Multiphoton microscopy is a powerful technique for deep in vivo imaging in scattering samples. However, it requires precise, sample-dependent increases in excitation power with depth in order to generate contrast in scattering tissue, while minimizing photobleaching and phototoxicity. We show here how adaptive imaging can optimize illumination power at each point in a 3D volume as a function of the sample's shape, without the need for specialized fluorescent labeling. Our method relies on training a physics-based machine learning model using cells with identical fluorescent labels imaged in situ. We use this technique for in vivo imaging of immune responses in mouse lymph nodes following vaccination. We achieve visualization of physiologically realistic numbers of antigen-specific T cells (~2 orders of magnitude lower than previous studies), and demonstrate changes in the global organization and motility of dendritic cell networks during the early stages of the immune response. We provide a step-by-step tutorial for implementing this technique using exclusively open-source hardware and software.

Multiphoton microscopy is a powerful technique for deep in vivo imaging in scattering samples. However, it requires precise, sample-dependent increases in excitation power with depth in order to maintain signal while minimizing photodamage. We show that cells with identical fluorescent labels imaged in situ can be used to train a physics-based machine learning model that solves this problem. After this training has been performed, the correct illumination power can be predicted and adaptively adjusted at each point in a 3D volume on subsequent samples as a function of the sample’s shape, without the need for specialized fluorescent labelling. We use this technique for in vivo imaging of immune responses in mouse lymph nodes following vaccination, with imaging volumes 2-3 orders of magnitude larger than previously reported. We achieve visualization of physiologically realistic numbers of antigen-specific T cells for the first time, and demonstrate changes in the global organization and motility of dendritic cell networks during the early stages of the immune response.

The maintenance of tissue homeostasis is critically dependent on the function of tissue-resident immune cells and the differentiation capacity of tissue-resident stem cells (SCs). How immune cells influence the function of SCs is largely unknown. Regulatory T cells (Tregs) in skin preferentially localize to hair follicles (HFs), which house a major subset of skin SCs (HFSCs). Here, we mechanistically dissect the role of Tregs in HF and HFSC biology. Lineage-specific cell depletion revealed that Tregs promote HF regeneration by augmenting HFSC proliferation and differentiation. Transcriptional and phenotypic profiling of Tregs and HFSCs revealed that skin-resident Tregs preferentially express high levels of the Notch ligand family member, Jagged 1 (Jag1). Expression of Jag1 on Tregs facilitated HFSC function and efficient HF regeneration. Taken together, our work demonstrates that Tregs in skin play a major role in HF biology by promoting the function of HFSCs.

The use of coumarin caged molecules has been well documented in numerous photocaging applications including for the spatiotemporal control of Cre-estrogen receptor (Cre-ERT2) recombinase activity. In this article, we report that 4-hydroxytamoxifen (4OHT) caged with coumarin via a conventional ether linkage led to an unexpected photo-Claisen rearrangement which significantly competed with the release of free 4OHT. The basis for this unwanted reaction appears to be related to the coumarin structure and its radical-based mechanism of uncaging, as it did not occur in ortho-nitrobenzyl (ONB) caged 4OHT that was otherwise linked in the same manner. In an effort to perform design optimization, we introduced a self-immolative linker longer than the ether linkage and identified an optimal linker which allowed rapid 4OHT release by both single-photon and two-photon absorption mechanisms. The ability of this construct to actively control Cre-ERT2 mediated gene modifications was investigated in mouse embryonic fibroblasts (MEFs) in which the expression of a green fluorescent protein (GFP) reporter dependent gene recombination was controlled by 4OHT release and measured by confocal fluorescence microscopy and flow cytometry. In summary, we report the implications of this photo-Claisen rearrangement in coumarin caged compounds and demonstrate a rational linker strategy for addressing this unwanted side reaction.

Myeloid-derived cells such as monocytes, dendritic cells (DCs), and macrophages are at the heart of the immune effector function in an inflammatory response. But because of the lack of an efficient imaging system to trace these cells live during their migration and maturation in their native environment at sub-cellular resolution, our knowledge is limited to data available from specific time-points analyzed by flow cytometry, histology, genomics and other immunological methods. Here, we have developed a ratiometric imaging method for measuring monocyte maturation in inflamed mouse lungs in situ using real-time using 2-photon imaging and complementary methods. We visualized that while undifferentiated monocytes were predominantly found only in the vasculature, a semi-differentiated monocyte/macrophage population could enter the tissue and resembled more mature and differentiated populations by morphology and surface phenotype. As these cells entered and differentiated, they were already selectively localized near inflamed airways and their entry was associated with changes in motility and morphology. We were able to visualize these during the act of differentiation, a process that can be demonstrated in this way to be faster on a per-cell basis under inflammatory conditions. Finally, our in situ analyses demonstrated increases, in the differentiating cells, for both antigen uptake and the ability to mediate interactions with T cells. This work, while largely confirming proposed models for in situ differentiation, provides important in situ data on the coordinated site-specific recruitment and differentiation of these cells and helps elaborate the predominance of immune pathology at the airways. Our novel imaging technology to trace immunogenic cell maturation in situ will complement existing information available on in situ differentiation deduced from other immunological methods, and assist better understanding of the spatio-temporal cellular behavior during an inflammatory response.

Live imaging of biological specimens using optical microscopy is limited by tradeoffs between spatial and temporal resolution, depth into intact samples, and phototoxicity. Two-photon laser scanning microscopy (2P-LSM), the gold standard for imaging turbid samples in vivo, has conventionally constructed images with sufficient signal-to-noise ratio (SNR) generated by sequential raster scans of the focal plane and temporal integration of the collected signals. Here, we describe spatiotemporal rank filtering, a nonlinear alternative to temporal integration, which makes more efficient use of collected photons by selectively reducing noise in 2P-LSM images during acquisition. This results in much higher SNR while preserving image edges and fine details. Practically, this allows for at least a four fold decrease in collection times, a substantial improvement for time-course imaging in biological systems.

Abstract The nature of an immune response is rarely defined by a unanimous decisions by the participating cells; cells with seemingly opposing functions pervade many immune sites and tumors are no exception. Multiple DC and Macrophage subsets exert push/pull on T cell responses in tumors. Fundamentals of this are gleaned by live-imaging in connection with conventional methods such as flow-cytometry and immunofluorescence. Here, we broadly treat real-time imaging as a discovery tool to understand the tumor microenvironment (TME) and the diversity of cellular interactions that comprise host-tumor interactions. We have advanced this technology from sites of primary, spontaneous and aggressive tumors into sites of metastasis. The results promote the concept of seeking “allies” for tumor therapies within the TME and provide a basis for considering the balance of push/pull signals that determine the consensus immune response. Citation Format: Edward W. Roberts, Mark B. Headley, Miranda Broz, Bijan Boldjipour, Kaitlin Corbin, Mikhail Binnewies, Adriaan Bins, Audrey Audrey Gerard, Matthew Krummel. Visualizing tumor immune interaction in real time [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA32.

We demonstrate the capabilities of μMagellan: a flexible, open source microscopy software for reproducible high throughput imaging of biological samples across heterogeneous scales of space and time. μMagellan provides a simple user interface for exploration and automated imaging of non-cuboidal regions. By utilizing the hardware abstraction layer of μMagellan, μMagellan provides a powerful and extensible platform for imaging heterogeneous biological samples on a wide range of existing microscopes.

We demonstrate the capabilities of μMagellan: a flexible, open source microscopy software for reproducible high throughput imaging of biological samples across heterogeneous scales of space and time. μMagellan provides a simple user interface for exploration and automated imaging of non-cuboidal regions. By utilizing the hardware abstraction layer of μManager, μMagellan provides a powerful and extensible platform for imaging heterogeneous biological samples on a wide range of existing microscopes.

In type 1 diabetes, the pancreatic islets are an important site for therapeutic intervention because immune infiltration of the islets is well established at diagnosis. Therefore, understanding the events that underlie the continued progression of the autoimmune response and islet destruction is critical. Islet infiltration and destruction is an asynchronous process, making it important to analyze the disease process on a single islet basis. To understand how T cell stimulation evolves through the process of islet infiltration, we analyzed the dynamics of T cell movement and interactions within individual islets of spontaneously autoimmune NOD mice. Using both intravital and explanted two-photon islet imaging, we defined a correlation between increased islet infiltration and increased T cell motility. Early T cell arrest was Ag dependent and due, at least in part, to Ag recognition through sustained interactions with CD11c+ APCs. As islet infiltration progressed, T cell motility became Ag independent, with a loss of T cell arrest and sustained interactions with CD11c+ APCs. These studies suggest that the autoimmune T cell response in the islets may be temporarily dampened during the course of islet infiltration and disease progression.

Multiphoton microscopy has become staple tool for tracking cells within tissues and organs due to superior depth of penetration, low excitation volumes, and reduced phototoxicity. Many factors, ranging from laser pulse width to relay optics to detectors and electronics, contribute to the overall ability of these microscopes to excite and detect fluorescence deep within tissues. However, we have found that there are few standard ways already described in the literature to distinguish between microscopes or to benchmark existing microscopes to measure the overall quality and efficiency of these instruments. Here, we discuss some simple parameters and methods that can either be used within a multiphoton facility or by a prospective purchaser to benchmark performance. This can both assist in identifying decay in microscope performance and in choosing features of a scope that are suited to experimental needs.