Time-gated single photon counting enables separation of CARS microscopy data from multiphoton-excited tissue autofluorescence

Ly, Sonny; McNerney, Gregory P.; Fore, Samantha; Chan, James W.; Huser, Thomas

doi:10.1364/oe.15.016839

Cited by 16 publications

(21 citation statements)

References 35 publications

(34 reference statements)

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“…The decay time of this smaller peak appears to be dramatically different from the one shown from ROI 1 or that shown in Figure 1(b). The slower decay would typically indicate that this signal is comprised of background contributions from autofluorescence in the sample [14]. In our case, however, the slope of the signal depends highly on the area of the selected ROI, indicating quite clearly that the varying slope is due to a convolution of the onset of the F-CARS peak with the weak residual E-CARS decay.…”

Section: Resultsmentioning

confidence: 66%

“…It should also be noted that all signals detected with our specific experimental configuration (E-CARS, F-CARS, and the nonresonant background signal) are significantly faster than the instrument response function of our system. The instrument response of our instrument is primarily limited by the timing jitter of the APD detectors and we have characterized this in a recent paper to be ~330 ps at 650 nm [14]. Thus, all decay curves exhibit very similar decay times representative of the instrument response function.…”

Section: Resultsmentioning

confidence: 97%

“…The TCSPC board is operated in Time-Tagged Time Resolved (TTTR) mode, which records both, the microscopic arrival time of each photon with picosecond time resolution, as well as the macroscopic (absolute) arrival time with 100 ns precision [11][12][13]. The instrument response function (IRF) was measured to be ~330 ps at ~ 650 nm [14].…”

Section: Time-correlated Single Photon Counting Detection Schemementioning

confidence: 99%

See 2 more Smart Citations

Simultaneous forward and epi-CARS microscopy with a single detector by timecorrelated single photon counting

Schie¹,

Weeks²,

McNerney³

et al. 2008

Opt. Express

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We present a novel scheme to simultaneously detect coherent anti-Stokes Raman scattering (CARS) microscopy signals in the forward (F) and backward (epi -E) direction with a single avalanche photodiode (APD) detector using time-correlated single photon counting (TCSPC). By installing a mirror at a well-defined distance above the sample the forwardscattered F-CARS signal is reflected back into the microscope objective leading to spatial overlap of the F and E-CARS signals. Due to traveling an additional distance the F-CARS signal is time delayed relative to the E-CARS signal. TCSPC then allows for the two signals to be resolved in the time domain. This results in an efficient, simple, and compact method of CARS signal detection. We demonstrate this technique by analyzing forward and backward CARS signals obtained by imaging living adipocyte cells derived from human mesenchymal stem cells.

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Section: Resultsmentioning

confidence: 66%

Section: Resultsmentioning

confidence: 97%

Section: Time-correlated Single Photon Counting Detection Schemementioning

confidence: 99%

See 1 more Smart Citation

Simultaneous forward and epi-CARS microscopy with a single detector by timecorrelated single photon counting

Schie¹,

Weeks²,

McNerney³

et al. 2008

Opt. Express

Self Cite

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show abstract

“…However, in highly autofluorescent samples such as plant tissues, the usually weak two-photon fluorescence (also blue shifted with respect to the excitation wavelengths) overwhelms the CARS signal. Consequently, CARS imaging in planta has only been applied to samples with reduced chlorophyll autofluorescence including dried tissues (Zeng et al, 2010;Ding et al, 2012), roots (Ly et al, 2007), and cuticle waxes after they have been stripped away from the leaf (Weissflog et al, 2010).…”

Section: Cars Microscopymentioning

confidence: 99%

In Vivo Chemical and Structural Analysis of Plant Cuticular Waxes Using Stimulated Raman Scattering Microscopy

et al. 2015

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The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles.

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“…Recently, Huser et al demonstrated the incorporation of CARS with TCSPC and FLIM. 8,9 Because CARS is a nearly instantaneous process (on the order of the laser pulse duration), it is indistinguishable from the instrument response function (IRF) of the TCSPC apparatus and can thus be separated from the nanosecond-timescale fluorescence decay by time-gated analysis. However, efforts to combine FLIM with CARS have been hampered by configurations where signals are collected in the descanned backward direction.…”

Section: Introductionmentioning

confidence: 99%

Forward-collected simultaneous fluorescence lifetime imaging and coherent anti-Stokes Raman scattering microscopy

et al. 2011

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Abstract. We demonstrate the simultaneous collection and separation of femtosecond-laser-based forwardcollected coherent anti-Stokes Raman scattering (F-CARS) and two-photon-excitation-induced fluorescence lifetime images (FLIM) using time-correlated single photon counting (TCSPC). We achieve this in a nondescanned geometry using a single multimode fiber without significant loss of light, field of view, and most importantly, TCSPC timing fidelity. In addition to showing the ability to separate CARS images from FLIM images using time gating, we also demonstrate composite multimodal epicollected FLIM imaging with fiber-collected F-CARS imaging in live cells. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).

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Time-gated single photon counting enables separation of CARS microscopy data from multiphoton-excited tissue autofluorescence

Cited by 16 publications

References 35 publications

Simultaneous forward and epi-CARS microscopy with a single detector by timecorrelated single photon counting

Simultaneous forward and epi-CARS microscopy with a single detector by timecorrelated single photon counting

In Vivo Chemical and Structural Analysis of Plant Cuticular Waxes Using Stimulated Raman Scattering Microscopy

Forward-collected simultaneous fluorescence lifetime imaging and coherent anti-Stokes Raman scattering microscopy

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