Vascular flow close to a lymph node in a live mouse harboring a metastatic tumor Vascular flow close to a lymph node in a live mouse harboring a metastatic tumor imaged by using intra-vital microscopy. Red blood cells and leucocytes are shown in black. In green, two tumoral cells in the process of entering the vasculature. The movie is played at 25X the real speed.
In vivo imaging of activated (yellow) and resting (green) platelets After a laser-induced injury
Thrombus formation after a ferric chloride injury Using the Cellvizio system
Using two-photon microscopy to visualise blood flow through the lungs Blood flow in animal lungs imaged by a two-photon microscope in the Thorn Lab, School of Biomedical Sciences, University of Queensland, Australia. 7.5x real time. Extracellular dyes are SRB and HPTS.
CD11c DTR GFP Qdot xyz 3D rotation of ankle, intravital green = CD11c langerhan cells blue = collagen red = Q dot for vasculature not the best movies but good proof of principle
Mucosal DCs and Blood Flow.avi CX3CR1 positive dendritic cells (DCs) are visualized in Peyers Patch (PP) by using a reporter in which a single allele for CX3CR1 is replaced with EGFP. While under anesthesia, a loop of the distal ileum was exposed, a single incision was made along the anti-mesenteric wall to expose the mucosal surface and visualized via single-photon intravital microscopy. Blood flow is confirmed by intravenous injection of a low molecular weight dextran conjugated to fluorescent Alexa-647 (red). Individual villi filled with DCs (green) are readily discernable while DCs are probing the surrounding the microenvironment (see examples in white circles). Also note that the low molecular weight dye diminishes over time since it is filtered out by the kidney.
A Dynamic Map of Antigen Recognition by CD4 T Cells at the Site of Leishmania major Infection Parasite-specific T cells (brown) display variable behaviour in mouse dermis with respect to phagocytic cells (green) infected with Leishmania major (red); one T cell (circled on left) decelerates upon contact, while another (circled on right) maintains high motility. Imaged using intravital two-photon microscopy. (Orchidée Filipe-Santos, Pascale Pescher, Béatrice Breart, Christoph Lippuner, Toni Aebischer, Nicolas Glaichenhaus, Gerald F. Späth, and Philippe Bousso)
PP T cell migration.avi Wild type donor T cells were harvested from mesenteric lymph node, labeled with cell tracker orange and transferred into animals expressing EGFP under control of the myeloid cell-specific promoter CX3CR1 (CX3CR1-EGFP). Following transfer, recipients were anesthetized, a loop of distal small bowel was exposed and Peyers Patch was visualized via single photon intravital microscopy. Sessile dendritic cells in the PP T cell zone (green), migratory vascular monocytes (green) and donor T cells (red) can be readily visualized. Although sessile, DCs extend and retract dendrites, probing for T cells which are actively trafficking through the tissue and transiently interacting with DCs.
Cross section of a large vein in a live mouse Cross section of a large vein in a live mouse imaged by using intra-vital microscopy. Circulating blood cells flowing through the vessel are shown in black. The movie is played at 9X the real speed.
Filtration of a fluorescent dye through the kidney of a live rat Filtration of a fluorescent dye through the kidney of a live rat imaged by using intra-vital microscopy. In red, a fluorescent dye that flows first through the distal and then to the proximal tubuli (both highlighted in green). The movie is played at 50X the real speed.
Liver-2 Vascular flow (Cascade Blue Dextran, blue) in the liver of a transgenic mouse expressing soluble GFP (Hepatocytes, green) and membrane-targeted Tomato (Cellular membranes, red). Imaging performed by intravital confocal microscopy.
(two-photon laser scanning microscopy) Intravital imaging of T-cellDC interactions in lymph nodes. Antigen-specificCD4+ T cells (red) and peptide-pulsed dendritic cells (DCs) (green) were imaged in the popliteal lymph node of an anesthetized mouse using two-photon laser scanning microscopy. Imaging was carried out at 33 hours, during the late phase of activation. T-cell activation by dendritic cells in the lymph node: lessons from the movies Philippe Bousso Nature Reviews Immunology 8, 675-684 (September 2008) doi:10.1038/nri2379
Imaging of cancer cells participating to a growing tumor Using the Cellvizio system
Real time thrombus formation after a laser-induced injury Platelets (green) are detected by infusion of a anti CD41-Alexa 488 antibody
Liver Vascular flow in the liver of a live rat imaged by using intra-vital microscopy. In red, Texas-Red Dextran, in cyan the hepatocytes revealed by endogenous fluorescence.
Confocal imaging of Platelets (red) and P-selectin (green) After a laser-induced injury
Vena4Y Flow Assay THP-1 monocytes adhering to VCAM-1 coated Vena4Y biochip at 0.5 dynes/cm2
Inflammation Control Experiment: HUT78 T-cells not adhering in BSA coated biochip at 0.5 dyne/cm² Inflammation Control Experiment: HUT78 T-cells not adhering in BSA coated biochip at 0.5 dyne/cm². **NO AUDIO WITH THIS VIDEO**
Endocytosis-1 Systemic injection of 70 KDa Texas Red-Dextran and 500 KDa FITC-Dextran in a live rat. Texas Red-dextran internalization in the stromal cells of the Salivary Glands is imaged using intravital two-photon microscopy.
Effect of BAPTA-AM on platelet accumulation After a laser-induced injury
Calcium Mobilization into platelets participating to a growi After a laser-induced injury
Intravital microscopy of HT1080-GFP tagged fibrosacroma tumour cells rolling Intravital microscopy of HT1080-GFP tagged fibrosacroma tumour cells rolling. Video courtesy of Prof. Robert Hoffman (Anti-Cancer Inc.) and Prof. William Gallagher (University College Dublin). **NO AUDIO WITH THIS VIDEO**
Dynamic imaging of cellular interactions with extracellular matrix From the Springer article: Dynamic imaging of cellular interactions with extracellular matrix by: Friedl, Peter; MV3CD44c3.avi (1.8 MB) Journal: Histochemistry and Cell Biology Vol. 122 Issue 3 DOI: 10.1007/s00418-004-0682-0 Published: 2004-09-01
Vasculature Segmentation in Intravital Microscopy Images Using a Physics Based Model Segmentation of vessels in biomedical images is important as it can provide insight on the changes of blood flow as well as assist in tracking of cells. The images of the liver vasculature obtained from IVM are ***yzed by manually measuring vessel properties in a few sample areas in the images. This is a tedious and time consuming process and is prone to variable technician bias. The automation of segmentation is challenging as the vessel images captured in intravital microscopy contain a high degree of noise. Also, a large number of vessels present in these images often results in a high computation requirement. We propose a new method of segmenting based on physical force model. "Vessel probes" placed in the image are moved toward the vessel boundary while spreading out from each other according to Newtonian Physics responding to the forces from image attributes as well as from each other. Some preliminary results will be discussed on this on-going project.
Multiphoton Microscope Alignment using Second Harmonics Generation Crystal Multiphoton Microscope Alignment using Second Harmonics Generation Crystal Mavericks femtosecond Cr:Forsterite laser is aligned into Olympus FluoView FV1000 using Second Harmonic Generation (SHG) crystal, in this case BBO crystal, placed in the focal plane of the microscope. The Mavericks-65 Cr:forsterite laser is tunable over wavelengths from 1230 to 1270 nm, making it ideal for imaging, condensed matter and biomedical applications. These wavelengths are less damaging to biological samples than the shorter wavelengths produced by Ti:sapphire and other femtosecond lasers. This allows in vivo imaging of cells and other biological samples. The MFM uses pulsed long-wavelength light to excite fluorophores within the specimen being observed. The fluorophore absorbs the energy from two long-wavelength photons which must arrive simultaneously in order to excite an electron into a higher energy state, from which it can decay, emitting a fluorescence signal. It differs from traditional fluorescence microscopy in which the excitation wavelength is shorter than the emission wavelength, as the summed energies of two long-wavelength exciting photons will produce an emission wavelength shorter than the excitation wavelength. Multiphoton fluorescence microscopy has similarities to confocal laser scanning microscopy. Both use focused laser beams scanned in a raster pattern to generate images, and both have an optical sectioning effect. Unlike confocal ...
(two-photon laser scanning microscopy) T cells establish short interactions with DCs no antigen Antigen-specificCD4+ T cells (red) and peptide-pulsed dendritic cells (DCs) (green) were imaged in the popliteal lymph node of an anesthetized mouse using two-photon laser scanning microscopy. Imaging was carried out at 33 hours, during the late phase of activation. T-cell activation by dendritic cells in the lymph node: lessons from the movies Philippe Bousso Nature Reviews Immunology 8, 675-684 (September 2008) doi:10.1038/nri2379
Multiphoton Microscopy - Mounted Artery of Mouse Mounted artery of the mouse. 3 channel overlay with autofluorescence of elastin (blue), syto for nuclei of cells in the vascular wall (green/white), Eosin auro-fluorescence (red) Imaging depth: 650 microns Movie acquired with Leica TCS SP5 MP - DM6000 CFS Courtesy of Marc von Zandvoort, Biophysics, University of Maastricht, Netherlands
Migration Macrophages migrating in a tumor implanted in the back of an immunocompromised mouse and imaged by intravital two-photon microscopy. The tissue is labeled by injection of Hoechst (cyan) to label the nuclei.
Dermal DC migration From: Ng LG, Hsu A, Mandell MA, Roediger B, Hoeller C, et al. (2008 ) Migratory Dermal Dendritic Cells Act as Rapid Sensors of Protozoan Parasites. PLoS Pathog 4(11): e1000222. doi:10.1371/journal.ppat.1000222 Dermal DC migration. A time-lapse sequence of maximum projection (21 µm stack) shows the migratory patterns of DDC in CD11c-YFP mouse ear skin. Time is shown as hh:mm:ss. CC-by
Platelet accumulation following a FeCl3 injury on mesentery Using the intravital microscope
GAS in vivo.avi This is a video clip of the skin flap of a wildtype mouse infected with Streptococcus pyogenes using intravital multiphoton microscopy. We injected 10^7 bacteria, then performed a skin flap surgery of the abdomen area, and then imaged the skin flap one hour post injection. We recorded a 15 minute movie that imaged from the surface of the collagen layer to 75µm into the tissue. The green dots represent the bacteria, Qdots was the name of the dye used to highlight the vasculature, and the blue color represents the collagen layer. We recorded the movie at 2X zoom and visualized a 158 x 158µm area. We used 10% laser power at 850nm wavelength.
Dissemination of cancer cells in vivo using the Cellvizio system
PP DCs and Blood Flow.avi CX3CR1 positive dendritic cells (DCs) are visualized in Peyers Patch (PP) by using a reporter in which a single allele for CX3CR1 is replaced with EGFP. While under anesthesia, a loop of the distal ileum was exposed and visualized via single-photon intravital microscopy. Both DC poor B cell zones and DC rich T cells zones can be discerned. DCs are seen probing (extending and retracting dendrites) the surrounding microenvironment, presumably interacting with migrating T cells. Blood flow is confirmed by intravenous injection of a low molecular weight dextran conjugated to fluorescent Alexa-647 (red).
Team Mobilife from USA Mobilife introduces innovative application technologies into the market of mobile medicine by pairing the widely-available Windows Mobile platform with computer-assisted intravital microscopy to provide on-field ***ysis of the human microcirculation to detect developing microangiopathy in children using a cellphone.
Ribbon Snake Iterations Robustly segmenting vessels even with less sharp boundary is important for achieving accurate biological ***ysis of blood vessels regulation within organs such as liver. This process is crucial for microvaculature reconstruction which is necessary for red blood cells flow distribution regulation ***ysis. The vessels with sharp edges are often used during manual ***ysis skewing the data toward a certain group of vessels. In this paper, we propose Bridging Vessel Snake (BVS) for segmenting a network of vessels (especially ones with less sharp boundary) in intravital microscopy images. Our method enables segmentation of vessels with varying diameter while imposing the structure of vessels by utilizing a ribbon snake and adding energies of width and region. The initialization achieved by the skeletonization is used to segment mostly sharper vessels. The ``bridges'' between the segmentation of sharper (thus higher confidence) vessels are used for hypothesizing less sharp vessels. A preliminary evaluation against a manual ground truth using a receiver operating characteristic (ROC) curve reveals that the algorithm was able to improve the area under ROC curve up to 20% on the vessels with lower sharpness and achieve the area under a ROC in all ground truthed vessels of 0.90.
X7cherry FUGW time lapse without info.avi Intravital microscopy of moving cells
Dynamic Fluorescence of Cancer in vivo - 2 AntiCancer Inc. uses fluorescent proteins for high-resolution, multiparameter in vivo imaging