Automatic segmentation of intravital fluorescence microscopy images by K-means clustering of FLIM phasors.

Fluorescence lifetime imaging microscopy (FLIM) provides additional contrast for fluorophores with overlapping emission spectra. The phasor approach to FLIM greatly reduces the complexity of FLIM analysis and enables a useful image segmentation technique by selecting adjacent phasor points and labeling their corresponding pixels with different colors. This phasor labeling process, however, is empirical and could lead to biased results.

Generating intravital super-resolution movies with conventional microscopy reveals actin dynamics that construct pioneer axons.

Super-resolution microscopy is broadening our in-depth understanding of cellular structure. However, super-resolution approaches are limited, for numerous reasons, from utilization in longer-term intravital imaging. We devised a combinatorial imaging technique that combines deconvolution with stepwise optical saturation microscopy (DeSOS) to circumvent this issue and image cells in their native physiological environment.

Generalized stepwise optical saturation enables super-resolution fluorescence lifetime imaging microscopy.

We present a novel super-resolution fluorescence lifetime microscopy technique called generalized stepwise optical saturation (GSOS) that generalizes and extends the concept of the recently demonstrated stepwise optical saturation (SOS) super-resolution microscopy [Biomed. Opt. Express9, 1613 (2018)].

Super-resolution fluorescence microscopy by stepwise optical saturation.

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing.

Skeletal cell YAP and TAZ combinatorially promote bone development.

The functions of the paralogous transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in bone are controversial. Each has been observed to promote or inhibit osteogenesis in vitro, with reports of both equivalent and divergent functions. Their combinatorial roles in bone physiology are unknown.

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.

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.

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.

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

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.

Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes.

We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H₂O₂) production in brain cells. For selective imaging of H₂O₂ over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H₂O₂ and mitochondria peroxy yellow 1 detects mitochondrial H₂O₂.

Frequency Multiplexed Multiphoton Phosphorescence Lifetime Microscopy.

Multiphoton microscopy (MPM) is widely used for optical sectioning deep in scattering tissue, [1-2]. Phosphorescence lifetime imaging microscopy (PLIM) [3] is a powerful technique for obtaining biologically relevant chemical information through Förster resonance energy transfer and phosphorescence quenching [4-5]. Point-measurement PLIM [6] of phosphorescence quenching probes has recently provided oxygen partial pressure measurements in small rodent brain vasculature identified by high-resolution MPM [7, 8].

In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems.

We characterize long (up to 285 mm) gradient index (GRIN) lens endoscope systems for multiphoton imaging. We fabricate a portable, rigid endoscope system suitable for imaging unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The portable device is capable of imaging a ~200 µm diameter field of view at 4 frames/s.

A quantum cascade laser cw cavity ringdown spectrometer coupled to a supersonic expansion source.

A new instrument has been constructed that couples a supersonic expansion source to a continuous wave cavity ringdown spectrometer using a Fabry-Perot quantum cascade laser (QCL). The purpose of the instrument is to enable the acquisition of a cold, rotationally resolved gas phase spectrum of buckminsterfullerene (C(60)). As a first test of the system, high resolution spectra of the nu(8) vibrational band of CH(2)Br(2) have been acquired at approximately 1197 cm(-1).

Low voltage-defect quantum cascade laser with heterogeneous injector regions.

We demonstrate an In(0.635)Al(0.356)As/In(0.

Negative refraction in semiconductor metamaterials.

An optical metamaterial is a composite in which subwavelength features, rather than the constituent materials, control the macroscopic electromagnetic properties of the material. Recently, properly designed metamaterials have garnered much interest because of their unusual interaction with electromagnetic waves. Whereas nature seems to have limits on the type of materials that exist, newly invented metamaterials are not bound by such constraints.