HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal-to-noise fluorescence.

The expansion of fluorescence bioimaging toward more complex systems and geometries requires analytical tools capable of spanning widely varying timescales and length scales, cleanly separating multiple fluorescent labels and distinguishing these labels from background autofluorescence. Here we meet these challenging objectives for multispectral fluorescence microscopy, combining hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). HyU is efficient and robust, capable of quantitative signal separation even at low illumination levels. In dynamic imaging of developing zebrafish embryos and in mouse tissue, HyU was able to cleanly and efficiently unmix multiple fluorescent labels, even in demanding volumetric timelapse imaging settings. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels. This enables coincident studies of tagged components, cellular behaviors and cellular metabolism within the same specimen, providing more accurate insights into the orchestrated complexity of biological systems.

Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell heterogeneity.

The transcription factor Bcl6 is essential for the development of germinal center (GC) B cells and follicular helper T (Tfh) cells. However, little is known about in vivo dynamics of Bcl6 protein expression during and after development of these cells. By using a Bcl6 reporter mouse strain, we found that antigen-engaged B cells upregulated Bcl6 before clustering in GCs. Two-photon microscopic analysis indicated that Bcl6 upregulation in pre-GC B cells contributed to sustaining their interactions with helper T cells and was required for their entry to GC clusters. Our data also suggested that Tfh cells gradually downmodulated Bcl6 protein over weeks after development. The Bcl6-low Tfh cells rapidly terminated proliferation and upregulated IL-7 receptor. These results clarify the role of Bcl6 in pre-GC B cell dynamics and highlight the modulation of Bcl6 expression in Tfh cells that persist in the late phase of the antibody response.

Imaging of the cross-presenting dendritic cell subsets in the skin-draining lymph node.

Dendritic cells (DCs) are antigen-presenting cells specialized for activating T cells to elicit effector T-cell functions. Cross-presenting DCs are a DC subset capable of presenting antigens to CD8(+) T cells and play critical roles in cytotoxic T-cell-mediated immune responses to microorganisms and cancer. Although their importance is known, the spatiotemporal dynamics of cross-presenting DCs in vivo are incompletely understood. Here, we study the T-cell zone in skin-draining lymph nodes (SDLNs) and find it is compartmentalized into regions for CD8(+) T-cell activation by cross-presenting DCs that express the chemokine (C motif) receptor 1 gene, Xcr1 and for CD4(+) T-cell activation by CD11b(+) DCs. Xcr1-expressing DCs in the SDLNs are composed of two different populations: migratory (CD103(hi)) DCs, which immigrate from the skin, and resident (CD8α(hi)) DCs, which develop in the nodes. To characterize the dynamic interactions of these distinct DC populations with CD8(+) T cells during their activation in vivo, we developed a photoconvertible reporter mouse strain, which permits us to distinctively visualize the migratory and resident subsets of Xcr1-expressing DCs. After leaving the skin, migratory DCs infiltrated to the deep T-cell zone of the SDLNs over 3 d, which corresponded to their half-life in the SDLNs. Intravital two-photon imaging showed that after soluble antigen immunization, the newly arriving migratory DCs more efficiently form sustained conjugates with antigen-specific CD8(+) T cells than other Xcr1-expressing DCs in the SDLNs. These results offer in vivo evidence for differential contributions of migratory and resident cross-presenting DCs to CD8(+) T-cell activation.

Differentiation of germinal center B cells and follicular helper T cells as viewed by tracking Bcl6 expression dynamics.

Development of germinal center (GC) B cells and follicular helper T (Tfh) cells requires the transcription factor B-cell lymphoma 6 (Bcl6). Expression of Bcl6 in B cells and helper T cells is regulated by complex signals including those generated through their antigen-specific interactions, which take place in various microenvironments depending on their activation/differentiation states. In the last several years, it has become possible to detect Bcl6 protein in individual B cells and T cells by intracellular staining with the newly developed antibodies or by using the reporter mice. Experiments using these reagents have started to clarify microanatomical location of early Bcl6 upregulation in B cells and T cells, and contributed to reveal the dispensability and indispensability of B cells in the early and late phase of Tfh differentiation. They also started to reveal the diversity, plasticity, and/or instability of Tfh cells. We summarize the recent findings made by tracking Bcl6 expression together with the updated knowledge about dynamics of antigen-engaged B cells and helper T cells and discuss them in relation to possible signaling requirements for the development of GC B cells and Tfh cells.

Imaging of Rab5 activity identifies essential regulators for phagosome maturation.

Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response. Rab5 is known as a key regulator of the early endocytic pathway and we have recently shown that Rab5 is also implicated in apoptotic cell engulfment; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominant-negative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.

A single-shot hyperspectral phasor camera for fast, multi-color fluorescence microscopy.

Hyperspectral fluorescence imaging improves multiplexed observations of biological samples by utilizing multiple color channels across the spectral range to compensate for spectral overlap between labels. Typically, spectral resolution comes at a cost of decreased detection efficiency, which both hampers imaging speed and increases photo-toxicity to the samples. Here, we present a high-speed, high-efficiency snapshot spectral acquisition method, based on optical compression of the fluorescence spectra via Fourier transform, that overcomes the challenges of discrete spectral sampling: single-shot hyperspectral phasor camera (SHy-Cam). SHy-Cam captures fluorescence spatial and spectral information in a single exposure with a standard scientific CMOS camera, with photon efficiency of over 80%, easily and with acquisition rates exceeding 30 datasets per second, making it a powerful tool for multi-color in vivo imaging. Its simple design, using readily available optical components, and its easy integration provide a low-cost solution for multi-color fluorescence imaging with increased efficiency and speed.

Spatiotemporal activation of Rac1 for engulfment of apoptotic cells.

The engulfment of apoptotic cells requires phagocytes to coordinately activate Rho family GTPases that regulate actin dynamics. Here, we used a FRET biosensor to visualize the spatiotemporal activation of Rac1 during engulfment of apoptotic cells. We report that apoptotic cells were usually engulfed by the phagocytes' lamellipodia, where Rac1 was activated. Often, apoptotic cells were engulfed successively at the same lamellipodial site, suggesting the presence of portals for apoptotic cells. At this location, the activated Rac1 was recruited to form phagocytic cups that were comprised of actin patches. When the phagocytic cup was closed, Rac1 was down-regulated, and the actin patches were abruptly broken down. The constitutively active Rac1 remained at phagocytic cup for a longer period than the wild-type Rac1, and the closure of the phagocytic cup was significantly delayed in cells expressing a constitutive active form of Rac1, resulting in inefficient engulfment. These results indicate that activated Rac1 is necessary to assemble F-actin, but closing the phagocytic cup requires Rac1 to be deactivated.

Pre-processing visualization of hyperspectral fluorescent data with Spectrally Encoded Enhanced Representations.

Hyperspectral fluorescence imaging is gaining popularity for it enables multiplexing of spatio-temporal dynamics across scales for molecules, cells and tissues with multiple fluorescent labels. This is made possible by adding the dimension of wavelength to the dataset. The resulting datasets are high in information density and often require lengthy analyses to separate the overlapping fluorescent spectra. Understanding and visualizing these large multi-dimensional datasets during acquisition and pre-processing can be challenging. Here we present Spectrally Encoded Enhanced Representations (SEER), an approach for improved and computationally efficient simultaneous color visualization of multiple spectral components of hyperspectral fluorescence images. Exploiting the mathematical properties of the phasor method, we transform the wavelength space into information-rich color maps for RGB display visualization. We present multiple biological fluorescent samples and highlight SEER's enhancement of specific and subtle spectral differences, providing a fast, intuitive and mathematical way to interpret hyperspectral images during collection, pre-processing and analysis.

[A case of axial spondyloarthritis acute onset as opportunity tonsil foci infection].

  A 49-year-old female with a chief complaints of arthralgia, and a medical history is Hashimoto's disease presented to us. She had been previously treated for Sjögren's syndrome at our hospital. She had anterior chest and polyarticular pain. On admission, her blood test results were as follows: white blood cells, 12700/µl; C reactive protein, 24.8 mg/dl; erythrocyte sedimentation rate 122 mm/h, Anti-streptolysin O, 1179 IU/ml;an, ASK, 10240. She had tenderness in both her hand and finger joints, recurrent episodes of tonsillitis and pustular eruption. Her imaging studies were remarkable for inflammation of the sacroiliac joint and bone erosion of the hand joint, among other findings. We considered a diagnosis of either axial spondyloarthritis or synovitis acne, pustulosis, hyperostosis and osteitis (SAPHO) syndrome due to an opportunistic tonsillar infection. The differential diagnosis between axial spondyloarthritis or SAPHO syndrome is difficult to make. We discuss this case in the context of previous literature.

Localization of activator protein-1 complex with DNA binding activity in mitochondria of murine brain after in vivo treatment with kainate.

To elucidate mechanisms underlying mitochondrial dysfunctions induced by glutamate, we have examined the effects of in vivo treatment with the ionotropic glutamate receptor agonist kainate on localization of the transcription factor activator protein-1 (AP-1) in mitochondria as well as nuclei of murine brain. A systemic administration of kainate dramatically enhanced AP-1 DNA binding in both mitochondrial and nuclear extracts of mouse cerebral cortex and hippocampus 1 hr to 3 d later. Unlabeled AP-1 probe selectively competed for AP-1 DNA binding in mitochondrial extracts of cortex and hippocampus obtained from mice injected with kainate. Supershift and immunoblotting analyses revealed participation of c-Fos, Fos-B, and Jun-B proteins in potentiation by kainate of mitochondrial AP-1 DNA binding in cortex and hippocampus. An immunohistochemical study demonstrated marked expression by kainate of c-Fos protein in the pyramidal and dentate granular layers, whereas an immunoelectron microscopic analysis showed localization of c-Fos protein within mitochondria, as well as nuclei, of the CA1 pyramidal and dentate granular cells in hippocampus obtained 2 hr after the administration of kainate. Mitochondrial AP-1 DNA binding was inhibited by particular unlabeled oligonucleotides containing sequences similar to the AP-1 site found in the noncoding region of mitochondrial DNA. Kainate markedly potentiated binding of radiolabeled oligonucleotide probes containing sequences effective in competing for AP-1 DNA binding in hippocampal mitochondrial extracts. These results suggest that kainate may facilitate expression of the AP-1 complex and subsequent translocation into mitochondria to participate in mechanisms associated with transcriptional regulation of mitochondrial DNA in murine hippocampus.

On-line capillary isoelectric focusing-mass spectrometry for quantitative analysis of peptides and proteins.

On-line capillary isoelectric focusing-mass spectrometry (cIEF-MS) was applied to determine concentrations of peptides and proteins using angiotensin II and human tetrasialo-transferrin as the model samples. The concentration of the carrier ampholyte was optimized for both resolution and ion intensity. cIEF-MS employing 1% Pharmalyte 3-10 and a sheath liquid containing water/methanol/acetic acid (50/49/1) resolved angiotensin I and II (5 microM each, DeltapI=0.2) at an Rs value of 2.29. The determined concentration of angiotensin II (0.1-5 microM) well correlated (R=0.999) with that obtained by the conventional RP-HPLC method. The limit of detection was 0.22 microM, which was about 10 times lower than that by UV detection (2 microM). The repeatability and accuracy were <15 and <11%, respectively. cIEF-MS was also applied to determine human tetrasialo-transferrin concentration. The good linearity (R2=0.998) was also observed between the transferrin concentration (0.5-1.2 g/L) and peak area ratio (IS; beta-lactoglobulin B) with acceptable accuracy (<1.9%) and repeatability ( approximately 10% at 1g/L).

Four-dimensional tracking of lymphocyte migration and interactions in lymph nodes by two-photon microscopy.

Two-photon microscopy of live tissue imaging provides insightful information about the four-dimensional dynamics of cell behavior and has contributed to the discoveries of new biological mechanisms including those in the immunology field. In recent years, it has become easier for many researchers to perform the tissue imaging experiments, due to the refinement of the commercially available microscope systems. However, it is still crucial for the efficient visualization of biological events to optimize the sample preparation by using the best available reagents. Further, it is equally important to elicit key information from the large quantity of imaging data, whose handling is often complex and laborious. Here, by taking as an example the two-photon analysis of lymphocyte migration and interaction in explanted lymph nodes, we describe the key points that need to be considered for the successful experimental design, sample preparation, image acquisition, and data analysis.

Transcription factor activator protein-1 expressed by kainate treatment can bind to the non-coding region of mitochondrial genome in murine hippocampus.

We have demonstrated previously that the transcription factor activator protein-1 (AP-1) complex is translocated into mitochondria into the nucleus in murine hippocampus after systemic kainate injection (Ogita et al. [2002] J. Neurosci. 22:2561-2570). The present study investigates whether the mitochondrial AP-1 complex translocated in response to kainate treatment binds to AP-1-like sites located at the non-coding region of the mitochondrial genome in mouse hippocampus. There are 10 sites with sequences similar to the nuclear AP-1 site in the non-coding region. Of 10 pieces (MT-1-MT-10) of synthesized double-stranded oligonucleotides, each containing a mitochondrial AP-1-like site, MT-3, MT-4, and MT-9 were effective in inhibiting mitochondrial AP-1 DNA binding enhanced by kainate. Electrophoresis mobility shift analysis using radiolabeled MT-3 and MT-9 probes demonstrated marked enhancement with binding of these 2 probes in hippocampal mitochondrial extracts prepared 2-6 hr after kainate treatment. Unlabeled AP-1 probe was more potent than unlabeled MT-9 probe in inhibiting the mitochondrial MT-9 binding. Supershift analysis revealed participation of particular Fos/Jun family proteins, such as c-Fos, Fos-B, c-Jun, Jun-B, and Jun-D, in MT-9 binding in hippocampal mitochondrial extracts prepared 4 hr after kainate treatment. Immunoprecipitation analysis using anti-c-Fos antibody demonstrated that c-Fos associated with the mitochondrial genome in hippocampal mitochondria prepared from kainate-treated animals. These results suggest that the AP-1 complex expressed by in vivo kainate treatment would bind to AP-1-like sites in the non-coding region of the mitochondrial genome after translocation into mitochondria from murine hippocampus.