Genevieve D. Vigil

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.

Automated bacterial identification by angle resolved dark-field imaging

We propose and demonstrate a dark-field imaging technique capable of automated identification of individual bacteria. An 87-channel multispectral system capable of angular and spectral resolution was used to measure the scattering spectrum of various bacteria in culture smears. Spectra were compared between various species and between various preparations of the same species. A 15-channel system was then used to prove the viability of bacterial identification with a relatively simple microscope system. A simple classifier was able to identify four of six bacterial species with greater than 90% accuracy in bacteria-by-bacteria testing.

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.

Photophysical characterization of sickle cell disease hemoglobin by multi-photon microscopy

The photophysical properties of human sickle cell disease (SCD) Hemoglobin (Hb) is characterized by multi-photon microscopy (MPM). The intrinsic two-photon excited fluorescence (TPEF) signal associated with extracted hemoglobin was investigated and the solidified SCD variant (HbS) was found to demonstrate broad emission peaking around 510 nm when excited at 800 nm. MPM is used to dynamically induce and image HbS gelling by photolysis of deoxygenated HbS. For comparison, photolysis conditions were applied to a healthy variant of human hemoglobin (HbA) and found to remain in solution not forming fibers. The use of this signal to study the mechanism of HbS polymerization associated with the sickling of SCD erythrocytes is discussed.

Skeletal Cell YAP And TAZ Redundantly Promote Bone Development By Regulation Of Collagen I Expression And Organization

The functions of the transcriptional co-activators YAP and TAZ in bone are controversial. Each has been observed to either promote or inhibit osteogenesis in vitro, while their roles in bone development are unknown. Here we report that combinatorial YAP/TAZ deletion from skeletal cells in mice caused osteogenesis imperfecta with severity dependent on targeted cell lineage and allele dosage. Osteocyte-conditional deletion impaired bone accrual and matrix collagen, while allele dosage-dependent deletion from all osteogenic lineage cells caused spontaneous fractures, with neonatal lethality only in dual homozygous knockouts. We identified putative target genes whose mutation in humans causes osteogenesis imperfecta and which contain promoter-proximate binding domains for the YAP/TAZ co-effector, TEAD4. Two candidates, Col1a1 and SerpinH1, exhibited reduced expression upon either YAP/TAZ deletion or YAP/TAZ-TEAD inhibition by verteporfin. Together, these data demonstrate that YAP and TAZ redundantly promote bone matrix development and implicate YAP/TAZ-mediated transcriptional regulation of collagen in osteogenesis imperfecta.

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.