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Leica TCS SP8 MP

Multiphoton Microscope Leica TCS SP8 MP

YFP mouse brain section, 720 µm z stack

Depth color coding.

Sample courtesy of Dr. Mark Lessard, The Jackson Laboratory, Bar Harbor, Maine, USA.

Zebrafish Development for 17 hours

Expression of cytoplasmic GFP in the notochord and prechordal plate, 980nm. Nuclei stain through RNA injection H2B-mCherry, 1030 nm.

Courtesy of BioEmergences USR3695 (IBiSA FBI), Gif-sur-Yvette, France.

In vivo Thy1-EYFP mouse, z stack

Adult Thy1-EYFP H line mouse, in vivo (cranial window), 800 µm z stack.

Courtesy of Dr. Masahiro Fukuda, Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.

Astyanax mexicanus surface fish development over 30 h

RNA injection for staining by farnesylated GFP (membranes, 980nm) and H2B-mCherry (nuclei, 1030nm).

Courtesy of BioEmergences USR3695 (IBiSA FBI), Dr. Sylvie Rétaux, Dr. Hélène Hinaux and Dr. Gaëlle Recher, CNRS, Gif-sur-Yvette, France.

Live imaging of Microglias in a live, awake mouse

Movement of Microglias in a live awake mouse, 4D animation of 217 slices and 180 sec.

Courtesy of Dr. Masahiro Fukuda, Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.

Simultaneous live rotation and IR excitation

Mouse testis section expressing dTomato (OPO).

Sample courtesy of Dr. Mark Lessard, The Jackson Laboratory, Bar Harbor, Maine, USA.

Tunicate (Phallusia mammillata) development over 6,5 h

Staining of the nuclei through RNA injection (H2B-eGFP, 980nm); membrane through bathing in FM4-64 (1030nm).

Courtesy of BioEmergences USR3695 (IBiSA FBI), Gif-sur-Yvette, France.

Calcium movement in GCaMP3 mouse after podocyte injury

Calcium movement after podocyte injury (bottom right) in a glomerulus of a GCaMP3 mouse. Vasculature labeling by Texas Red.

Courtesy of Dr. Matthias Hackl, University Clinic Cologne, Germany.

Multicolor image of a Zebrafish embryo

Lateral line primordium labeled with GFP (Green), neurons with DsRed (red), nucleus with BFP (blue). SHG signal from muscles (grey).

Courtesy of Dr. Lionel Newton, EMBL Heidelberg, Germany.

SHG signal and a parasympathetic neuronal marker in cleared pancreas

Tyrosine Hydroxylase positive neurons (red), SHG at 1080nm (green), Dapi (blue).

Courtesy of Dr. Rafael Drigo, Per-Olof Berggren Laboratory, Lee Kong Chian School of Medicine (LKCSoM), Singapore.

Zebrafish embryo development over 21h

4D time lapse, 420 stacks, 3 channel overlay: RNA injection H2B (blue), mCherry (red), EGFPras (green).

Courtesy of BioEmergences USR3695 (IBiSA FBI), Gif-sur-Yvette, France.

Zebrafish (gastrulation) RNA injection for Neptune fluorescent protein cytoplasmic staining

THG (in blue); 20x1NA; InSight at 1140 nm.

Courtesy of BioEmergences USR3695 (IBiSA FBI), Gif-sur-Yvette, France.

Mouse lymph node with GFP-labelled macrophages

SHG signal from collagen (grey).

Thy1-YFP mouse brain treated with CLARITY

Animation generated with LAS X 3D Visualization.

Courtesy of Prof. Karl Deisseroth and Dr. Raju Tomer, Stanford University, Palo Alto, CA, USA.

See-through brains

Nature Video reveals how Karl Deisseroth and his team created 3D visualizations of mouse brains by using CLARITY and fluorescent labelling.  

3D animation of a Mouse olfactory bulb

Depth of 1.5mm.

Courtesy of Dr. Günter Giese and Annemarie Scherbarth, MPI Heidelberg, Germany.

Second harmonic generation (SHG)

Second harmonic generation (SHG) - also called frequency doubling - is a nonlinear optical process that can be observed in mediums without an inversion symmetry. Two photons with the same frequency interacting with a nonlinear material are transformed into one that has exactly half of the wavelength of the incident light. In other words the energy and the frequency of the emitted photon are doubled compared to the one entering the sample.

Deep Tissue Imaging

Ureteric bud from an embryonic day 16 kidney from a HoxB7 EGFP mouse.

Courtesy of Deborah Hyink, Mount Sinai School of Medicine, New York, NY, USA

Simultanous excitation with OPO and Ti:Sa

Mouse mammary gland (left) and spleen (right). Blood vessels labelled with 70kD-Texas Red excited with OPO at 1150 nm (red). Simultaneous excitation at 800 nm results in second harmonic generation  (SHG) signal of type I collagen (purple) and autofluorescence of single cells (green).

Courtesy of Evelyne Beerling, Jacco van Rheenen, Hubrecht Institute, Utrecht, The Netherlands

Rapid acquisition of z-stacks in living animals

3D reconstructions of representative 50 µm z-stacks from timelapse acquisitions. Excitation at 910 nm, spectral unmixing was performed using the LAS AF software.

Left: Microglia labelled with GFP (green) are shown in relation to blood vessels injected with 655 nm quantum dots (red) residing in the brain parenchyma. Some microglia have their processes wrapped around blood vessels. Second harmonic generation (SHG) of skull bone (blue). 

Right: Spleen seven days following infection with lymphocytic choriomeningitis virus (LCMV). Anti-viral CD8-CFP (red) and CD4-YFP (green) T cells.

Courtesy of Debasis Nayak, Bernd Zinselmeyer and Dorian McGavern, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA

Ultarapid imaging of embryonic blood flows

Zebrafish embryonic heart,100 µm deep. Blood cells labelled with DsRed (red), SHG of muscle (gray).

The timelapse on the right was acquired with the resonant scanner at 167 frames/second at 512 x 64 pixels. Multiphoton excitation at 1100 nm with OPO.

Courtesy of Julien Vermot, IGBMC, Strasbourg-Illkirch, France

Mouse diaphragm

Second Harmonic Generation signal from muscle (red), motor neurons (green). 

Courtesy of Rüdiger Rudolf, Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Germany.

Principle of Multiphoton Microscopy

The probability of excitation in one-photon excitation (left) linearly depends on the amount of photons from the light source. In two-photon excitation (right) it is proportional to the square of the intensity of the light source and limited to the vicinity of the focal plane.

Principle of Multiphoton Microscopy

Energy levels of a fluorophore in one-photon (left) and two-photon (right) excitation. Colors correspond to the absorbed and emitted wavelengths.