Multiphoton microscopy with near infrared contrast agents

Abstract

Abstract. While multiphoton microscopy ͑MPM͒ has been performed with a wide range of excitation wavelengths, fluorescence emission has been limited to the visible spectrum. We introduce a paradigm for MPM of near-infrared ͑NIR͒ fluorescent molecular probes via nonlinear excitation at 1550 nm. This all-NIR system expands the range of available MPM fluorophores, virtually eliminates background autofluorescence, and allows for use of fiber-based, turnkey ultrafast lasers developed for telecommunications. Multiphoton microscopy ͑MPM͒ has become an indispensable optical imaging modality due to its intrinsic threedimensional localization, low photobleaching and photodamage outside the focal volume, and improved imaging depth. 1MPM typically uses near-infrared ͑NIR͒ excitation at 700 to 1000 nm to generate fluorescence in the visible wavelengths.2 Although the excitation wavelength of MPM has been extended 3-8 beyond 1000 nm, to our knowledge no imaging has been performed with NIR-emitting contrast agents. Conventional ͑linear͒ fluorescence imaging has shown that molecular imaging with NIR ͑ Ͼ 700 nm͒ agents minimizes the contribution from endogenous fluorescence and increases penetration depth.9 Since conventional MPM efficiently images endogenous molecules such as collagen, elastin, and reduced nicotinamide adenine dinucleotide ͑NADH͒, 10 the signal from these and other endogenous fluorophores restricts molecular imaging with exogenous contrast agents. This is particularly problematic when target concentrations are low, and strategies to reduce autofluorescence become critical to image contrast. Here, we measure twophoton-induced fluorescence of NIR dyes and demonstrate their use for autofluorescence-free biological imaging.Heptamethine cyanine dyes were selected as NIR contrast agents because of their biocompatibility and the availability of diverse structures with single photon ͑1P͒ absorption and emission between 700 and 850 nm. Cypate, a derivative of indocyanine green, was prepared as previously reported, 11 and 3,3Ј-diethylthiatricarbocyanine iodide ͑DTTCI͒ were purchased from a commercial source ͑Sigma-Aldrich, Saint Louis, Missouri͒. These dyes exhibit an absorption/emission peak of 799/ 817 nm and 771/ 800 nm, respectively. Fortuitously, these absorption maxima of NIR dyes correspond to roughly half the telecommunications spectral window at 1550 nm. As this overlaps with the gain spectrum of erbium, we perform all-NIR MPM using a simple, turnkey erbiumdoped fiber laser. A mode-locked femtosecond fiber laser ͑Mercury 1000, PolarOnyx, Sunnyvale, California͒ provided 100 mW of excitation light at ϳ1550 nm through a ϳ1-m optical fiber ͑Fig. 1͒. At the output of the fiber, the pulse duration was nominally 100 fs with a repetition rate of 50 MHz, although positive material dispersion of the optical components in the system leads to pulse broadening and reduces excitation efficiency at the sample. These losses can be recovered if the pulses arriving at the sample are transformlimited by precompensating for material...

Multispectral imaging in the extended near-infrared window based on endogenous chromophores

“…18 Overall, the improvement in the depth penetration for the exNIR spectral range is expected to be substantially larger than in optical window 1 as predicted by several authors [19][20][21] and which was recently explored with single-wall carbon nanotubes emitting at >1000 nm 16,17,22 and two-photon excitation of NIR fluorescent reporters probes at 1550 nm. 23,24 Here, we demonstrated that water and lipids dominate the spectral landscape in exNIR by a set of characteristic absorption bands, and we explored the feasibility of imaging in exNIR by focusing on water and lipids as endogenous absorbers. We hypothesized that the position and the relative intensity of the peaks in exNIR are tissue specific and reflect tissue composition.…”

All-near-infrared multiphoton microscopy interrogates intact tissues at deeper imaging depths than conventional single- and two-photon near-infrared excitation microscopes

“…6,12,14 A vexing question addressed in this study is the relationship between the different NIR excitation and emission wavelengths and the imaging depth in tissue. To assess the enhancement of imaging depth with the two different all-NIR microscopic methods relative to the traditional MPM with detection in the visible wavelengths, we used LS288, DTTCI, and Cy3 dyes.…”

“…Jose, California); a galvanometric mirror pair (6215H, Cambridge Technology, Lexington, Massachusetts); a dichroic mirror (LP02-980RS, Semrock, Rochester, New York); the XLUMPLFLN 20XW objective lens; a thermoelectrically cooled, red-enhanced GaAs PMT and photon counting head (H7421-50, Hamamatsu); and a multifunction data acquisition card (PCI-6229, National Instruments, Austin, Texas). 12 The laser is centered at 1575 AE 20 nm with a frequency of 43.7 MHz, and it provides up to 200 mW of transform-limited (<150-fs-wide) pulses at the output of a 1-m single-mode fiber patch cord. The optical fiber was shortened (to ∼0.75 m) to partially compensate for material dispersion in the system.…”

“…A detailed discussion of this instrument, including data acquisition, point spread function measurements, and confirmation of the square-law relationship between the signal and incident power, is available elsewhere. 12 During image acquisition, a 3-mm-thick glass filter (RG9, Schott, Elmsford, New York) was used to remove the residual excitation light and capture fluorescence of >700 nm, with peak transmission at 780 nm. Custom-designed Labview software controls the image acquisition process.…”

“…[12][13][14] Owing to the low autofluorescence in the NIR, this all-NIR MPM approach (both excitation and emission are in the NIR wavelengths) has the potential to further enhance deep-tissue imaging. However, the absorption coefficient of water at 1550 nm is substantially higher than at the typical excitation wavelengths used in state-of-the-art MPM systems.…”