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White Confocal

Confocal microscopy has brought a major turning point for the imaging techniques in biology and medicine. In contrast to material applications, biomedical research uses, in most cases, fluorescent stains to visualize specific structures and components of biological samples. Typically, three to five different fluorochromes are used for simultaneous detection. The fluorochromes vary significantly, to match the specific requirements for the given experiment. Consequently, the excitation and emission colors also vary and call for a maximum of spectral flexibility. A series of ingenious inventions has finally lead to a fully tunable confocal microscope. The design concept leaves no spectral limitations, and is hence called the “White Confocal”. It combines a white light laser source, tunable excitation filtering, tunable beam-splitting and tunable emission-detection – with full access to the keys for all colors.

  • Primary Beam Splitting Devices for Confocal Microscopes

    Current fluorescence microscopy employs incident illumination which requires separation of illumination and emission light. The classical device performing this separation is a color-dependent beam splitting mirror which has fixed spectral parameters and transmits the emission usually between 90% and 98% within the designated bands. Transmission is wavelength dependent and also differs by technology, requirements and design. An alternative is the acousto optical beam splitter which has freely tunable reflection notches and transmits the emission on average at 95% between these notches.
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  • The Apical Ectodermal Ridge of the Mouse Model of Ectrodactyly Dlx5;Dlx6−/− Shows Altered Stratification and Cell Polarity, which are Restored by Exogenous Wnt5a Ligand

    The congenital malformation split hand/foot (SHFM) is characterized by missing central fingers and dysmorphology or fusion of the remaining ones. During limb development, the apical ectodermal ridge (AER) is a key-signaling center responsible for early proximal–distal growth and patterning. In search for the mechanism, we examined the non-canonical Wnt signaling, considering that Dwnt-5 is a target of distalless in Drosophila and the knockout of Wnt5, Ryk, Ror2 and Vangl2 in the mouse causes severe limb malformations.
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  • Contributions of Microbiome and Mechanical Deformation to Intestinal Bacterial Overgrowth and Inflammation in a Human Gut-on-a-Chip

    A human gut-on-a-chip microdevice was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation. This in vitro model replicated results from past animal and human studies, including demonstration that probiotic and antibiotic therapies can suppress villus injury induced by pathogenic bacteria.
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  • The Oncogenic Triangle of HMGA2, LIN28B and IGF2BP1 Antagonizes Tumor-Suppressive Actions of the let-7 Family

    The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3′-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3′ UTR shortening.
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  • ERK2 and Akt are Negative Regulators of Insulin and Tumor Necrosis Factor-α Stimulated VCAM-1 Expression in Rat Aorta Endothelial Cells

    Diabetes is quickly becoming the most widespread disorder in the Western world. Among the most prevalent effects of diabetes is atherosclerosis, which in turn is driven in part by inflammation. Both insulin and Tumor Necrosis Factor-alpha (TNFα) increase the presence of Vascular Cellular Adhesion Molecule-1 (VCAM-1) expression. The aim of this study is to determine the effects of downregulating Extracellular signal-Regulated Kinase-2 (ERK2) and Akt on insulin and TNFa-stimulated VCAM-1 expression.
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  • Time Gating of Chloroplast Autofluorescence Allows Clearer Fluorescence Imaging In Planta

    Chloroplast, an organelle facilitating photosynthesis, exhibits strong autofluorescence, which is an undesired background signal that restricts imaging experiments with exogenous fluorophore in plants. In this study, the autofluorescence was characterized in planta under confocal laser microscopy, and it was found that the time-gated imaging technique completely eliminates the autofluorescence. As a demonstration of the technique, a clearer signal of fluorescent protein-tagged phototropin, a blue-light photoreceptor localized at the chloroplast periphery, was visualized in planta.
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  • Video: The White Light Laser – How to Effectively Excite Multiple Fluorophores with a Single Light Source

    The Leica White Light Laser produces a continuous spectral output between the wavelengths of 470 and 670 nm. It allows you to select 8 excitation lines from 3 trillion unique combinations for simultaneous imaging of multiple fluorophores. The white light laser source of the Leica TCS SP8 X perfectly matches the wavelength of any fluorophore.
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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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  • Acousto Optics in True Confocal Spectral Microscope Systems

    Acousto-optical elements have successfully replaced planar filters in many positions. The white confocal, regarded as the fully spectrally tunable confocal microscope, was not possible without this technique. Acousto-optical elements are highly transparent, quickly tunable and allow many colors to be managed simultaneously. As they show a strong dependence in polarization and have comparably small dimensions, their active part is used to modify and guide the laser illumination light, thereby leaving the principal beam (0th order) unaffected. Excitation color selection and attenuation (excitation filtering), as well as separation of illumination and detection light (beam splitting) are the main fields of application.
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  • Spectral Detection – How to Define the Spectral Bands that Collect Probe-specific Emission

    To specifically collect emission from multiple probes, the light is first separated spatially and then passes through a device that defines a spectral band. Classically, this is a common glass-based bandpass filter. More recent approaches employ arrays for fixed-band detection or moving mirror sliders for fully tunable band characteristics.
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  • Multiple Microscopy Modes in a Single Sweep with Supercontinuum White Light

    Lasers have been critical to the advancement on confocal microscopy, and the white light laser (WLL) offers particular advantages. Finessing WLL output for bioimaging is a complex task, though, and traditional approaches retain key limitations. But acousto-optical beamsplitting enables smoother operation, leading to enhanced microscopy capabilities.
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  • The White Confocal – Spectral Gaps Closed

    This article summarizes the development and differences in design and functionality of confocal technology as far as spectral properties are concerned, from classical filter-based excitation and emission color selection to fully flexible spectral excitation and emission tuning. All three major components: light source with excitation color selection, beam splitting for incident illumination and detector emission filtering have been completely transformed.
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  • The White Confocal

    Modern biomedical research is currently dominated by imaging and measuring with optical microscopes. One branch of the microscopy technology is confocal microscopy. For correlation purposes, multiparameter fluorescence imaging is particularly of unique interest. This article is concerned with the spectral performance of the various modules in a confocal point-scanning microscope ("True Confocal System"), and how these modules have evolved to allow for tunability and flexibility in excitation and emission collection in multiple bands (channels).
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  • Gates Open for Improved Confocal Fluorescence and Super-Resolution STED

    True confocal microscope systems feature single-point illumination and single-point detection. The method is called "optical sectioning" since the generated image contains only information from the focal plane. The serial detection offers highly efficient and low-noise sensors for signal conversion. Although the nonparallel detection is not conducive to high-speed imaging, modern scanning concepts allow frame rates above 400 frames per second at reasonable noise levels. This is by far enough for most applications, including the monitoring of fast ion-transport phenomena in living material.
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  • Sensors for True Confocal Scanning

    In this article, advantages and disadvantages of different types of sensors for single point true confocal scanning devices are discussed. Traditionally, photomultiplier tubes have been employed in such systems. For some cases, avalanche photodiodes have proven to fit best. A new development uniting vacuum and silicon technology has led to chimeric sensors, called hybrid detectors (HyD). They benefit from both technologies.
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  • White Light Laser

    The perfect light source for confocal microscopes in biomedical applications has sufficient intensity, tunable color and is pulsed for use in lifetime fluorescence. Furthermore, it should offer means to avoid reflection of excitation light, and the coupling into the beam path must be efficient and homogeneous throughout the full visible spectrum. Such a source has been invented and implemented: the white light laser in combination with acousto-optical beam splitting.
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  • How to Study Protein Recruitment to DNA Lesions by a Combination of UV Laser and White Light Laser

    Understanding how DNA lesions are optimally repaired is of functional significance, especially from the view of genome karyotype stability.
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  • Beam Splitting

    Fluorescence Microscopy usually employs incident light illumination. This requires a device that directs the light for illumination into the sample and transmits the light emitted by the sample to the detection system. In the past, various types of mirrors were the only option. Today, the acousto optical beam splitter serves best for the task.
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  • Spectral Imaging

    To separate fractions of the emission for recording channels that reproduce the emission of individual fluorochromes, it is necessary to spatially disperse the emission spectrally. This is possible by employing dichroic mirrors or a genuine dispersive element like a prism or a grating.
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  • Confocal Excitation

    Fluorescence excitation needs specifically colored light. In confocal microscopy, multiline lasers or laser batteries are classically used. This requires devices that pick the requested lines fitting the currently employed fluorochromes. Intensity control is a second task that must be accomplished.
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  • Choose Your Excitation Wavelength

    Although time correlated single photon counting (TCSPC) is the method of choice for fluorescence lifetime quantification, it requires dedicated instrumentation including a pulsed laser source, a photon counting card, and a fast detector.
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  • The First Supercontinuum Confocal that Adapts to the Sample

    Until now, biological and medical research fluorescence imaging in multi-user facilities or institutes has been limited by the type or number of dyes that could be excited. The Leica TCS SP5 X supercontinuum confocal unites the broadband capabilities of the Leica TCS SP5 AOBS® and the freedom and flexibility to select any excitation line within the continuous range of 470 to 670 nm.
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  • FRET Measurements on Fuzzy Fluorescent Nanostructures

    In the last decade, fluorescence resonance energy transfer (FRET) has become a useful technique for studying intermolecular interactions applied to the analysis of biological systems. Although FRET measurements may be very helpful in the comprehension of different cellular processes, it can be difficult to obtain quantitative results, hence the necessity of studying FRET on controllable systems.
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Useful Links

Communities and Web Sources

www.researchgate.net/
Social network for scientists

www.lsoft.com/scripts/wl.exe?SL1=CONFOCALMICROSCOPY&H=LISTS.UMN.EDU
Confocal Microscopy Mailing List, University of Minnesota

www.ibiology.org/
Teaching tools, video lectures on biology and microscopy

bitesizebio.com
Online magazine and community for molecular and cell biology researchers

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

Search Engines and Data Bases

www.cellimagelibrary.org
Public resource database of images, videos, and animations of cells

harvester.fzk.de/harvester
Bioinformatic meta search engine for genes and proteins

www.gopubmed.com
Search interface for pubmed

en.wikipedia.org/wiki/List_of_academic_databases_and_search_engines
List of academic databases and search engines

scholar.google.com
Beta 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.org
CBE-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.xml
SPIE - peer-reviewed journals on applied research in optics and photonics

onlinelibrary.wiley.com/journal/10.1002/(ISSN)1864-0648
Journal of Biophotonics

www.plosone.org/home.action
International, 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.html
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