No abstract

Two-color multiphoton microscopy through wavelength mixing of synchronized lasers has been shown to increase the spectral window of excitable fluorophores without the need for wavelength tuning. However, most currently available dual output laser sources rely on the costly and complicated optical parametric generation approach. In this report, we detail a relatively simple and low cost diamond Raman laser pumped by a ytterbium fiber amplifier emitting at 1055 nm, which generates a first Stokes emission centered at 1240 nm with a pulse width of 100 fs. The two excitation wavelengths of 1055 and 1240 nm, along with the effective two-color excitation wavelength of 1140 nm, provide an almost complete coverage of fluorophores excitable within the range of 1000–1300 nm. When compared with 1055 nm excitation, two-color excitation at 1140 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (for example, tdKatushka2). We demonstrate multicolor imaging of tdKa-tushka2 and Hoechst 33342 via simultaneous two-color two-photon, and two-color three-photon microscopy in engineered 3D multicellular spheroids. We further discuss potential benefits and applications for two-color three-photon excitation. In addition, we show that this laser system is capable of in vivo imaging in mouse cortex to nearly 1 mm in depth with two-color excitation.

The recent proliferation of platforms designed to handle arrays of nano- and picolitre volumes is in response to the need to perform biological assays on discrete entities, such as single cells. However, a critical challenge associated with this trend for in vitro compartmentalization is the need for highly sensitive, yet low-volume detection platforms. In this paper, we coupled confocal fluorescence detection with recirculating microfluidic control to perform single particle DNA assays within five nL chambers. The performance of this low-volume assay was shown to match that of traditional single molecule detection platforms. However, volume requirements per measurement were nearly 3 orders of magnitude less than conventional systems, enabling future integration with lab-on-a-chip systems that require discrete or digitalized sample processing.