Silicon nitride (SiN) is a promising candidate material for becoming a standard high-performance solution for integrated
biophotonics applications in the visible spectrum. As a key feature, its compatibility with the complementary-oxidemetal-
semiconductor (CMOS) technology permits cost reduction at large manufacturing volumes that is particularly
advantageous for manufacturing consumables. In this work, we show that the back-end deposition of a thin SiN film
enables the large light-cladding interaction desirable for biosensing applications while the refractive index contrast of the
technology (Δn ≈ 0.5) also enables a considerable level of integration with reduced waveguide bend radii. Design and experimental validation also show that several advantages are derived from the moderate SiN/SiO<sub>2</sub> refractive index contrast, such as lower scattering losses in interconnection waveguides and relaxed tolerances to fabrication
imperfections as compared to higher refractive index contrast material systems. As a drawback, a moderate refractive
index contrast also makes the implementation of compact grating couplers more challenging, due to the fact that only a
relatively weak scattering strength can be achieved. Thereby, the beam diffracted by the grating tends to be rather large
and consequently exhibit stringent angular alignment tolerances. Here, we experimentally demonstrate how a proper
design of the bottom and top cladding oxide thicknesses allows reduction of the full-width at half maximum (FWHM)
and alleviates this problem. Additionally, the inclusion of a CMOS-compatible AlCu/TiN bottom reflector further
decreases the FWHM and increases the coupling efficiency. Finally, we show that focusing grating designs greatly
reduce the device footprint without penalizing the device metrics.
Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by ensuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow. Two-photon excitation has the advantages that it enables simultaneous excitation of multiple dyes and achieves a very high SNR through simplified filtering and fluorescence background reduction. We demonstrate that two-photon excitation in conjunction with a targeted multidye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or in vivo. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling cells with targeted nanoparticles containing multiple fluorophores enables normalization of the fluorescence signal and thus quantitative measurements under nonuniform excitation. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both in vitro in a glass capillary and in vivo in the blood stream of live mice. The technique also enables us to monitor the fluorescent dye labeling dynamics in vivo. In addition, we present a unique two-beam scanning method to conduct cell size measurement in nonuniform flow.
We have developed a new two-photon system for <i>in vivo</i> flow cytometry, thereby allowing us to
simultaneously quantify different circulating populations in a single animal. The instrument was able to resolve
minute-by-minute depletion dynamics of injected fluorescent microspheres at finer time scales than conventional
flow cytometry. Also observed were the circulation dynamics of human MCF-7 and MDA-MB-435 breast cancer
cells, which have low and high metastatic potential, respectively. After co-injection of both cell types into mice,
markedly greater numbers of MCF-7 cells were present in the circulation at early time points. While low metastatic
MCF-7 cells were cleared from the vascular system within 24 hours, detectable numbers of metastatic MDA-MB-
435 cells in the circulation remained constant over time. When we replace the commercial (80-MHz) NIR
excitation laser with a reduced-repetition-rate (20-MHz) mode-locked oscillator, the signal is enhanced four-fold,
enabling superior detection in blood of cell lines expressing fluorescent proteins tdTomato and mPlum (crosslabeled
with DiI and DiD). Detection sensitivity versus incident laser power is understood in terms of detected
event photon count distribution, which can be predicted with simple fluorophore distribution assumptions. The
technique of two-color, two-photon flow cytometry greatly enhances the capabilities of ex vivo flow cytometry to
investigate dynamics of circulating cells in cancer and other important diseases.
Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantization is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two-channel detection and two-photon excitation flow cytometry (T<sup>3</sup>FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or <i>in vivo</i>.