Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.
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...
The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales.
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