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.
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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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
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.Read article -
Order versus Disorder
In modern biomaterial design the generation of an environment mimicking some of the extracellular matrix features is envisaged to support molecular cross-talk between cells and scaffolds during tissue formation/remodeling. In bone substitutes chemical biomimesis has been particularly exploited; conversely, the relevance of pre-determined scaffold architecture for regenerated bone outputs is still unclear.Read article -
Good Vibrations
In recent years, new molecular imaging techniques, such as coherent anti-Stokes Raman scattering microscopy (CARS), have been developed for rapid vibrational imaging of living cells.Read article
Interactive Tutorials
Useful Links
Communities and Web Sources
www.researchgate.net/Social network for scientists
www.lsoft.com/scripts/wl.exe?SL1=CONFOCALMICROSCOPY&H=LISTS.UMN.EDUConfocal Microscopy Mailing List, University of Minnesota
www.ibiology.org/Teaching 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
harvester.fzk.de/harvesterBioinformatic meta search engine for genes and proteins
www.gopubmed.comSearch interface for pubmed
en.wikipedia.org/wiki/List_of_academic_databases_and_search_enginesList of academic databases and search engines
scholar.google.comBeta of Google's search engine for scientific article abstracts
Journals
www.doaj.org/Directory of open access journals
emboj.embopress.org/The EMBO Journal
www.lifescied.orgCBE-Life Sciences Education – an ASCB online journal
www.sciencemag.org/Science
www.nature.com/Nature
www.cell.com/Biweekly publication of exceptional research articles
jcs.biologists.org/Journal of Cell Science
dev.biologists.org/Development
jeb.biologists.org/The Journal of Experimental Biology
dmm.biologists.org/DMM Disease Models & Mechanisms
www.biotechniques.com/International Journal of Life Science Methods
www.opticsinfobase.org/Collection of Journals and Proceedings in Optics and Photonics
spie.org/x576.xmlSPIE - peer-reviewed journals on applied research in optics and photonics
onlinelibrary.wiley.com/journal/10.1002/(ISSN)1864-0648Journal of Biophotonics
www.plosone.org/home.actionInternational, peer-reviewed, open-access, online publication
rspb.royalsocietypublishing.org/Proceedings B - the Royal Society's biological research journal
www.microscopy-analysis.com/International Journal for microscopists
Organizations
www.microscopy.org/Microscopy Society of America
www.eurmicsoc.org/European Microscopy Society
www.rms.org.uk/Royal Microscopical Society
www.ascb.org/ASCB American Society of Cell Biology
www.biologists.com/cob_activities.htmlthe company of biologists
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