Stokes polarimetry-based second harmonic generation microscopy for collagen and skeletal muscle fiber characterization.

The complete polarization state of second harmonic (SH) light was measured and characterized by collagen type I and skeletal muscle fiber using a Stokes vector-based SHG microscope. The polarization states of the SH signal are analyzed in a pixel-by-pixel manner and displayed through two dimensional (2D) Stokes vector images. Various polarization parameters are reconstructed using Stokes values to quantify the polarization properties of SH light. Also, the measurements are extended for different input polarization states to investigate the molecular structure of second harmonic generation (SHG) active molecules such as collagen type I and myosin.

Synchronized subharmonic modulation in stimulated emission microscopy.

In this work, we have demonstrated a stimulated emission (SE)-based pump-probe microscopy with subharmonic fast gate synchronization, which allows over an order of magnitude improvement in signal-to-noise ratio. Critically, the alternative way of modulation is implemented with the highest possible frequency that follows the lasers' repetition rate. Its working is based on a homemade frequency divider that divides the repetition frequency (76 MHz) of the Ti:sapphire (probe) laser to half of the repetition frequency, 38 MHz, which is used to synchronously drive the pump laser and to provide the reference signal for the ensuing lock-in detection. In this way, SE can be detected with sensitivity reaching the theoretical (shot noise) limits, with a much lower time constant (0.1 ms) for faster image acquisition.

Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO2 transport.

Human CO2 respiration requires rapid conversion between CO2 and HCO3- Carbonic anhydrase II facilitates this reversible reaction inside red blood cells, and band 3 [anion exchanger 1 (AE1)] provides a passage for HCO3- flux across the cell membrane. These 2 proteins are core components of the CO2 transport metabolon. Intracellular H2O is necessary for CO2/HCO3- conversion. However, abundantly expressed aquaporin 1 (AQP1) in erythrocytes is thought not to be part of band 3 complexes or the CO2 transport metabolon. To solve this conundrum, we used Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging (FLIM-FRET) and identified interaction between aquaporin-1 and band 3 at a distance of 8 nm, within the range of dipole-dipole interaction. Notably, their interaction was adaptable to membrane tonicity changes. This suggests that the function of AQP1 in tonicity response could be coupled or correlated to its function in band 3-mediated CO2/HCO3- exchange. By demonstrating AQP1 as a mobile component of the CO2 transport metabolon, our results uncover a potential role of water channel in blood CO2 transport and respiration.-Hsu, K., Lee, T.-Y., Periasamy, A., Kao, F.-J., Li, L.-T., Lin, C.-Y., Lin, H.-J., Lin, M. Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO2 transport.

Polarization resolved second harmonic microscopy.

Second harmonic (SH) microscopy has proven to be a powerful imaging modality over the past years due to its intrinsic advantages as a multiphoton process with endogenous contrast specificity, which allows pinhole-less optical sectioning, non-invasive observation, deep tissue penetration, and the possibility of easier signal detection at visible wavelengths. Depending on the relative orientation between the polarization of the incoming light and the second-order susceptibility of non-centrosymmetric structures, SH microscopy provides the unique capacity to probe the absolute molecular structure of a broad variety of biological tissues without the necessity for additional labeling. In addition, SH microscopy, when working with polarimetry, provides clear and in-depth insights on the details of molecular orientation and structural symmetry. In this review, the working principles of the polarization resolving techniques and the corresponding implements of SH microscopy are elucidated, with focus on Stokes vector based polarimetry. An overview of the advancements on SH anisotropy measurements are also presented. Specifically, the recent progresses on the following three topics in polarization resolved SH microscopy will be elucidated, which include Stokes vector resolving for imaging molecular structure and orientation, 3-D structural chirality by SH circular dichroism, and correlation with fluorescence lifetime imaging (FLIM) for in vivo wound healing diagnosis. The potentials and challenges for future researches in exploring complex biological tissues are also discussed.

Revealing molecular structure and orientation with Stokes vector resolved second harmonic generation microscopy.

We report on measurements and characterization of polarization properties of Second Harmonic (SH) signals using a four-channel photon counting based Stokes polarimeter. In this way, the critical polarization parameters can be obtained concurrently without the need of repeated image acquisition. The critical polarization parameters, including the degree of polarization (DOP), the degree of linear polarization (DOLP), and the degree of circular polarization (DOCP), are extracted from the reconstructed Stokes vector based SH images in a pixel-by-pixel manner. The measurements are further extended by varying the polarization states of the incident light and recording the resulting Stokes parameters of the SH signal. In turn this allows the molecular structure and orientation of the samples to be determined. Use of Stokes polarimetry is critical in determination of the full polarization state of light, and enables discrimination of material properties not possible with conventional crossed-polarized detection schemes. The combination of SHG microscopy and Stokes polarimeter hence makes a powerful tool to investigate the structural order of targeted specimens.

Three-dimensional trace measurements for fast-moving objects using binary-encoded fringe projection techniques.

A fringe projection technique to trace the shape of a fast-moving object is proposed. A binary-encoded fringe pattern is illuminated by a strobe lamp and then projected onto the moving object at a sequence of time. Phases of the projected fringes obtained from the sequent measurements are extracted by the Fourier transform method. Unwrapping is then performed with reference to the binary-encoded fringe pattern. Even though the inspected object is colorful, fringe orders can be identified. A stream of profiles is therefore retrieved from the sequent unwrapped phases. This makes it possible to analyze physical properties of the dynamic objects. Advantages of the binary-encoded fringe pattern for phase unwrapping also include (1) reliable performance for colorful objects, spatially isolated objects, and surfaces with large depth discontinuities; (2) unwrapped errors only confined in a local area; and (3) low computation cost.

Investigating cartilage-related diseases by polarization-resolved second harmonic generation (P-SHG) imaging.

Establishing quantitative parameters for differentiating between healthy and diseased cartilage tissues by examining collagen fibril degradation patterns facilitates the understanding of tissue characteristics during disease progression. These findings could also complement existing clinical methods used to diagnose cartilage-related diseases. In this study, cartilage samples from normal, osteoarthritis (OA), and rheumatoid arthritis (RA) tissues were prepared and analyzed using polarization-resolved second harmonic generation (P-SHG) imaging and quantitative image texture analysis. The enhanced molecular contrast obtained from this approach is expected to aid in distinguishing between healthy and diseased cartilage tissues. P-SHG image analysis revealed distinct parameters in the cartilage samples, reflecting variations in collagen fibril arrangement and organization across different pathological states. Normal tissues exhibited distinct χ33/χ31 values compared with those of OA and RA, indicating collagen type transition and cartilage erosion with chondrocyte swelling, respectively. Compared with those of normal tissues, OA samples demonstrated a higher degree of linear polarization, suggesting increased tissue birefringence due to the deposition of type-I collagen in the extracellular matrix. The distribution of the planar orientation of collagen fibrils revealed a more directional orientation in the OA samples, associated with increased type-I collagen, while the RA samples exhibited a heterogeneous molecular orientation. This study revealed that the imaging technique, the quantitative analysis of the images, and the derived parameters presented in this study could be used as a reference for disease diagnostics, providing a clear understanding of collagen fibril degradation in cartilage.

Fluorescence lifetime imaging with pulsed diode laser enabled stimulated emission.

We present here a stimulated emission based fluorescence lifetime imaging (FLIM) scheme using a pair of synchronized diode lasers operating at gain switched pulse mode. The two semiconductor lasers, with wavelengths at 635 nm and 700 nm, serve as the excitation and the stimulation light sources for the ATTO647N labeled sample, respectively. FLIM is readily achieved with their relative time delay controlled electronically. The coherent nature of the stimulated emission signal also allows FLIM at long working distance. In this way, a high performance all-semiconductor FLIM module is realized in a flexible, compact, and cost effective configuration.

Long working distance fluorescence lifetime imaging with stimulated emission and electronic time delay.

In this work, long working distance fluorescence lifetime imaging is realized with stimulated emission in combination with electronic time delay control. Spatial coherence, as a result of stimulated emission, supports unattenuated fluorescence detection at extended distance, using low NA optics. An electronic time delayed trigger provides an advantageous way in adjusting the pulse separation and probing the fluorescence lifetime in the nanosecond ranges. The fluorescence lifetime of selected fluorophores is accurately determined through the pump-probe configuration. The characteristics and applications in fluorescence lifetime measurement of stimulated emission are investigated and summarized succinctly here.

Polarization-resolved second harmonic generation microscopy with a four-channel Stokes-polarimeter.

We developed a four-channel photon counting based Stokes-polarimeter for spatial characterization of polarization effects in second harmonic generation (SHG). We have implemented a calibration technique allowing quantitative measurement of polarization parameters, such as the degree of polarization (DOP), degree of linear polarization (DOLP), degree of circular polarization (DOCP), as well as anisotropy from the acquired Stokes parameters. The technique is used as contrast mechanism to characterize the polarization properties from two potassium dihydrogen phosphate (KDP) micro-crystals and collagen type-I in SHG microscopy.

An insight into optical beam induced current microscopy: Concepts and applications.

Laser scanning optical beam induced current (OBIC) microscopy has become a powerful and nondestructive alternative to other complicated methods like electron beam induced current (EBIC) microscopy, for high resolution defect analysis of electronic devices. OBIC is based on the generation of electron-hole pairs in the sample due to the raster scanning of a focused laser beam with energy equal or greater than the band gap energy and synchronized detection of resultant current profile with respect to the beam positions. OBIC is particularly suitable to localize defect sites caused by metal-semiconductor interdiffusion or electrostatic discharge (ESD). OBIC signals, thus, are capable of revealing the parameters/factors directly related to the reliability and efficiency of the electronic device under test (DUT). In this review, the basic principles of OBIC microscopy strategies and their notable applications in semiconductor device characterization are elucidated. An overview on the developments of OBIC microscopy is also presented. Specifically, the recent progresses on the following three OBIC measurement strategies have been reviewed, which include continuous laser based single photon OBIC, pulsed laser based single photon OBIC, and multiphoton OBIC microscopy for three-dimensional mapping of photocurrent response of electronic devices at high spatiotemporal resolution. Challenges and future prospects of OBIC in characterizing complex electronic devices are also discussed. HIGHLIGHTS: Characterization of electronic device quality is of paramount importance. Optical beam induced current (OBIC) microscopy offers spatially resolved mapping of local electronic properties. This review presents the principle and notable applications of OBIC microscopy.

Alcohol patch test with hue-saturation-value model analysis predicts ALDH2 genetic polymorphism.

BACKGROUND: The alcohol patch test (APT) can detect aldehyde dehydrogenase (ALDH) genetic polymorphisms used to diagnose cutaneous erythema. However, the subjective results can vary owing to confounding factors. The hue-saturation-value (HSV) model provides an objective means of image analysis with APT. METHODS: This study enrolled 57 participants (27.7 ± 9.0 years, 52.6% females) with ALDH2*1/*1, ALDH2*1/*2, and ALDH2*2/*2 percentages of 50.9%, 43.8%, and 5.3%, respectively. In total, 56 APT protocols were applied and analyzed employing both visual inspection and the HSV model. The value of the delta standard deviation (SD) of the hue histogram, which manifests the difference between the APT reaction and the baseline skin color, was obtained using the HSV model. The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to predict the ALDH2*2 allele with the HSV model. RESULTS: Upon visual inspection, a maximal Youden index with a sensitivity of 82.1% and a specificity of 96.6% was determined for the ALDH2 genetic mutation. Using the delta SD of hue obtained in the HSV model, a maximal Youden index with 85.7% sensitivity and 96.6% specificity was determined using the ROC curve analysis (AUC = 0.948, p < 0.001). Thus, the use of the HSV model analysis with APT resulted in equal specificity, but better sensitivity, compared to those obtained upon visual inspection. CONCLUSION: The HSV model took into account the potential confounding factors, and thus, could help in the prediction of ALDH2 genetic polymorphisms.

Phafin2 modulates the structure and function of endosomes by a Rab5-dependent mechanism.

By regulating the amount of protein receptors on the cell membrane and the metabolisms of receptor-bound ligands, endocytosis represents one of the fundamental biological activities that regulate how cells respond to the environment. We report here that a Fab1-YotB-Vac1p-EEA1 (FYVE) domain-containing lipid associated protein, called Phafin2, is preferentially expressed in the human hepatocellular carcinoma (HCC) and is involved in the biogenesis of endosomes. Over-expression of Phafin2 or its FYVE domain results in the formation of enlarged endosomes that are still functional for endocytosis; the biogenesis of such abnormal organelles is mediated by phosphoinositide 3-kinases (PI3K) and Rab5 signaling. Using fluorescence resonance energy transfer measured by fluorescence lifetime imaging microscopy (FLIM-FRET), we further demonstrate in live cells that Phafin2 can directly activate Rab5. By modulating the receptor internalization/recycling and Rab5 activation, Phafin2 affects the density of membranous insulin receptors, and regulates the transcriptional activity of AP-1 that is downstream of the insulin signaling pathway. These results provide a vivid example that an endosome modulator, such as Phafin2, may control the cells' responses to the extracellular cues.

Enhancing fluorescence of quantum dots by silica-coated gold nanorods under one- and two-photon excitation.

Nano-composites of quantum dots (QDs) and gold nanorods (GNRs) or silica-coated GNRs (GNRs_SiO2) were synthesized. The attached GNRs modify the excitation intensity and spontaneous emission of QDs through the surface plasmonic effects. The fluorescence from QDs is enhanced and can be optimized by modifying the thickness of silica coated on GNRs, under both one- and two-photon excitations. The measurements of fluorescence intensity and lifetime demonstrate that the enhancement may be attributed to the matching of the localized surface plasmon resonance of GNR to the excitation wavelength. In addition to enhancing QD-fluorescence in QD-GNR@SiO2, GNRs also present as an effective contrast agent for bio-imaging, through light scattering and or two-photon emission, as well as for photo-thermal therapy. The composite's multifunctional characteristics are highly valuable and to be exploited in bio-applications.

Visualization of the Orai1 homodimer and the functional coupling of Orai1-STIM1 by live-cell fluorescence lifetime imaging.

The Orai1-STIM1 constructed store-operated Ca2+ channels (SOCs) have been found to exert several essential Ca2+ entry/signaling cascades, e.g., the generation of immune response in T lymphocytes. Although biochemical and novel imaging evidence appear to indicate that Orai1 and STIM1 interact with each other to achieve store-operated Ca2+ entry (SOCE), the detailed mechanism of functional SOCE in situ has yet to be fully understood. In this study, green fluorescence protein (EGFP as donor) targeted to either the N- or C-terminal of Orai1 (wild type or delta1-90+delta267-301 double deletion type) and mOrange (as acceptor) tagged STIM1 were used to comprise a fluorescence resonance energy transfer (FRET) pair within living PC12 cells. The fluorescence lifetime map and histogram/distribution of each single cell, determined by one-photon excitation fluorescence lifetime imaging microscopy (FLIM), was used to visualize FRET and show the Orai1 homodimer and Orai1-STIM1 binding. Both the color-coded lifetime map and the distribution of EGFP-tagged Orai1 significantly changed after the administration of thapsigargin, the SOCE stimulating agent. The FRET efficiency from each experimental set was also calculated and compared using double exponential analysis. In summary, we show the detailed interactions Orai1-Orai1 and Orai1-STIM1 within intact living cells by using the FLIM-FRET technique.

High temporal resolution and polarization resolved fluorescence lifetime measurements through stimulated emission.

We have implemented polarization-resolved fluorescence lifetime measurement through stimulated emission based pump-probe technique, which promises much higher temporal resolution (∼4 ps) than conventional time-correlated single-photon counting (TCSPC). The depolarization of ATTO 647N fluorescent dye is resolved through anisotropy fluorescence lifetime measurements, with variable time delay introduced between the pump and the probe beams. Importantly, the polarization anisotropy measurement and the corresponding rotational correlation time characterization of the fluorescent dye are carried out at various temperatures. We have also demonstrated the need of high temporal resolution via hetero Förster energy transfer (Hetero-FRET) through the interaction between the gold nanorods (GNRs) and the fluorescent dye ATTO 647N. Notably, our results compare highly favorably with conventional TCSPC method, which is rather limited in temporal resolution, for the above characterization. Additionally, this technique is applicable even under ambient light while being very cost-effective and robust.

Spontaneous loss versus stimulation gain in pump-probe microscopy: a proof of concept demonstration.

SIGNIFICANCE: The large background, narrow dynamic range, and detector saturation have been the common limiting factors in stimulated emission (SE)-based pump-probe microscopy, attributed to the very small signal overriding the very intense laser probe beam. To better differentiate the signal of interest from the background, lock-in detection is used to measure the fluorescence quenching, which is termed spontaneous loss (SL). The advantages are manifold. The spontaneous fluorescence signal can be well separated from both the pump and the probe beams with filters, thus eliminating the background, enlarging the dynamic range, and avoiding the saturation of the detector. AIM: We propose and demonstrate an integrated pump-probe microscopy technique based on lock-in detection for background removal and dynamic range enhancement through SL detection. APPROACH: The experimental setup is configured with a pulsed diode laser at a wavelength λpu = 635 nm, acting as a pump (excitation) and a mode-locked Ti:sapphire laser at a central wavelength λpr = 780 nm, serving as the probe beam (stimulation). Both pulse trains are temporally synchronized through high precision delay control by adjusting the length of the triggering cables. The pump and probe beams are alternatively modulated at different frequencies f1 and f2 to extract the stimulated gain (SG) and SL signal. RESULTS: SG signal shows saturation due to the irradiation of the intense probe beam onto the photodetector. However, the detector saturation does not occur at high probe beam power for SL detection. The fluorescence lifetime images are acquired with reduced background. The theoretical signal-to-noise ratios for SG and SL are also estimated by photon statistics. CONCLUSION: We have confirmed that the detection of SL allows the elimination of the large background without photodetector saturation, which commonly exists in SG configuration. This modality would allow unprecedented manipulation and investigation of fluorophores in fluorescence imaging.

Label-free characterization of collagen fibers in cancerous esophagus tissues using ratiometric nonlinear optical microscopy.

IMPACT STATEMENT: The issue of classifying esophageal cancer at various developmental stages is crucial for determining the optimized treatment protocol for the patients, as well as the prognosis. Precision improvement in staging esophageal cancer keeps seeking quantitative and analytical imaging methods that could augment histopathological techniques. In this work, we used nonlinear optical microscopy for ratiometric analysis on the intrinsic signal of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) from single collagen fibers only in submucosa of esophageal squamous cell carcinoma (ESCC). The blind tests of TPEF/SHG and forward (F)/backward (B) SHG were demonstrated to compare with the histology conclusion. The discussion of sensitivity and specificity was provided via statistical comparison between the four stages of esophageal cancer. To the best of our knowledge, this is the first study of using these two ratios in combination for staging ESCC.

Colorimetry provides a rapid objective measurement of de novo hair growth rate in mice.

BACKGROUND: Depilated mice have been used as a test platform for hair growth-regulating agents. However, currently available assessment tools for hair growth in mice are less than ideal. METHODS: Tristimulus colorimetry of the fur color of depilated agouti, albino, and black mice with L*, a*, and b* values were performed daily until the full growth of pelage. Using light-emitting diode (LED) irradiation (650 and 890 nm) with a daily dose of 3.5 J/cm(2) as hair growth regulators, the hair growth rates observed by the global assessment were compared with those derived from colorimetry. RESULTS: In contrast to a* and b* values, L* values changed more drastically over time in the anagen phase regardless of fur color. Unlike the inhibitory effect of 650 nm irradiation, LED of 890 nm promoted de novo hair regrowth in mice. The difference in hair growth rates detected by colorimetry paralleled the observation made by the global assessment. CONCLUSION: The L* value of fur color obtained by tristimulus colorimetry was a sensitive yet quantitative indicator of de novo hair growth, and could be used to project the hair growth rate in mice.

Detection of the interaction between SNAP25 and rabphilin in neuroendocrine PC12 cells using the FLIM/FRET technique.

Exocytosis has been proposed to contain four sequential steps, namely docking, priming, fusion, and recycling, and to be regulated by various proteins-protein interactions. Synaptosomal-associated protein of 25 kDa (SNAP25) has recently been found to bind rabphilin, the Rab3A specific binding protein, in vitro. However, it is still unclear whether SNAP25 and rabphilin interact during exocytosis within cells in vivo. This problem was addressed by the integration of fluorescence resonance energy transfer (FRET) with high sensitivity fluorescence lifetime imaging microscopy (FLIM) to observe this protein-protein interaction. Enhanced green fluorescence protein-labeled SNAP25 (donor) and red fluorescence protein-labeled rabphilin (acceptor) were expressed in neuroendocrine PC12 cells as a FRET pair and ATP stimulation was carried out for various durations. With 10 s stimulation, a 0.17-ns left shift of the lifetime peak was found when compared with donor only. Analysis of the lifetime image further suggested that the lifetime recovered to a similar level as the donor only in a time dependent manner. Four-dimensional (4D) images by FLIM provided useful information indicating that the interaction of SNAP25 and rabphilin occurred particularly within optical sections near cell membrane. Together the results suggest that SNAP25 bound rabphilin loosely at docking step before exocytosis and the binding became tighter at the very start of exocytosis. Finally, these two proteins dissociated after stimulation. To our knowledge, this is the first report to demonstrate the interaction of SNAP25 and rabphilin in situ using the FLIM-FRET technique within neuroendocrine cells.