Stimulated parametric emission microscopy

Self Cite

We demonstrate how the resolution and imaging depth limitations of nonlinear optical microscopy can be overcome by modulating the spatial overlap between two-color pulses. We suppress out-of-focus signals, which limit the imaging depth, by a factor of 100, and enhance the lateral and axial resolution by factors of 1.6 and 1.4–1.8 respectively. Using spatial overlap modulation, we demonstrate background-free three-dimensional imaging of fixed mouse brain tissue at depths for which the signals of the conventional technique are swamped by background noise from out-of-focus regions.

Section: Introductionmentioning

confidence: 99%

Background-free deep imaging by spatial overlap modulation nonlinear optical microscopy

Isobe

Kawano

Takeda

et al. 2012

Self Cite

“…In addition to 2PEF imaging of exogenous labels and fluorescent proteins, multiphoton microscopy can be used to probe the intrinsic nonlinear optical properties of tissues. A growing literature shows that physiologically and/or structurally relevant information is obtained through the detection of coherent signals such as second-harmonic generation (SHG) [2], third-harmonic generation (THG) [3, 4, 5], or four-wave mixing (FWM) processes [6] such as coherent Raman scattering (CARS, etc) [7, 8] or stimulated parametric emission (SPE) [9]. In particular, THG imaging detects spatial variations of the electronic part of the third-order nonlinear susceptibility χ (3) (–3 ω;ω , ω , ω ) [10, 11], such as lipid inclusions in an aqueous environment [12], and has proven useful for embryo and tissue imaging applications [3, 4, 5, 13, 14].…”

Section: Introductionmentioning

confidence: 99%

Combined third-harmonic generation and four-wave mixing microscopy of tissues and embryos

Mahou¹,

Olivier²,

Labroille³

et al. 2011

Nonlinear microscopy can be used to probe the intrinsic optical properties of biological tissues. Using femtosecond pulses, third-harmonic generation (THG) and four-wave mixing (FWM) signals can be efficiently produced and detected simultaneously. Both signals probe a similar parameter, i.e. the real part of the third-order nonlinear susceptibility χ(3). However THG and FWM images result from different phase matching conditions and provide complementary information. We analyze this complementarity using calculations, z-scan measurements on water and oils, and THG-FWM imaging of cell divisions in live zebrafish embryos. The two signals exhibit different sensitivity to sample size and clustering in the half-wavelength regime. Far from resonance, THG images reveal spatial variations |Δχ(3)(−3ω;ω,ω,ω)| with remarkable sensitivity while FWM directly reflects the distribution of χ(3)(−2ω1 + ω2;ω1, –ω2, ω1). We show that FWM images provide χ(3) maps useful for proper interpretation of cellular THG signals, and that combined imaging carries additional structural information. Finally we present simultaneous imaging of intrinsic THG, FWM, second-harmonic (SHG) and two-photon-excited fluorescence (2PEF) signals in live Caenorhabditis elegans worms illustrating the information provided by multimodal nonlinear imaging of unstained tissue.

“…Nonlinear optical microscopy based on a variety of nonlinear optical techniques, such as twophoton excitation fluorescence (TPEF) [1][2][3], second-harmonic generation (SHG) [4], thirdharmonic generation [5,6], four-wave mixing [7][8][9][10], two-photon absorption (TPA) [11,12], stimulated Raman scattering (SRS) [13][14][15] and cross-phase modulation [16,17], has been developed for applications in physics, chemistry, and biology. Nonlinear optical microscopy offers several advantages over linear optical microscopy, which include three-dimensional resolution without a confocal pinhole, high penetration depth with near-infrared light excitation, less out-of-focus photon-induced damage and photobleaching.…”

Section: Introductionmentioning

confidence: 99%

Implementation of spatial overlap modulation nonlinear optical microscopy using an electro-optic deflector

Isobe

Kawano

Kumagai

et al. 2013

Self Cite

A spatial overlap modulation (SPOM) technique is a nonlinear optical microscopy technique which enhances the three-dimensional spatial resolution and rejects the out-of-focus background limiting the imaging depth inside a highly scattering sample. Here, we report on the implementation of SPOM in which beam pointing modulation is achieved by an electro-optic deflector. The modulation and demodulation frequencies are enhanced to 200 kHz and 400 kHz, respectively, resulting in a 200-fold enhancement compared with the previously reported system. The resolution enhancement and suppression of the out-of-focus background are demonstrated by sum-frequency-generation imaging of pounded granulated sugar and deep imaging of fluorescent beads in a tissue-like phantom, respectively.