Multicolor two-photon tissue imaging by wavelength mixing

Abstract

We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.

One Plus One Equals Three

“…3). This process has already been demonstrated with a different technical setup, see e. g. [5]. The fluorophor simultaneously absorbs one 780 nm and one 1030 nm photon.…”

Unleashing Optics and Optoacoustics for Developmental Biology

“…In [8] an optical parametric oscillator is used to generate a beam with a tunable wavelength above 1080 nm, allowing excitation in the NIR. Simultaneous two-photon excitation of three fluorophores has also been achieved [9] by combining two beams of different wavelengths, generating the third excitation wavelength through the spatiotemporal overlap between both beams. The simultaneous excitation of three distinct fluorophores enabled imaging of Brainbow-labeled mouse cortical tissue [10], allowing the differentiation of individual neurons within the tissue.…”

“…Elongated and periodic structures such as myofibrils or motitic spindles, for example, are capable of SHG, generating light of exactly half the wavelength (or one third of the original wavelength for THG) [11]. The structural contrast obtained by SHGM and THGM can provide complementary information to the images obtained in 2PM, which is why the two methods are often combined [7,9,12]. Although both SHG and THG microscopy have been frequently used since the turn of the millennium, only recent developments have applied them to cell lineage tracking in early zebrafish embryos [13] or to live brain imaging combined with targeted patch-clamp recordings of single neurons, allowing both recording and influencing the membrane potential of single cells [12] while performing volumetric imaging.…”

“…At this point a hybrid system combining two or more imaging modalities may be the solution. Optical techniques with similar geometries can be combined fairly easily and provide additional contrast [7,9,12]. However, the combination of two similar optical techniques will usually not improve the imaging depth significantly.…”

Multimodal Nonlinear Optical Microscopy for Simultaneous 3-D Label-Free and Immunofluorescence Imaging of Biological Samples

“…Recently, multicolor imaging techniques in TPEF microscopy have been studied [14][15][16]. Kawano et al [14] and Tillo et al [15] have proposed the combination of two fluorescent proteins having a single excitation wavelength and well-separated emission spectra; various fluorescent proteins and dyes have high two-photon absorption cross-sections between 720 nm and 860 nm and may emit fluorescence at each emission wavelength region corresponding to single-photon excitation.…”