All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging

Wokosin, David L.; Centonze, V.; White, John; Armstrong, D.; Robertson, G. N.; Ferguson, A. I.

doi:10.1109/2944.577337

Cited by 35 publications

(21 citation statements)

References 60 publications

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“…In two-photon excitation imaging, the photobleaching and autofluorescence are considerably reduced since the infrared pulsed laser light illumination occurs only at the focal plane (Denk et al, 1990;Goppert-Mayer, 1931;Hell et al, 1996;Konig et al, 1996;Lakowicz, 1997;Periasamy, 1999;Piston et al, 1994;Potter et al, 1996;So et al, 1995;Wokosin et al, 1996). In DDM and LSCM, however, one-photon UV or visible light illuminates the specimen throughout the whole field of view and a considerable amount of photobleaching occurs above and below the focal plane.…”

Section: Introductionmentioning

confidence: 97%

An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in xenopus morphogenesis

Periasamy

Skoglund

Noakes

et al. 1999

Microsc. Res. Tech.

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Section: Introductionmentioning

confidence: 97%

An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in xenopus morphogenesis

Periasamy

Skoglund

Noakes

et al. 1999

Microsc. Res. Tech.

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“…The TPLSM was designed and used at the Integrated Microscopy Resource at the University of Wisconsin-Madison. It has been described in detail 16,37 . Briefly, this system uses a solid-state, diode-pumped, neodymiumdoped:yttrium lithium fluoride-based (Nd:YLF) laser (DPM 1000-PC, Microlase Optical Systems, Glasgow, UK) with a fixed wavelength of 1,047 nm, a maximum mean power of 550 mW, and a pulse duration of 175 fs.…”

mentioning

confidence: 99%

“…Two-photon laser scanning microscopy (TPLSM) is a fluorescence imaging technique that has been promoted as possibly less harmful to living cells 14 . Two (or more) infrared photons simultaneously excite a fluorophore rather than a single visible photon [14][15][16] . Therefore, the lower energy per photon of the longer wavelength, as well as the restriction of fluorophore excitation to the focal plane, should reduce the total photodamage to the specimen compared with conventional LSCM.…”

mentioning

confidence: 99%

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

et al. 1999

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A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H 2 O 2 may account, in part, for this inhibition. Thus, twophoton microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.Keywords two-photon microscopy; laser scanning confocal microscopy; live cell fluorescence imaging; embryo; mitochondrial dynamics; mammal; hamster The detection of specific cellular components by imaging techniques such as wide-field epifluorescence or laser scanning confocal microscopy (LSCM) requires exposure to high intensity light that can cause cellular damage 1 . Consequently, the quantity or quality of images that can be collected is limited or, even worse, the reliability of the images may be compromised. This is a particular problem when imaging events that occur over periods of time ranging from hours to days, such as embryonic development. For this reason, much of our current understanding of subcellular morphological changes during mammalian embryonic development is based on images of fixed or static specimens at different developmental stages [2][3][4][5][6] . Thus, it can be difficult to interpret dynamic processes accurately, because the continuity of events must be inferred. The establishment of long-term fluorescence imaging methods that maintain the viability of live specimens is critical for advancing our understanding of cell biology and embryonic development in areas such as ion dynamics 7 , cytoplasmic reorganization, compaction and blastocoel formation, embryonic development in exotic species (where specimens are heterogeneous and difficult * Corresponding author (jsquirre@students.wisc.edu). to obtain), use of fluorescent tags for the preselection of embryos for subsequent embryo transfer 8 , and studies of protein expression in living cells using green fluorescent protein 9 . HHS Public AccessEmbryos of some mammals, particular hamsters, are very sensitive to culture conditions 10 . Furthermore, studies suggest that mammalian oocytes and embryos are adversely affected by exposure to visible light [11][12][13] . Because of this sensitivity, mammalian embryos are ideal to test live-cell imaging techniques. In addition, there are obvious morphological changes associated with differentiation, namely compaction and blastocoel formation, which can be used to assess viability. The embryo must undergo cell division during and after imaging as well as maintain a level of developmental competence that allows it to initiate dif...

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“…In addition, the high lasing bandwidth (700-1000 nm) of the tisapphire lasers make them versatile light sources for two-photon microscopy. In addition to the ti-sa lasers, other femtosecond sources such as the Cr:LiSA.F and Nd-YLF (pulse compressed) lasers can be used for two-photon excitation 8 ,' 9 . In addition, picosecond and continuous-wave (cw) lasers can also be used for two-photon excitation.…”

Section: Laser Sources For Two-photon Excitationmentioning

confidence: 99%

Applications of two-photon fluorescence microscopy in deep-tissue imaging

Dong

Hsu

et al. 2000

Optical Sensing, Imaging, and Manipulation for Biological and Biomedical Applications

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Based on non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near-infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

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All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging

Cited by 35 publications

References 60 publications

An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in xenopus morphogenesis

An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in xenopus morphogenesis

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

Applications of two-photon fluorescence microscopy in deep-tissue imaging

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