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Multiphoton Microscopy

With multiphoton microscopy we can image several hundreds of micrometers deep into biological samples as scattering of red-shifted light in tissues is highly reduced compared to visible light.  For multiphoton excitation, pulsed infrared lasers with wavelengths of up to 1300 nm in case of the OPO (optical-parametric oscillator, What is an OPO?) are used.

Deep tissue imaging of thick specimens or in whole animals allows to observe cellular processes in their natural context. Especially intravital imaging with multiphoton excitation plays a growing role in many biomedical research areas.

  • High-Resolution 3D Imaging of Whole Organ after Clearing

    Zebrafish testis has become a powerful model for reproductive biology of teleostean fishes and other vertebrates and encompasses multiple applications in applied and basic research. Many studies have focused on 2D images, which is time consuming and implies extrapolation of results. Three-dimensional imaging of whole organs recently became an important challenge to better understand their architecture and allow cell enumeration.
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  • Multiphoton Microscopy – a Satisfied Wish List

    The colorful picture shows colon tumor cells, fluorescently labelled and lineage traced from a multicolor tracer. The gray color codes for the second harmonic generation (SHG) signal from Collagen 1. Lineage traced tumor cells are shown in magenta, blue, green, yellow and red. All channels were recorded with two-photon excitation, using the SP8 DIVE by Leica Microsystems. Sample and image were kindly provided by J. van Rheenen, H. Snippert, Utrecht (the Nederlands,) and I. Steinmetz, Leica Microsystems Mannheim.
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  • Mission Impossible Accomplished: Tunable Colors for Non-descanning Detection

    Leica Microsystems’ 4Tune detector, the key component of the SP8 DIVE Deep In Vivo Explorer, provides spectrally tunable image recording with non-descanning detection. An innovative solution for multiparameter multiphoton microscopy. The colorful image on the right shows multiphoton microscopy of an unstained mouse skin section acquired using the 4Tune detector. The green color codes for autofluorescence of muscle tissue. Red shows second harmonic generation of fibers upon illumination with 900 nm. The blue pattern is generated by third harmonic generation at lipid boundaries from illumination at 1230 nm.
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  • Laser Beam Shaping for Multicolor Multiphoton Microscopy

    Multiphoton Microscopy is one of the current hot topics in life science research. The new Leica TCS SP8 DIVE from Leica Microsystems presents a series of beneficial new innovations, including a freely tunable non-descanning detector and an ingenious beam manipulating device VBE. The variable beam expander offers free tuning of both beam diameter and axial IR-correction for up to four IR beams simultaneously.
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  • Five Questions Asked: Prof. Dr. Jacco van Rheenen speaks about the most important considerations when imaging deep into mouse tissue

    When operating a confocal microscope, or when discussing features and parameters of such a device, we inescapably mention the pinhole and its diameter. This short introductory document is meant to explain the significance of the pinhole for those, who did not want to spend too much time to dig into theory and details of confocal microscopy but wanted to have an idea about the effect of the pinhole.
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  • Chronic Stress in Mice Remodels Lymph Vasculature to Promote Tumour Cell Dissemination

    Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer.
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  • Clarifying Tissue Clearing

    Biological specimens are intrinsically three dimensional; however because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by “clearing” the tissue have been ongoing for over a century, there have been a large number of recent innovations. This review introduces the physical basis for light-scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue.
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  • Multiphoton Microscopy Publication List

    Multiphoton Microscopy is an advanced technique for imaging thick samples. Applications range from the visualization of the complex architecture of the whole brain to the study of tumor development and metastasis or the responses of the immune system in living animals. On this regularly updated reference list you can find selected publications on reseach using multiphoton microscopy.
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  • BABB Clearing and Imaging for High Resolution Confocal Microscopy: Counting and Sizing Kidney Cells in the 21st Century

    Multipohoton microscopy experiment using Leica TCS SP8 MP and Leica 20x/0.95 NA BABB immersion objective. Understanding kidney microanatomy is key to detecting and identifying early events in kidney disease. Improvements in tissue clearing and imaging have been crucial in this field, and now we report on a novel, time-efficient method to study podocyte depletion in renal glomeruli using a combination of immunofluorescence, optical clearing, confocal microscopy and 3D analysis.
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  • Clearing of Fixed Tissue: A Review from a Microscopist’s Perspective

    Chemical clearing of fixed tissues is becoming a key instrument for the three-dimensional reconstruction of macroscopic tissue portions, including entire organs. Indeed, the growing interest in this field has both triggered and been stimulated by recent advances in high-throughput microscopy and data analysis methods, which allowed imaging and management of large samples.
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  • 4Pi-RESOLFT Nanoscopy

    Here we apply the 4Pi scheme to RESOLFT nanoscopy using two-photon absorption for the on-switching of fluorescent proteins. We show that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible. Our method thus offers robust access to densely packed, axially extended cellular regions that have been notoriously difficult to super-resolve. Our approach also entails a fluorescence read-out scheme that translates molecular sensitivity to local off-switching rates into improved signal-to-noise ratio and resolution.
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  • From Light to Mind: Sensors and Measuring Techniques in Confocal Microscopy

    This article outlines the most important sensors used in confocal microscopy. By confocal microscopy, we mean "True Confocal Scanning", i.e. the technique that illuminates and measures one single point only. The aim is not to impart in-depth specialist knowledge, but to give the user a small but clear overview of the differences between the various technologies and to advise on which sensor may be most suitable for which applications.
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  • Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

    Cancer research unsing multiphoton microscopy and 3D electron microscopy. Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task.
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  • Third Harmonic Generation Microscopy – Label-Free 3D-Tissue Imaging and Blood Flow Characterization

    THG microscopy as special variants of multiphoton microscopy. Third Harmonic Generation (THG) microscopy is a non-fluorescent multi-photon technique that combines the advantages of label-free imaging with restriction of signal generation to the focal spot of the scanning laser. It allows three-dimensional imaging of refraction index mismatches and of hemoglobin.
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  • Interview with Dr. Gertrude Bunt and Prof. Fred S. Wouters on the FOM 2015

    Only a few days to go before the start of Focus on Microscopy 2015 in Göttingen, Germany. This year’s FOM is being organized by Dr. Gertrude Bunt and Prof. Dr. Fred S. Wouters from the University Medical Center, Göttingen, in cooperation with Prof. Dr. G.J. (Fred) Brakenhoff, University of Amsterdam, The Netherlands.
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  • How to Choose the Right Confocal Microscope for Your Lab?

    Confocal microscopy has come a very long way since its invention more than a half-century ago. Today, with novel technology driven by leading imaging companies, it has become the standard for fluorescence microscopy. Choosing the right confocal microscope for your specific research requires the appropriate mix of features related to resolution, sensitivity, and speed.
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  • Deeper Insights in Transparent Animals

    CLARITY clearing derivatives for multiphoton microscopy. Transparent organisms help us to identify spatial arrangements and connections of cells and tissues, especially neuronal circuits can easily be identified and characterized. CLARITY is on everyone's lips.
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  • Clearing Procedures for Deep Tissue Imaging

    Multi-channel multiphoton microscopy with dedicated optics for CLARITY. Why clearing? Curiosity is human nature. And nothing attracts as much curiosity as the inside of living organisms. While in ancient times those who cut human bodies open to do research were put to death, and modern anatomy started only after Pope Clement VII allowed dissection, we can now watch brains working in living animals – and have a good chance of soon being able to interfere with the observed activities for healing (or control) purposes.
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  • Webinar: Exploring Neurons and Synapses: Imaging Tools and Techniques

    In the coming years, considerable effort and resources will be directed at understanding the neural connections of the brain. During this webinar, we will examine many of the tools being used to study how neurons interact with one another at their synapses.
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  • Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing

    Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity.
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  • The Environment Makes the Stem Cell

    A recent publication in Nature shows that all stem cells divide and compete for niche space by passively "kicking out" others so that eventually one stem cell takes over the whole niche. Jacco van Rheenen and Saskia Ellenbroek talk about a new method of intravital imaging, which allows following the fate of individual stem cells over time in vivo and explains the new paradigm for stem cell development in the intestinal stem cell niche.
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  • Smart Control for Resonant Galvo Scanners

    High time-resolution confocal microscopy (HTRCLSM) requires fast scanning devices. Whereas non-resonant galvo scanners allow full position control, but only at slow speed, resonant scanners allow ~25,000 lines per second, but offer much less positioning freedom. To still allow zoom and pan functions, several approaches have been tried, with varying success. The Leica confocal microscopes od the TCS series use a very smart solution that enables stepless zooming with short switching times.
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  • Tracking Glomerular Fate Over Long Time Distances

    Multi-channel multiphoton microscopy with dedicated optics for CLARITY. The glomerular filtration barrier (GFB) is a complex spatial structure within the kidney glomerulis where ultrafiltration takes place. Podocytes are critical elements of the GFB and take part in the filtration process. They have been shown to be involved in the development of kidney diseases. However, due to technical limitations, the mechanism of glomerular pathology is not well understood.
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  • Map the Brain with CLARITY

    Imaging whole brains with CLARITY and multiphoton microscopy. Image a whole brain without sectioning? Investigate neuronal circuits without reconstruction? Perform molecular phenotyping without destroying subcellular structures? Understanding the brain with molecular resolution and global scope has always been challenging. The novel CLARITY method, developed by the Deisseroth laboratory at Stanford University, USA, pushes the barrier of deep tissue imaging a big step ahead.
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  • Single-Wavelength Two-Photon Excitation-stimulated Emission Depletion (SW2PE-STED) Superresolution Imaging

    Two-photon microscopy, multiphoton microcopy and super-resolution imaging. We developed a new class of two-photon excitation–stimulated emission depletion (2PE-STED) optical microscope. In this work, we show the opportunity to perform superresolved fluorescence imaging, exciting and stimulating the emission of a fluorophore by means of a single wavelength.
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  • Direct In Vivo Evidence for Tumor Propagation by Glioblastoma Cancer Stem Cells

    Cancer research using multiphoton microscopy. High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions.
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  • Webinar: Confocal and Two-Photon Microscopy Methods for Imaging the Brain

    Brain research using confocal and multiphoton microscopy. A deeper understanding of brain function requires visualization of the complex architecture of neurons and their connections. In this special webinar, two researchers working at the cutting-edge of brain imaging will discuss their applications of the latest confocal and two-photon microscopy techniques. From fluorescent labeling and imaging of neural circuits using the Brainbow system to in vivo imaging of brain structure, function, and blood flow, this webinar will give researchers a deeper appreciation of the potential for new microscopy methods to unlock the secrets of the cellular world. Unique insights into sample handling and processing of multicolor images will also be presented, and attendees will have the opportunity to ask questions.
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  • Detectors for Sensitive Detection: HyD

    This article discusses detectors (more precisely: sensors), that are employed in single point, i.e. true confocal scanning microscopes. The sensors in such systems are usually photomultiplier tubes. Also, the silicon pendants of PMTs are used for particular applications, especially single-molecule measurements. A new development has led to chimeric devices called hybrid detector (HyD) which unite benefits of both technologies.
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  • Developments in Multiphoton Excitation Microscopy

    Basics, history, and applications of multiphoton microscopy. Honouring Goeppert-Mayer’s prediction of simultaneous two-photon absorption by an atom or molecule reported in the 1930s in her PhD dissertation thesis, we can state that multiphoton excitation (MPE) microscopy, more frequently identified with two-photon excitation (2PE) fluorescence microscopy, is a key microscopy method in many areas from medicine to biology, from biophysics to materials science.
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  • Principles of Multiphoton Microscopy for Deep Tissue Imaging

    Basics of multiphoton microscopy. This interactive tutorial explains the principles of multiphoton microscopy for deep tissue imaging. Multiphoton microscopy uses excitation wavelengths in the infrared taking advantage of the reduced scattering of longer wavelengths. This makes multiphoton imaging the perfect tool for deep tissue imaging in thick sections and living animals.
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Interactive Tutorials

Useful Links

Communities and Web Sources network for scientists Microscopy Mailing List, University of Minnesota tools, video lectures on biology and microscopy

bitesizebio.comOnline magazine and community for molecular and cell biology researchers

www.somersault1824.comResource for high-end scientific illustrations, images and animations

Search Engines and Data Bases

www.cellimagelibrary.orgPublic resource database of images, videos, and animations of cells meta search engine for genes and proteins

www.gopubmed.comSearch interface for pubmed of academic databases and search engines of Google's search engine for scientific article abstracts

Journals of open access journals EMBO Journal

www.lifescied.orgCBE-Life Sciences Education – an ASCB online journal publication of exceptional research articles of Cell Science Journal of Experimental Biology Disease Models & Mechanisms Journal of Life Science Methods of Journals and Proceedings in Optics and Photonics - peer-reviewed journals on applied research in optics and photonics of Biophotonics, peer-reviewed, open-access, online publication B - the Royal Society's biological research journal Journal for microscopists

Organizations Society of America Microscopy Society Microscopical Society American Society of Cell Biology company of biologists

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