morphologically similar to that seen in the human. We report here the structural and functional disruption of smooth muscle cells (SMCs) in the walls of pial vessels affected by amyloid deposition in the Tg2576 mouse. We demonstrate, using multiphoton imaging, that the arrangement of SMCs becomes disorganized before the onset of cell death, and that these disorganized SMCs are unable to respond appropriately to application of endothelial-dependent and endothelial-independent vasodilators in a closedcranial window preparation.

We quantitatively analyzed, using laser scanning confocal microscopy, the three-dimensional structure of individual senile plaques in Alzheimer disease. We carried out the quantitative analysis using statistical methods to gain insights about the processes that govern A␤ peptide deposition. Our results show that plaques are complex porous structures with characteristic pore sizes. We interpret plaque morphology in the context of a new dynamical model based on competing aggregation and disaggregation processes in kinetic steady-state equilibrium with an additional diffusion process allowing A␤ deposits to diffuse over the surface of plaques.Although the mechanism whereby A␤ deposition may lead to dementia in Alzheimer disease (AD) is unknown, compelling genetic evidence suggests that aggregation of A␤ to form senile plaques (SP) is an essential component of AD pathophysiology (1-3). Biochemical studies suggest that these A␤ deposits are insoluble, and their formation process is viewed as irreversible. From inspection of AD tissue samples, it is evident that a wide variety of morphologies and textures of SP are present in the AD brain. Their morphologies cannot be explained by known aggregation models (4)(5)(6)(7)(8).A␤ is a Ϸ39-to 42-amino acid amphipathic peptide derived from a portion of the transmembrane domain and extracellular region of the A␤ precursor protein (9). A␤ is a normal cellular product and is present in nanomolar concentrations in biological fluids (10,11). In vitro, at higher concentrations, it is extremely insoluble and precipitates to form aggregates (12-15). In the AD brain, A␤ deposits form ␤-pleated, sheets which are the major constituent of SP. Racemized amino acids have been found in A␤, suggesting that at least some of the deposits are long-lived (16). Given the insoluble nature of A␤, it is reasonable to predict that plaques would continue to grow in size and number as the disease progresses. However, experimental data show that this is not the case; instead, plaque size and A␤ burden (total percentage) appear to remain relatively constant over a wide range of disease durations (17)(18)(19).Optical microscopy of the AD brain reveals innumerable A␤ deposits of various sizes and shapes. In an effort to understand how A␤ deposition occurs and evolves over time, we have examined the fine structure of the SP using confocal scanning laser microscopy (20, 21) and immunofluorescence techniques for A␤ immunostaining. The confocal microscope is able to obtain optical sections that are Ϸ0.3-to 0.5-m thick, allowing the reconstruction of the three-dimensional fine structure of a plaque with a resolution close to the theoretical limit of the order of the wavelength of visible light.Standard immunostaining in thick sections might suggest that plaques are relatively solid (Fig. 1a), but our examination of individual cross-sections using confocal microscopy reveals cavities and inner structure, suggesting that the threedimensional structure of A␤ aggregates in SPs is porous [ Fig. 1b]. By step...

No abstract

No abstract

No abstract