Two-photon fluorescence microscopy with a diode-pumped Cr:LiSAF laser

Robertson, G. N.; Armstrong, D.; Dymott, M. J. P.; Ferguson, A. I.; Hogg, G.

doi:10.1364/ao.36.002481

Cited by 14 publications

(5 citation statements)

References 3 publications

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“…2.1.2 for an overview on these sources) have been produced. Some of these have been employed for nonlinear TPEF imaging [7–11] and although average powers of up to 500mW have been demonstrated [6], they are often limited in their ability to sufficiently scale their average power.…”

Section: Introductionmentioning

confidence: 99%

Compact ultrafast semiconductor disk laser: targeting GFP based nonlinear applications in living organisms

Aviles-Espinosa

Filippidis

Hamilton

et al. 2011

Biomed. Opt. Express

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We present a portable ultrafast Semiconductor Disk Laser (SDL) (or vertical extended cavity surface emitting laser—VECSELs), to be used for nonlinear microscopy. The SDL is modelocked using a quantum-dot semiconductor saturable absorber mirror (SESAM), delivering an average output power of 287 mW, with 1.5 ps pulses at 500 MHz and a central wavelength of 965 nm. Specifically, despite the fact of having long pulses and high repetition rates, we demonstrate the potential of this laser for Two-Photon Excited Fluorescence (TPEF) imaging of in vivo Caenorhabditis elegans (C. elegans) expressing Green Fluorescent Protein (GFP) in a set of neuronal processes and cell bodies. Efficient TPEF imaging is achieved due to the fact that this wavelength matches the peak of the two-photon action cross section of this widely used fluorescent marker. The SDL extended versatility is shown by presenting Second Harmonic Generation images of pharynx, uterus, body wall muscles and its potential to be used to excite other different commercial dyes. Importantly this non-expensive, turn-key, compact laser system could be used as a platform to develop portable nonlinear bio-imaging devices.

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Section: Introductionmentioning

confidence: 99%

Compact ultrafast semiconductor disk laser: targeting GFP based nonlinear applications in living organisms

Aviles-Espinosa

Filippidis

Hamilton

et al. 2011

Biomed. Opt. Express

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“…After the first demonstration with a colliding pulse mode‐locked dye laser ( Denk et al ., 1990 ) featuring τ ≈100 fs and f ≈100 MHz, the tunable titanium–sapphire laser (τ ≈100 fs and f ≈80 MHz) became the light source of choice for routine two‐photon biological microscopy ( Curley et al ., 1992 ; Stelzer et al ., 1994 ; Denk, 1996). All‐solid state approaches such as the Nd:YLF laser (τ ≈350 fs and f ≈100 MHz) ( Wokosin et al ., 1996 ), the Cr:LiSaF laser ( Robertson et al ., 1997 ) and the Cr:LiSrAlF laser ( Svoboda et al ., 1996 ) showed very good performance, but their operational parameters τ and f did not significantly deviate from those of the standard Ti:sapphire laser.…”

Section: Introductionmentioning

confidence: 99%

Picosecond pulsed two‐photon imaging with repetition rates of 200 and 400 MHz

Bewersdorf

Hell

1998

Journal of Microscopy

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SummaryWe show the viability of high-resolution two-photon fluorescence imaging of fixed and live cells by exciting the fluorophores with a train of near-infrared pulses with duration in the picosecond range. This is exemplified with a compact, diode-pumped Nd:YVO 4 laser, emitting trains of 7-ps pulses at a wavelength of 1064 nm, with a repetition rate of 200 MHz at two separate outputs. Incoherent combination of the outputs enabled two-photon excitation with a repetition rate of 400 MHz. For a numerical aperture of 1·4 (oil), we used an average illumination power of up to 20-40 mW at the sample. The pulses were coupled into a beam scanning microscope, either directly or through a single mode glass fibre. Compared with standard femtosecond titanium-sapphire excitation conditions, our experiments were performed with a 2·5 or 5 times higher repetition rate, 30-70 times longer pulses and 10-35 times lower pulse peak intensity. The experiments indicate the possibility of significantly relaxing the temporal pulse width constraints for a series of applications.

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“…Three-photon excited fluorescence was subsequently demonstrated by Singh and Bradley in 1964 on naphthalene crystals , in tissue, and in whole blood, it has been used as a detection method in liquid chromatography , and capillary separations, it has allowed remarkable advances in high-resolution fluorescence microscopy, − and it is able to quantifying fluorescent species in dilute solutions − even at the single-molecule level . Multiphoton excited fluorescence has also been used to determine multiphoton absorption cross sections − and multiphoton excited excitation spectra and perform steady-state fluorescence measurements on complex biological samples. − More recently, multiphoton excited fluorescence has been expanded with time-resolved spectroscopy for imaging and excited-state fluorescence anisotropy and intensity decay measurements. − The latter area is of particular interest here.…”

mentioning

confidence: 99%

A Parallel Multiharmonic Frequency-Domain Fluorometer for Measuring Excited-State Decay Kinetics Following One-, Two-, or Three-Photon Excitation

Baker

et al. 1998

Anal. Chem.

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We report on the performance of a new, multiharmonic frequency-domain instrument that uses the high harmonic content of a passively mode-locked, pulse-picked femto-second Ti-sapphire laser as the excitation source for the determination of one-, two-, or three-photon excited time-resolved fluorescence anisotropy and intensity decay kinetics. In operation, the new instrument can provide a complete frequency-domain data set at 100 modulation frequencies in less than 1 min. The new instrument exhibits 5-10-ps measurement precision and it can rapidly and accurately recover complex excited-state fluorescence anisotropy and intensity decay kinetics under one-, two-, or three-photon excitation for dilute or optically dense samples that exhibit single or multiexponential decay kinetics. This latter aspect of the instrument is demonstrated by successfully determining the excited-state intensity decay kinetics for a dilute aqueous solution of rhodamine 6G dissolved in a high concentration of bromocresol green. This approach is extended by determining the excited-state fluorescence intensity decay kinetics of dilute fluorescein directly in undiluted, whole blood as a function of pH under two-photon excitation conditions. The high-speed capabilities of the new instrument are exploited by performing two-photon excited fluorescence anisotropy decay experiments on the fly for site-selectively labeled bovine serum albumin as it undergoes enzymatic digestion by trypsin.

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Two-photon fluorescence microscopy with a diode-pumped Cr:LiSAF laser

Cited by 14 publications

References 3 publications

Compact ultrafast semiconductor disk laser: targeting GFP based nonlinear applications in living organisms

Compact ultrafast semiconductor disk laser: targeting GFP based nonlinear applications in living organisms

Picosecond pulsed two‐photon imaging with repetition rates of 200 and 400 MHz

A Parallel Multiharmonic Frequency-Domain Fluorometer for Measuring Excited-State Decay Kinetics Following One-, Two-, or Three-Photon Excitation

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