Nonlinear magic: multiphoton microscopy in the biosciences

Zipfel, Warren R.; Williams, Rebecca M.; Webb, Watt W.

doi:10.1038/nbt899

Cited by 3,499 publications

(2,728 citation statements)

References 134 publications

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“…The multiphoton microscope used has been described previously (Kloppenburg et al, 2000;Zipfel et al, 2003b). The illumination source consisted of a Ti:sapphire laser (Milenia/Tsunami combination; Spectra Physics, Mountain View, CA) directed into a Bio-Rad MRC 600 laser scanner interfaced with a modified Olympus AX-70 upright microscope.…”

Section: Materials and Methods In Vivo Multiphoton Microscopymentioning

confidence: 99%

“…There are caveats to the interpretation of images generated from MPM (Williams et al, 2001;Zipfel et al, 2003b). The image at a given focal plane is affected by the nature of the material penetrated in all regions superficial to it.…”

Section: Caveats To Interpretation Of Mpm Images In These Experimentsmentioning

confidence: 99%

“…MPM allows imaging under stable physiological conditions, with analysis of delivery of fluoresceinated molecules of different molecular weight to the growth plate in vivo and in real time (Williams et al, 2001;Zipfel et al, 2003aZipfel et al, , 2003bNiell and Smith, 2004;Rubart, 2004). Our hypothesis is that tracers of different molecular weight reach the growth plate through different vascular routes, and that they arrive at different times and in different amounts.…”

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In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopy

et al. 2005

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Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4-to 5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da) or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa), vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few percent of vascular concentration), 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes. Key words: growth plate; vasculature; multiphoton microscopy; endochondral ossificationBone elongation in children occurs by endochondral ossification in cartilaginous growth plates at the ends of long bones. Chondrocytic differentiation in the growth plate begins with clonal expansion of stem cells, continues during cellular proliferation, and ends with cellular enlargement, followed by death and replacement by bone. The kinetics of chondrocytic activity in each stage and of the regulatory transitions between them determine the rate of bone elongation achieved. As in any cellular growth system, nutrients are required to sustain the process, and multiple endocrine and paracrine inputs regulate at each stage. For growth plate cartilage, the role of the extracellular matrix also must be considered, not only as a substantive contributor to elongation per se, but through its potential role as a communication link among chondrocytes of the growth plate, and between growth plate chondrocytes and cells of surrounding tissues such as the peri-

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Section: Materials and Methods In Vivo Multiphoton Microscopymentioning

confidence: 99%

Section: Caveats To Interpretation Of Mpm Images In These Experimentsmentioning

confidence: 99%

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In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopy

et al. 2005

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“…It is therefore particularly exciting to track virus particles in live tissues and animals to see how viruses break host defence barriers to reach target cells for infection and how viral infection is transmitted from cell to cell. With the rapid development of in vivo imaging methods, such as non-linear optical microscopy 78,79 and endoscopy, this dream might be realized in the not-too-distant future.…”

Section: Future Perspectivesmentioning

confidence: 99%

Virus trafficking – learning from single-virus tracking

2007

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What could be a better way to study virus trafficking than 'miniaturizing oneself' and 'taking a ride with the virus particle' on its journey into the cell? Single-virus tracking in living cells potentially provides us with the means to visualize the virus journey. This approach allows us to follow the fate of individual virus particles and monitor dynamic interactions between viruses and cellular structures, revealing previously unobservable infection steps. The entry, trafficking and egress mechanisms of various animal viruses have been elucidated using this method. The combination of single-virus trafficking with systems approaches and state-of-the-art imaging technologies should prove exciting in the future.

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“…Modern confocal approaches include scanning slits or microlens arrays to increase time resolution for large image areas (instruments are available from major microscope manufacturers). Multiphoton approaches eliminate the need for a pinhole in FCS-and confocal-type experiments and allow a greater penetration depth, but they do not reduce the spatial and temporal limitations of the techniques [25]. TIRF microscopy takes advantage of the evanescent wave that is developed when light is totally internally reflected at the interface between two media having dissimilar refractive indices, such as that between a glass coverslip surface and an aqueous sample.…”

Section: Motivationmentioning

confidence: 99%

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

Yang

Musser

2006

Methods

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The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and ∼15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells.

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Nonlinear magic: multiphoton microscopy in the biosciences

Cited by 3,499 publications

References 134 publications

In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopy

In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopy

Virus trafficking – learning from single-virus tracking

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

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