Aamir A. Khan

Easily prepared ruthenium-complex nanomicelle probes for two-photon quantitative imaging of oxygen in aqueous media

We present a simple and inexpensive technique to enable 3D, high-resolution, and quantitative imaging of dissolved oxygen in aqueous media using a commercially available hydrophobic dye, [Ru(dpp)3]2+. The dye is encapsulated in nanomicelles formed by a poloxamer (a biocompatible surfactant), allowing for uniform dispersion in aqueous media without long, complex, and expensive chemical synthesis procedures. The nanomicelle probes are tested and found to remain stable for several months in water and for several hours in biological media. The probes are sufficiently large in size for vasculature retention and enable intravenous oxygen imaging in vivo. The two-photon cross-section of the [Ru(dpp)3]2+ nanomicelle probes surpass that of the well-established and widely used oxygen-sensitive probes in multiphoton microscopy. We also characterize the oxygen-sensitivity of the probes as a proof of their viability as low cost and easily prepared markers for multiphoton quantitative oxygen imaging in vivo and for other applications in aqueous media.

Investigation of signal-to-noise ratio in frequency-domain multiphoton fluorescence lifetime imaging microscopy

Multiphoton microscopy (MPM) combined with fluorescence lifetime imaging microscopy (FLIM) has enabled three-dimensional quantitative molecular microscopy in vivo. The signal-to-noise ratio (SNR), and thus the imaging rate of MPM-FLIM, which is fundamentally limited by the shot noise and fluorescence saturation, has not been quantitatively studied yet. In this paper, we investigate the SNR performance of the frequency-domain (FD) MPM-FLIM with two figures of merit: the photon economy in the limit of shot noise, and the normalized SNR in the limit of saturation. The theoretical results and Monte Carlo simulations find that two-photon FD-FLIM requires 50% fewer photons to achieve the same SNR as conventional one-photon FLIM. We also analytically show that the MPM-FD-FLIM can exploit the DC and higher harmonic components generated by nonlinear optical mixing of the excitation light to improve SNR, reducing the required number of photons by an additional 50%. Finally, the effect of fluorophore saturation on the experimental SNR performance is discussed.

Super-sensitivity multiphoton frequency-domain fluorescence lifetime imaging microscopy

We present a series of experiments that demonstrate a super-sensitive chemical imaging technique based on multiphoton frequency-domain fluorescence lifetime imaging microscopy (MPM-FD-FLIM) that shows a 2× improvement in imaging speed compared to the theoretical limit of conventional MPM-FD-FLIM. Additionally, this technique produces unprecedented sensitivity over a large range of fluorescence lifetimes. These results are achieved through simple modifications to data analysis in a conventional MPM-FD-FLIM microscope and are based on an analytical model describing the signal-to-noise ratio (SNR) of a MPM-FD-FLIM system [J. Opt. Soc. Am. A33, B1 (2016)]. Here we experimentally validate this model.

Saturation-compensated measurements for fluorescence lifetime imaging microscopy

Fluorophore saturation is the key factor limiting the speed and excitation range of fluorescence lifetime imaging microscopy (FLIM). For example, fluorophore saturation causes incorrect lifetime measurements when using conventional frequency-domain FLIM at high excitation powers. In this Letter, we present an analytical theoretical description of this error and present a method for compensating for this error in order to extract correct lifetime measurements in the limit of fluorophore saturation. We perform a series of simulations and experiments to validate our methods. The simulations and experiments show a 13.2× and a 2.6× increase in excitation range, respectively. The presented method is based on algorithms that can be easily applied to existing FLIM setups.

Label-free and depth resolved optical sectioning of iron-complex deposits in sickle cell disease splenic tissue by multiphoton microscopy.

Abstract. Multiphoton microscopy (MPM) imaging of intrinsic two-photon excited fluorescence (TPEF) is performed on humanized sickle cell disease (SCD) mouse model splenic tissue. Distinct morphological and spectral features associated with SCD are identified and discussed in terms of diagnostic relevance. Specifically, spectrally unique splenic iron-complex deposits are identified by MPM; this finding is supported by TPEF spectroscopy and object size to standard histopathological methods. Further, iron deposits are found at higher concentrations in diseased tissue than in healthy tissue by all imaging methods employed here including MPM, and therefore, may provide a useful biomarker related to the disease state. These newly characterized biomarkers allow for further investigations of SCD in live animals as a means to gain insight into the mechanisms impacting immune dysregulation and organ malfunction, which are currently not well understood.

Silica-coated ruthenium-complex nanoprobes for two-photon oxygen microscopy in biological media

Multiphoton microscopy (MPM) allows for three-dimensional in vivo microscopy in scattering tissue with submicron resolution and high signal-to-noise ratio. MPM combined with fluorescence lifetime measurements further enables quantitative imaging of molecular concentrations, such as dissolved oxygen, with the same optical resolution as MPM, in vivo. However, biocompatible oxygen-sensitive MPM probes are not available commercially and are difficult to synthesize. Here we present a simple MPM oxygen imaging probe compatible with aqueous biological media based on a water-soluble ruthenium-complex nanomicelle. By adding a layer of silica shell to the nanomicelle assembly, oxygen sensitivity and probe stability in biological media increases dramatically. While uncoated probes are unusable in the presence of serum albumin, photophysical characterization shows that the silica coating enables quantitative oxygen measurements in biological media and increases probe stability by more than an order of magnitude.

Description of deep saturated excitation multiphoton microscopy for super-resolution imaging

Here we recount the standard two-level model that describes saturated excitation (SAX) in multiphoton microscopy (MPM), a new technique for super-resolution fluorescence microscopy in scattering tissue, which requires no special chemistry and only simple modifications to a commercial MPM microscope. We use the model to study conditions required for improvements in MPM SAX resolution and experimental implementation strategies. Simulation results find zeros, or nodes, in the frequency response, which generate highly irregular point-spread functions (PSFs), such as rings and ripples, that contain spatial frequency content >3× larger than allowed by diffraction. These PSFs are a direct result of zeros in the frequency response of saturated fluorophores under specific excitation conditions. The impact on image quality is discussed using simulations of targets imaged with SAX PSFs. Further, we explore engineering sets of irregular PSFs by varying the excitation power and reconstructing super-resolution images from the set of captured images.

Label free detection of phospholipids by infrared absorption spectroscopy

We present our study on compact, label-free dissolved lipid sensing by combining capillary electrophoresis separation in a PDMS microfluidic chip online with mid-infrared (MIR) absorption spectroscopy for biomarker detection. On-chip capillary electrophoresis is used to separate the biomarkers without introducing any extrinsic contrast agent, which reduces both cost and complexity. The label free biomarker detection could be done by interrogating separated biomarkers in the channel by MIR absorption spectroscopy. Phospholipids biomarkers of degenerative neurological, kidney, and bone diseases are detectable using this label free technique. These phospholipids exhibit strong absorption resonances in the MIR and are present in biofluids including urine, blood plasma, and cerebrospinal fluid. MIR spectroscopy of a 12-carbon chain phosphatidic acid (PA) (1,2-dilauroyl-snglycero- 3-phosphate (sodium salt)) dissolved in N-methylformamide, exhibits a strong amide peak near wavenumber 1660 cm-1 (wavelength 6 μm), arising from the phosphate headgroup vibrations within a low-loss window of the solvent. PA has a similar structure to many important phospholipids molecules like phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylserine (PS), making it an ideal molecule for initial proof-of-concept studies. This newly proposed detection technique can lead us to minimal sample preparation and is capable of identifying several biomarkers from the same sample simultaneously.

FDTD modeling of chip-to-chip waveguide coupling via optical quilt packaging

We present Finite-Difference Time-Domain (FDTD) simulations to explore feasibility of chip-to-chip waveguide coupling via Optical Quilt Packaging (OQP). OQP is a newly proposed scheme for wide-bandwidth, highly-efficient waveguide coupling and is suitable for direct optical interconnect between semiconductor optical sources, optical waveguides, and detectors via waveguides. This approach leverages advances in quilt packaging (QP), an electronic packaging technique wherein contacts formed along the vertical faces are joined to form electrically-conductive and mechanically-stable chip-to-chip contacts. In OQP, waveguides of separate substrates are aligned with sub-micron accuracy by protruding lithographically-defined copper nodules on the side of a chip. With OQP, high efficiency chip-to-chip optical coupling can be achieved by aligning waveguides of separate chips with sub-micron accuracy and reducing chip-to-chip distance. We used MEEP (MIT Electromagnetic Equation Propagation) to investigate the feasibility of OQP by calculating the optical coupling loss between butt coupled waveguides. Transmission between a typical QCL ridge waveguide and a single-mode Ge-on-Si waveguide was calculated to exceed 65% when an interchip gap of 0.5 μm and to be no worse than 20% for a gap of less than 4 μm. These results compare favorably to conventional off-chip coupling. To further increase the coupling efficiency and reduce sensitivity to alignment, we used a horn-shaped Ge-on-Si waveguide and found a 13% increase in coupling efficiency when the horn is 1.5 times wider than the wavelength and 2 times longer than the wavelength. Also when the horizontal misalignment increases, coupling loss of the horn-shaped waveguide increases at a slower rate than a ridge waveguide.

Theoretical Analysis of the Signal-to-Noise Ratio of Two-Photon Oxygen Imaging Probes

SNR performance of optical chemical sensors is described in the presence of phosphorescence saturation by using Poisson statistics and Stern-Volmer kinetics. The described framework can be applied across wide ranges of sensitivites and lifetimes.