High-throughput multiphoton-induced three-dimensional ablation and imaging for biotissues.
Ontology highlight
ABSTRACT: In this study, a temporal focusing-based high-throughput multiphoton-induced ablation system with axially-resolved widefield multiphoton excitation has been successfully applied to rapidly disrupt biotissues. Experimental results demonstrate that this technique features high efficiency for achieving large-area laser ablation without causing serious photothermal damage in non-ablated regions. Furthermore, the rate of tissue processing can reach around 1.6 × 10(6) μm(3)/s in chicken tendon. Moreover, the temporal focusing-based multiphoton system can be efficiently utilized in optical imaging through iterating high-throughput multiphoton-induced ablation machining followed by widefield optical sectioning; hence, it has the potential to obtain molecular images for a whole bio-specimen.
Project description:To understand the function and mechanism of biological systems, it is crucial to observe the cellular dynamics at high spatiotemporal resolutions within live animals. The recent advances in genetically encoded function indicators have significantly improved the response rate to a near millisecond time scale. However, the widely employed in vivo imaging systems often lack the temporal solution to capture the fast biological dynamics. To broadly enable the capability of high-speed in vivo deep-tissue imaging, we developed an optical gearbox. As an add-on module, the optical gearbox can convert the common multiphoton imaging systems for versatile multiscale high-throughput imaging applications. In this work, we demonstrate in vivo 2D and 3D function imaging in mammalian brains at frame rates ranging from 50 to 1000 Hz. The optical gearbox's versatility and compatibility with the widely employed imaging components will be highly valuable to a variety of deep tissue imaging applications.
Project description:The dynamic ability of neuronal dendrites to shape and integrate synaptic responses is the hallmark of information processing in the brain. Effectively studying this phenomenon requires concurrent measurements at multiple sites on live neurons. Substantial progress has been made by optical imaging systems that combine confocal and multiphoton microscopy with inertia-free laser scanning. However, all of the systems developed so far restrict fast imaging to two dimensions. This severely limits the extent to which neurons can be studied, as they represent complex three-dimensional structures. Here we present a new imaging system that utilizes a unique arrangement of acousto-optic deflectors to steer a focused, ultra-fast laser beam to arbitrary locations in three-dimensional space without moving the objective lens. As we demonstrate, this highly versatile random-access multiphoton microscope supports functional imaging of complex three-dimensional cellular structures such as neuronal dendrites or neural populations at acquisition rates on the order of tens of kilohertz.
Project description:Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level.
Project description:BackgroundThe applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed.MethodsGFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored using the META detector for spectral emission scanning. Backward scattered or reflected SHG (SHG-B) was detected using a conventional confocal detector with maximum aperture and forward scattered or transmitted SHG (SHG-F) detected using a non-descanned detector.ResultsWe show here that the developing mammary gland is encased in a thin but dense layer of collagen fibers. Sparse collagen layers are also interspersed between stromal layers of fat cells surrounding TEBs. At the margins, TEBs approach the outer collagen layer but do not penetrate it. Abnormal mammary glands from an HAI-1 transgenic FVB mouse model were found to contain TEBs with abnormal pockets of cells forming extra lumens and zones of continuous lateral bud formation interspersed with sparse collagen fibers.ConclusionsCollagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen. In addition to providing a bright signal, Carmine Alum stained whole mount slides can be imaged retrospectively such as performed for the HAI-1 mouse gland revealing new aspects of abnormal TEB morphology. These studies demonstrated the intimate contact, but relatively sparse abundance of collagen fibrils adjacent to normal and abnormal TEBS in the developing mammary gland and the ability to obtain these high resolution details subject to the discussed limitations. Our studies demonstrated that the TEB architecture is essentially unchanged after processing.
Project description:We have developed a miniaturized 3D cell-culture array (the Data Analysis Toxicology Assay Chip or DataChip) for high-throughput toxicity screening of drug candidates and their cytochrome P450-generated metabolites. The DataChip consists of human cells encapsulated in collagen or alginate gels (as small as 20 nl) arrayed on a functionalized glass slide for spatially addressable screening against multiple compounds. A single DataChip containing 1,080 individual cell cultures, used in conjunction with the complementary human P450-containing microarray (the Metabolizing Enzyme Toxicology Assay Chip or MetaChip), simultaneously provided IC(50) values for nine compounds and their metabolites from CYP1A2, CYP2D6, and CYP3A4 and a mixture of the three P450s designed to emulate the human liver. Similar responses were obtained with the DataChip and conventional 96-well plate assays, demonstrating that the near 2,000-fold miniaturization does not influence the cytotoxicity response. The DataChip may therefore enable toxicity analyses of drug candidates and their metabolites at throughputs compatible with the availability of compounds at early-stage drug discovery.
Project description:Analytical probes capable of mapping molecular composition at the nanoscale are of critical importance to materials research, biology and medicine. Mass spectral imaging makes it possible to visualize the spatial organization of multiple molecular components at a sample's surface. However, it is challenging for mass spectral imaging to map molecular composition in three dimensions (3D) with submicron resolution. Here we describe a mass spectral imaging method that exploits the high 3D localization of absorbed extreme ultraviolet laser light and its fundamentally distinct interaction with matter to determine molecular composition from a volume as small as 50 zl in a single laser shot. Molecular imaging with a lateral resolution of 75 nm and a depth resolution of 20 nm is demonstrated. These results open opportunities to visualize chemical composition and chemical changes in 3D at the nanoscale.
Project description:Traditional histologic methods are limited in their ability to detect pathologic changes of CKD, of which cisplatin therapy is an important cause. In addition, poor reproducibility of available methods has limited analysis of the role of fibrosis in CKD. Highly labor-intensive serial sectioning studies have demonstrated that three-dimensional perspective can reveal useful morphologic information on cisplatin-induced CKD. By applying the new technique of multiphoton microscopy (MPM) with clearing to a new mouse model of cisplatin-induced CKD, we obtained detailed morphologic and collagen reconstructions of millimeter-thick renal sections that provided new insights into pathophysiology. Quantitative analysis revealed that a major long-term cisplatin effect is reduction in the number of cuboidal cells of the glomerular capsule, a change we term the "uncapped glomerulus lesion." Glomerulotubular disconnection was confirmed, but connection remnants between damaged tubules and atubular glomeruli were observed. Reductions in normal glomerular capsules corresponded to reductions in GFR. Mild increases in collagen were noted, but the fibrosis was not spatially correlated with atubular glomeruli. Glomerular volume and number remained unaltered with cisplatin exposure, but cortical tubulointerstitial mass decreased. In conclusion, new observations were made possible by using clearing MPM, demonstrating the utility of this technique for studies of renal disease. This technique should prove valuable for further characterizing the evolution of CKD with cisplatin therapy and of other conditions.
Project description:The advent of optogenetics has revolutionized experimental research in the field of Neuroscience and the possibility to selectively stimulate neurons in 3D volumes has opened new routes in the understanding of brain dynamics and functions. The combination of multiphoton excitation and optogenetic methods allows to identify and excite specific neuronal targets by means of the generation of cloud of excitation points. The most widely employed approach to produce the points cloud is through a spatial light modulation (SLM) which works with a refresh rate of tens of Hz. However, the computational time requested to calculate 3D patterns ranges between a few seconds and a few minutes, strongly limiting the overall performance of the system. The maximum speed of SLM can in fact be employed either with high quality patterns embedded into pre-calculated sequences or with low quality patterns for real time update. Here, we propose the implementation of a recently developed compressed sensing Gerchberg-Saxton algorithm on a consumer graphical processor unit allowing the generation of high quality patterns at video rate. This, would in turn dramatically reduce dead times in the experimental sessions, and could enable applications previously impossible, such as the control of neuronal network activity driven by the feedback from single neurons functional signals detected through calcium or voltage imaging or the real time compensation of motion artifacts.
Project description:Impact statementSelf-assembled tissues have potential to serve both as implantable grafts and as tools for disease modeling and drug screening. For these applications, tissue production must ultimately be scaled-up and automated. Limited technologies exist for precisely manipulating self-assembled tissues, which are fragile early in culture. Here, we presented a method for automatically stacking self-assembled smooth muscle cell rings onto mandrels, using a custom-designed well plate and robotic punch system. Rings then fuse into tissue-engineered blood vessels (TEBVs). This is a critical step toward automating TEBV production that may be applied to other tubular tissues as well.