Lightening the way of hematopoiesis: Infrared laser-mediated lineage tracing with high spatial-temporal resolution.

Hematopoiesis refers to the developmental process generating all blood lineages. In vertebrates, there are multiple waves of hematopoiesis, which emerge in distinct anatomic locations at different times and give rise to different blood lineages. In the last decade, numerous lineage-tracing studies have been conducted to investigate the hierarchical structure of the hematopoietic system. Yet, the majority of these lineage-tracing studies are not able to integrate the spatial-temporal information with the developmental potential of hematopoietic cells. With the newly developed infrared laser-evoked gene operator (IR-LEGO) microscope heating system, it is now possible to improve our understanding of hematopoiesis to spatial-temporal-controlled single-cell resolution. Here, we discuss the recent development of the IR-LEGO system and its applications in hematopoietic lineage tracing in vivo.

IL-33-PU.1 Transcriptome Reprogramming Drives Functional State Transition and Clearance Activity of Microglia in Alzheimer's Disease.

Impairment of microglial clearance activity contributes to beta-amyloid (Aß) pathology in Alzheimer's disease (AD). While the transcriptome profile of microglia directs microglial functions, how the microglial transcriptome can be regulated to alleviate AD pathology is largely unknown. Here, we show that injection of interleukin (IL)-33 in an AD transgenic mouse model ameliorates Aß pathology by reprogramming microglial epigenetic and transcriptomic profiles to induce a microglial subpopulation with enhanced phagocytic activity. These IL-33-responsive microglia (IL-33RMs) express a distinct transcriptome signature that is highlighted by increased major histocompatibility complex class II genes and restored homeostatic signature genes. IL-33-induced remodeling of chromatin accessibility and PU.1 transcription factor binding at the signature genes of IL-33RM control their transcriptome reprogramming. Specifically, disrupting PU.1-DNA interaction abolishes the microglial state transition and Aß clearance that is induced by IL-33. Thus, we define a PU.1-dependent transcriptional pathway that drives the IL-33-induced functional state transition of microglia, resulting in enhanced Aß clearance.

In vivo single-cell lineage tracing in zebrafish using high-resolution infrared laser-mediated gene induction microscopy.

Heterogeneity broadly exists in various cell types both during development and at homeostasis. Investigating heterogeneity is crucial for comprehensively understanding the complexity of ontogeny, dynamics, and function of specific cell types. Traditional bulk-labeling techniques are incompetent to dissect heterogeneity within cell population, while the new single-cell lineage tracing methodologies invented in the last decade can hardly achieve high-fidelity single-cell labeling and long-term in-vivo observation simultaneously. In this work, we developed a high-precision infrared laser-evoked gene operator heat-shock system, which uses laser-induced CreERT2 combined with loxP-DsRedx-loxP-GFP reporter to achieve precise single-cell labeling and tracing. In vivo study indicated that this system can precisely label single cell in brain, muscle and hematopoietic system in zebrafish embryo. Using this system, we traced the hematopoietic potential of hemogenic endothelium (HE) in the posterior blood island (PBI) of zebrafish embryo and found that HEs in the PBI are heterogeneous, which contains at least myeloid unipotent and myeloid-lymphoid bipotent subtypes.

Animals begin life as a single cell that then divides to become a complex organism with many different types of cells. Every time a cell divides, each of its two daughter cells can either stay the same type as their parent or adopt a different identity. Once a cell acquires an identity, it usually cannot 'go back' and choose another. Eventually, this process will produce daughter cells with the identity of a specific tissue or organ and that cannot divide further. Multipotent cells are cells that can produce daughter cells with different identities, including other multipotent cells. These cells can usually give rise to different cell types in a specific organ, and generate more cells to replace any cells that die in that organ. Tracking the cells descended from a multipotent cell in a specific tissue can provide information about how the tissue develops. Hemogenic endothelium cells produce the multipotent cells that give rise to two types of white blood cells: myeloid cells and lymphoid cells. Myeloid cells include innate immune cells that protect the body from infection non-specifically; while lymphoid cells include T cells and B cells with receptors that detect specific bacteria or viruses. It remains unclear whether each of these two cell types originate from a single population of hemogenic endothelium cells or from two distinct subpopulations. He et al. have now developed a new optical technique to label a single hemogenic endothelium cell in a zebrafish and track the cell and its descendants. This method revealed that there are at least two distinct populations of hemogenic endothelium cells. One of them can give rise to both lymphoid and myeloid cells, while the other can only give rise to myeloid cells. These findings shed light on the mechanisms of blood formation, and potentially could provide useful tools to study the development of diseases such as leukemia. Additionally, the single-cell labeling technology He et al. have developed could be applied to study the development of other tissues and organs.

New fluorescent compounds produced by femtosecond laser surgery in biological tissues: the mechanisms.

The femtosecond laser ablation in biological tissue produces highly fluorescent compounds that are of great significance for intrinsically labelling ablated tissue in vivo and achieving imaging-guided laser microsurgery. In this study, we analyzed the molecular structures of femtosecond laser-ablated tissues using Raman spectroscopy and transmission electron microscopy. The results showed that though laser ablation caused carbonization, no highly fluorescent nanostructures were found in the ablated tissues. Further, we found that the fluorescence properties of the newly formed compounds were spatially heterogeneous across the ablation site and the dominant fluorescent signals exhibited close similarity to the tissue directly heated at a temperature of 200 °C. The findings of our study indicated that the new fluorescent compounds were produced via the laser heating effect and their formation mechanism likely originated from the Maillard reaction, a chemical reaction between amino acids and reducing sugars in tissue.

Adult zebrafish Langerhans cells arise from hematopoietic stem/progenitor cells.

The origin of Langerhans cells (LCs), which are skin epidermis-resident macrophages, remains unclear. Current lineage tracing of LCs largely relies on the promoter-Cre-LoxP system, which often gives rise to contradictory conclusions with different promoters. Thus, reinvestigation with an improved tracing method is necessary. Here, using a laser-mediated temporal-spatial resolved cell labeling method, we demonstrated that most adult LCs originated from the ventral wall of the dorsal aorta (VDA), an equivalent to the mouse aorta, gonads, and mesonephros (AGM), where both hematopoietic stem cells (HSCs) and non-HSC progenitors are generated. Further fine-fate mapping analysis revealed that the appearance of LCs in adult zebrafish was correlated with the development of HSCs, but not T cell progenitors. Finally, we showed that the appearance of tissue-resident macrophages in the brain, liver, heart, and gut of adult zebrafish was also correlated with HSCs. Thus, the results of our study challenged the EMP-origin theory for LCs.

The first wave of T lymphopoiesis in zebrafish arises from aorta endothelium independent of hematopoietic stem cells.

T lymphocytes are key cellular components of the adaptive immune system and play a central role in cell-mediated immunity in vertebrates. Despite their heterogeneities, it is believed that all different types of T lymphocytes are generated exclusively via the differentiation of hematopoietic stem cells (HSCs). Using temporal-spatial resolved fate-mapping analysis and time-lapse imaging, here we show that the ventral endothelium in the zebrafish aorta-gonad-mesonephros and posterior blood island, the hematopoietic tissues previously known to generate HSCs and erythromyeloid progenitors, respectively, gives rise to a transient wave of T lymphopoiesis independent of HSCs. This HSC-independent T lymphopoiesis occurs early and generates predominantly CD4 Tαß cells in the larval but not juvenile and adult stages, whereas HSC-dependent T lymphopoiesis emerges late and produces various subtypes of T lymphocytes continuously from the larval stage to adulthood. Our study unveils the existence, origin, and ontogeny of HSC-independent T lymphopoiesis in vivo and reveals the complexity of the endothelial-hematopoietic transition of the aorta.

In Vivo nonlinear optical imaging of immune responses: tissue injury and infection.

In this study, we demonstrate a noninvasive imaging approach based on multimodal nonlinear optical microscopy to in vivo image the responses of immune cells (neutrophils) to the tissue injury and bacterial infection in a zebrafish model. Specifically, the second harmonic generation from myosin thick filaments in sarcomere enabled a clear visualization of the muscle injury and infection. Two-photon excited fluorescence was used to track the behavior of the neutrophils that were transgenically labeled by red fluorescent protein. The corresponding reduced nicotinamide adenine dinucleotide (NADH) two-photon excited fluorescence images revealed a detailed morphological transformation process of individual neutrophils during muscle tissue injury and bacterial infection. The analysis of time-resolved NADH signals from the neutrophils provided important biological insights of the cellular energy metabolism during the immune responses. We found a significant increase of free/protein-bound NADH ratios in activated neutrophils in bacterial-infected tissue. In this study, we also discovered that, under 720 nm excitation, two wild-type strains (DH5? and BL21) of bacteria Escherichia coli emitted distinct endogenous fluorescence of double-peak at ?450 and ?520 nm, respectively. We demonstrated that the double-peak fluorescence signal could be used to differentiate the E. coli from surrounding tissues of dominant NADH signals, and to achieve label-free tracking of E. coli bacteria in vivo.

Multimodal nonlinear optical microscopy improves the accuracy of early diagnosis of squamous intraepithelial neoplasia.

We explore diagnostic utility of a multicolor excitation multimodal nonlinear optical (NLO) microscopy for noninvasive detection of squamous epithelial precancer in vivo. The 7,12-dimenthylbenz(a)anthracene treated hamster cheek pouch was used as an animal model of carcinogenesis. The NLO microscope system employed was equipped with the ability to collect multiple tissue endogenous NLO signals such as two-photon excited fluorescence of keratin, nicotinamide adenine dinucleotide, collagen, and tryptophan, and second harmonic generation of collagen in spectral and time domains simultaneously. A total of 34 (11 controlled and 23 treated) Golden Syrian hamsters with 62 in vivo spatially distinct measurement sites were assessed in this study. High-resolution label-free NLO images were acquired from stratum corneum, stratum granulosum-stratum basale, and stroma for all tissue measurement sites. A total of nine and eight features from 745 and 600 nm excitation wavelengths, respectively, involving tissue structural and intrinsic biochemical properties were found to contain significant diagnostic information for precancers detection (p<0.05). Particularly, 600 nm excited tryptophan fluorescence signals emanating from stratum corneum was revealed to provide remarkable diagnostic utility. Multivariate statistical techniques confirmed the integration of diagnostically significant features from multicolor excitation wavelengths yielded improved diagnostic accuracy as compared to using the individual wavelength alone.

Two-photon excitation chlorophyll fluorescence lifetime imaging: a rapid and noninvasive method for in vivo assessment of cadmium toxicity in a marine diatom Thalassiosira weissflogii.

We demonstrate that a two-photon excitation fluorescence lifetime imaging technology can rapidly and noninvasively assess the cadmium (Cd)-induced toxic effects in a marine diatom Thalassiosira weissflogii. The chlorophyll, an intrinsic fluorophore, was used as a contrast agent for imaging of cellular structures and for assessment of cell toxicity. The assessment is based on an imaging-guided statistical analysis of chlorophyll fluorescence decay. This novel label-free imaging method is physically based and free of tedious preparation and preprocessing of algal samples. We first studied the chlorophyll fluorescence quenching induced by the infrared two-photon excitation laser and found that the quenching effects on the assessment of Cd toxicity could be well controlled and calibrated. In the toxicity study, chlorophyll fluorescence lifetime images were collected from the diatom samples after exposure to different concentrations of Cd. The alteration of chloroplast structure at higher Cd concentration was clearly identified. The decay of chlorophyll fluorescence extracted from recorded pixels of high signal-to-noise ratio in the fluorescence lifetime image was analyzed. The increase of average chlorophyll fluorescence lifetime following Cd treatment was observed, indicating the Cd inhibition effect on the electron transport chain in photosynthesis system. The findings of this study show that the temporal characteristics of chlorophyll fluorescence can potentially be utilized as a biomarker for indicating Cd toxicity noninvasively in algal cells.

Diagnostic value of nonlinear optical signals from collagen matrix in the detection of epithelial precancer.

During precancer development in epithelium, neoplastic cells remodel the underlying stroma, for example, the basement membrane, capillaries, fibroblasts, and extracellular matrix. The purpose of this study is to investigate the relationship between the nonlinear optical signals from the collagen matrix in stroma and the progression of early epithelial carcinogenesis. Two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) signals were measured from the stroma of hamster oral cheek pouch in vivo. We found that three features, including the intensity ratio of TPEF over SHG, the spatial frequency distribution, and the texture feature of SHG images, provide the quantitative identification of epithelial precancer at different pathologic stages. We demonstrated that the combination of all three features by using a support vector machine algorithm can significantly improve the accuracy in the detection of epithelial precancer.

Monitoring changes of cellular metabolism and microviscosity in vitro based on time-resolved endogenous fluorescence and its anisotropy decay dynamics.

Reduced nicotinamide adenine dinucleotide (NADH) is a well-known metabolic coenzyme and endogenous fluorophore. In this study, we develop a system that simultaneously measures time- and wavelength-resolved fluorescence to extract free and protein-bound NADH signals from total cellular fluorescence. We analyze temporal characteristics of NADH fluorescence in a mixture of NADH and lactate dehydrogenase (LDH) as well as in living cell samples. The results show that in both the NADH/LDH mixture and cell samples, a fraction of free NADH and protein-bound components can be identified. The extracted free and bound NADH signals are confirmed by time-resolved measurement of anisotropy decay of NADH fluorescence, based on the fact that free NADH is a small fluorescent molecule with much shorter rotational diffusion time than bound NADH. The ratio of free NADH signal to bound NADH signal is very different between normal and cancer cervical epithelial cells. In addition, the ratio changes significantly when the cell samples are treated with a mitochondrial inhibitor or uncoupler, demonstrating that the method is sensitive to monitor cellular metabolic activity. Finally, we demonstrate that the microviscosity for relatively small molecules such as NADH in cells could be extracted from wavelength- and time-resolved NADH fluorescence of living cell samples.

Conserved motif of CDK5RAP2 mediates its localization to centrosomes and the Golgi complex.

As the primary microtubule-organizing centers, centrosomes require gamma-tubulin for microtubule nucleation and organization. Located in close vicinity to centrosomes, the Golgi complex is another microtubule-organizing organelle in interphase cells. CDK5RAP2 is a gamma-tubulin complex-binding protein and functions in gamma-tubulin attachment to centrosomes. In this study, we find that CDK5RAP2 localizes to the Golgi complex in an ATP- and centrosome-dependent manner and associates with Golgi membranes independently of microtubules. CDK5RAP2 contains a centrosome-targeting domain with its core region highly homologous to the Motif 2 (CM2) of centrosomin, a functionally related protein in Drosophila. This sequence, referred to as the CM2-like motif, is also conserved in related proteins in chicken and zebrafish. Therefore, CDK5RAP2 may undertake a conserved mechanism for centrosomal localization. Using a mutational approach, we demonstrate that the CM2-like motif plays a crucial role in the centrosomal and Golgi localization of CDK5RAP2. Furthermore, the CM2-like motif is essential for the association of the centrosome-targeting domain to pericentrin and AKAP450. The binding with pericentrin is required for the centrosomal and Golgi localization of CDK5RAP2, whereas the binding with AKAP450 is required for the Golgi localization. Although the CM2-like motif possesses the activity of Ca(2+)-independent calmodulin binding, binding of calmodulin to this sequence is dispensable for centrosomal and Golgi association. Altogether, CDK5RAP2 may represent a novel mechanism for centrosomal and Golgi localization.

Clinical study of quantitative diagnosis of early cervical cancer based on the classification of acetowhitening kinetics.

A quantitative colposcopic imaging system for the diagnosis of early cervical cancer is evaluated in a clinical study. This imaging technology based on 3-D active stereo vision and motion tracking extracts diagnostic information from the kinetics of acetowhitening process measured from the cervix of human subjects in vivo. Acetowhitening kinetics measured from 137 cervical sites of 57 subjects are analyzed and classified using multivariate statistical algorithms. Cross-validation methods are used to evaluate the performance of the diagnostic algorithms. The results show that an algorithm for screening precancer produced 95% sensitivity (SE) and 96% specificity (SP) for discriminating normal and human papillomavirus (HPV)-infected tissues from cervical intraepithelial neoplasia (CIN) lesions. For a diagnostic algorithm, 91% SE and 90% SP are achieved for discriminating normal tissue, HPV infected tissue, and low-grade CIN lesions from high-grade CIN lesions. The results demonstrate that the quantitative colposcopic imaging system could provide objective screening and diagnostic information for early detection of cervical cancer.

Study of cadmium-induced cytotoxicity using two-photon excitation endogenous fluorescence microscopy.

We demonstrate that using time-resolved two-photon excitation endogenous fluorescence microscopy, the cadmium (Cd)-induced cellular toxic level can be assessed by the free-to protein-bound reduced nicotinamide adenine dinucleotide (free/bound NADH) ratio in a living cell. NADH fluorescence excited at 730 nm is captured at different times following exposure to cadmium at a variety of concentrations. The temporal characteristics of NADH fluorescence from mitochondrial and nuclear compartments are analyzed, respectively. The results show that cadmium induces a significant increase of the free/bound NADH ratio in mitochondria and nucleus, caused by the inhibition effect on the electron transport chain (ETC) and the stimulating effect on the glycolysis pathway, respectively. It is found that induction of metallothionein (MT) in cells occurs after 4 h of exposure to a sublethal concentration of Cd and reaches a peak at 6 h. More importantly, the increase in MT level can effectively suppress the elevation of the free/bound NADH ratio caused by a subsequent exposure to a higher concentration of Cd, indicating that MT plays a key role in protecting cells from Cd-induced toxicity. Our findings show that the free/bound NADH ratio can potentially be used as a sensitive indicator of toxic and carcinogenic actions induced by Cd.

Two-photon autofluorescence microscopy of multicolor excitation.

We developed a two-photon autofluorescence lifetime imaging system with excitations selected from the supercontinuum generated from a photonic crystal fiber. The system excites multiple endogenous fluorophores, such as nicotinamide adenine dinucleotide (NADH) and tryptophan, simultaneously and produces coregistered two-photon autofluorescence images of a biological sample. The technology provides a unique approach to investigate the cellular metabolic activity and protein expression in cells that are potentially important for noninvasive precancer diagnostics. We demonstrated that by taking the tryptophan fluorescence as a reference the ratio of NADH to the tryptophan signal serves as a sensitive indicator of cellular metabolism. The ratio can also clearly differentiate normal cells from cancer cells. The tryptophan fluorescence lifetime images of cells shows that the lifetime of tryptophan fluorescence, varying over a wide range, may be highly dependent on the expression and structure of the protein that tryptophan is packed in.

Optical imaging of cervical precancerous lesions based on active stereo vision and motion tracking.

A 3-D optical imaging system based on active stereo vision and motion tracking is built to track the motion of patient and to register the time-sequenced images of cervix recorded during colposcopy. The imaging system is evaluated by examining human subjects in vivo before routine colposcopy examination procedures. The system tracks the motion of patient accurately. The temporal kinetics of the acetowhitening process in the area of interest can be quantitatively measured. The results demonstrate that the kinetics of acetowhitening may be potentially used for accurately differentiating the precancerous lesions from the normal and benign lesions, and grading the precancerous lesions.

Unified Mie and fractal scattering by cells and experimental study on application in optical characterization of cellular and subcellular structures.

A unified Mie and fractal model for light scattering by biological cells is presented. This model is shown to provide an excellent global agreement with the angular dependent elastic light scattering spectroscopy of cells over the whole visible range (400 to 700 nm) and at all scattering angles (1.1 to 165 deg) investigated. Mie scattering from the bare cell and the nucleus is found to dominate light scattering in the forward directions, whereas the random fluctuation of the background refractive index within the cell, behaving as a fractal random continuous medium, is found to dominate light scattering at other angles. Angularly dependent elastic light scattering spectroscopy aided by the unified Mie and fractal model is demonstrated to be an effective noninvasive approach to characterize biological cells and their internal structures. The acetowhitening effect induced by applying acetic acid on epithelial cells is investigated as an example. The changes in morphology and refractive index of epithelial cells, nuclei, and subcellular structures after the application of acetic acid are successfully probed and quantified using the proposed approach. The unified Mie and fractal model may serve as the foundation for optical detection of precancerous and cancerous changes in biological cells and tissues based on light scattering techniques.

Assessment of the relative contribution of cellular components to the acetowhitening effect in cell cultures and suspensions using elastic light-scattering spectroscopy.

We aim to investigate the mechanism of acetowhitening upon which the colposcopic diagnosis of cervical cancer is based. The changes in light scattering induced by acetic acid in intact cervical cancer cells and cellular components were studied using elastic light-scattering spectroscopy. After adding acetic acid to intact cancer cell culture samples (cell suspensions and attached monolayer cell cultures), a slight decrease in small-angle forward scattering was observed, while the large-angle scattering increased by a factor of 5-9, indicating that acetowhitening signals are mainly contributed from small-sized intracellular scattering structures. The cellular components of different sizes and masses were isolated to investigate their individual contribution to the changes of light scattering induced by acetic acid. The study provided the evidence that the cellular components of diameter smaller than 0.2 microm in the cytoplasm are the major contributors to the acetowhitening effect in whole cells, while the light scattering from the mitochondria are not sensitive to the acetic acid.