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Rolf T. Borlinghaus, Dr.

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Rolf Borlinghaus was born 1956  in Grötzingen, Germany. After his diploma in Biology he worked on electrogenic steps of the Na/K-ATPase by laser-induced release of ATP from a caged compound at Peter Läuger’s Laboratory in the Biophysics Department, University Konstanz, Germany from where he was promoted to Dr.rer.nat. in 1988. He started working as a Product Manager for research Fluorescence and confocal Microscopes with Carl Zeiss, Oberkochen in 1990 and continued to tackle this challenge at Leica in 1997 (at that time Leica Lasertechnik, Heidelberg). For personal insights, in 2007, Rolf Borlinghaus dispensed his managerial responsibilities and is now supporting the confocal marketing group as senior scientist in a half-time position. The other half is dedicated to relations, food, music, books and botany.

  • 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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  • 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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  • Pinhole Effect in Confocal Microscopes

    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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  • HyVolution – Super-Resolution Imaging with a Confocal Microscope

    Since the invention of the microscope, there has been continual discussion about the possibility of showing more detailed features of specimens as compared to just magnifying them. In this article we describe the HyVolution concept and how the combination of confocal multiparameter fluorescence imaging at the confocal super-resolution regime with psf-based real deconvolution allows high-speed multicolor imaging with a resolution down to 140 nm.
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  • HyVolution – the Smart Path to Confocal Super-Resolution

    Super-resolution refers to any device or method that can resolve better than the classical Abbe limit. Apart from infinite super-resolution techniques such as STED (stimulated emission depletion) and SMLM (single-molecule localization methods) that can theoretically resolve to any detail, there are also methods for limited super-resolution. Here we present HyVolution by Leica, which merges optical super-resolution and computational super-resolution. The optical part is provided by confocal microscopy, and the computational part by deconvolution. Lateral resolution of 140 nm is demonstrated. HyVolution offers multiple fluorescence recording in truly simultaneous mode.
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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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  • Confocal and Digital Light Sheet Imaging

    Optical imaging instrumentation can magnify tiny objects, zoom in on distant stars and reveal details that are invisible to the naked eye. But it notoriously suffers from an annoying problem: the limited depth of field. Our eye-lens (an optical imaging instrument) has the same trouble, but our brain smartly removes all not-in-focus information before the signal reaches conscious cognition.
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  • Super-Resolution – On a Heuristic Point of View About the Resolution of a Light Microscope

    Since super-resolution has become one of the most favored methods in biomedical research, the term has become increasingly popular. Still, there is much of confusion about what is super-resolution and what is resolution at all. Here, the classical view of microscopic resolution is discussed and some techniques that resolve better than classical are briefly introduced. The picture on the right shows the intensity distribution of an image of two points whose distance is just the Rayleigh criterion (false color coding).
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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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  • 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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  • Super-resolution Microscopy and the Third Dimension

    Optical imaging devices have a finite depth of field and diffraction limited resolution. The depth of field problem was tackled first with confocal microscopes, diffraction unlimited resolution is available since a few years with super-resolution microscopes. Super-resolution microscopes with a solved depth of field problem are now available.
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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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  • Pinhole Geometry: Four Corners are Perfect

    Square and hexagonal pinholes provide identical image signal levels, if the geometries are compared in a sensible manner. The amount of light passing the pinhole depends on the area of that aperture, consequently the area is the parameter that must be compared when discussing brightness of focus images. The use of incommensurable edge lengths is meant to confuse the reader and thus dishonest and reprehensible. In this article, the signal level as a function of geometry and size in confocal microscopes is described.
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  • Unmask the Hidden without Probes: CARS

    Fluorescence microscopy assumed a pivotal role in cell biology once it was possible to stain cell components selectively by fluorescing dyes. One of the first explorers of targeted stainings, Paul Ehrlich, had the idea that something that stains specifically should also kill specifically – which was associated with the term “magic bullet”, the essential idea of chemotherapy. His group discovered Salvarsan, a tailored drug against syphilis – though not specific enough not to cause substantial side effects. Screening many fluorescent dyes led to a long list of stainings which are used in histology, including dyes like DAPI or hematoxylin and eosin.
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  • STED and GSDIM: Diffraction Unlimited Resolution for all Types of Fluorescence Imaging

    This article gives an overview of two different types of superresolution techniques. Stimulated emission depletion (STED) microscopy is a versatile and fast method that is based on point scanning microscopy – usually an extension of a confocal microscope. Ground state depletion imaging (GSDIM) is a parallel recording widefield approach that explores inherent switching of fluorochromes and typically comes with a TIRF microscope. The two methods use very different approaches to reach the same goal: to see more details in light microscopes than possible when diffraction limited.
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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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  • 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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  • 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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  • Optogenetics

    Optogenetics is a technique that allows light-controlled responses of transfected cells. The cells are genetically modified by introduction of genes that code for light-induced channels or ion pumps. The term optogenetics denotes the light control feature introduced by genetic engineering.
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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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