Comparative Evaluation of Genetically Encoded Voltage Indicators

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Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Deep-skin multiphoton microscopy in vivo excited at 1600 nm: A comparative investigation with silicone oil and deuterium dioxide immersion

Multiphoton microscopy (MPM) excited at the 1700-nm window has enabled deep-tissue penetration in biological tissue, especially brain. MPM of skin may also benefit from this deep-penetration capability. Skin is a layered structure with varying refractive index (from 1.34 to 1.5). Consequently, proper immersion medium should be selected when imaging with high numerical aperture objective lens. To provide guidelines for immersion medium selection for skin MPM, here we demonstrate comparative experimental investigation of deep-skin MPM excited at 1600 nm in vivo, using both silicone oil and deuterium dioxide (D₂O) immersion. We specifically characterize imaging depths, signal levels and spatial resolution. Our results show that both immersion media give similar performance in imaging depth and spatial resolution, while signal levels are slightly better with silicone oil immersion. We also demonstrate that local injection of fluorescent beads into the skin is a viable technique for spatial resolution characterization in vivo.      

Dissection and 2-photon imaging of peripheral lymph nodes in mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response (1). Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 (2). Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37 degrees C.     

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.     

Small Networks Encode Decision-Making in Primary Auditory Cortex

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Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.     

Self-phase-modulated femtosecond laser source at 1603 nm and its application to deep-brain 3-photon microscopy in vivo

3-photon microscopy (3PM) excited at the 1700 nm window enables deep-tissue imaging in vivo, especially in brain. PC rod soliton source has previously been exclusively used as the excitation source, which is rather costly and difficult to align. Here we demonstrate a novel nonlinear optical technique to build femtosecond laser source at the 1700 nm window, based on self-phase modulation (SPM) in a short span of large-mode-area fiber. The spectral broadening experienced by the pump pulse leads to the generation of a red-shifted sidelobe at 1603 nm. After spectral filtering, this sidelobe corresponds to 170-fs, 167-nJ pulses at 1603 nm. Using this SPM source, we further demonstrate deep-brain 3 PM to a depth of 1500 μm below the mouse brain surface in vivo. Our SPM femtosecond laser source thus provides a cost effective and easy-to-align alternative excitation source to the PC rod soliton source.     

Intravital imaging of the mouse thymus using 2-photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus. Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation. Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KI(gfp/gfp) mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.     

Silk Fibroin Films Facilitate Single-Step Targeted Expression of Optogenetic Proteins

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A cerebellar-thalamocortical pathway drives behavioral context-dependent movement initiation

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Interstitial Migration of CD8αβ T Cells in the Small Intestine Is Dynamic and Is Dictated by Environmental Cues

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Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 ¹. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established ². Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Lepr^db (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.     

植物水孔蛋白的功能

水孔蛋白是由多基因编码的介导水分快速跨膜转运的膜内在蛋白。植物水孔蛋白分为4类,具有多功能性,包括介导水分的快速跨膜转运,参与气孔运动,参与叶肉内CO_2的运输,调节植物对中性分子(甘油、NH_3、尿素)和营养元素(硼、硅)的吸收,参与植物体内的氧化应激及信号的跨膜转导等。