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What is Confocal Laser Scanning Microscopy?

Confocal Laser Scanning Microscopy (CLSM) is one of a series of methods to generate slices from microscopic samples by means of optics. The sample stays intact, and the slicing may be repeated many times. True Confocal Scanning (TCS) is a technique, where only a single, diffraction limited spot is illuminated and observed at a time. The benefit of confocal imaging is a dramatically increased contrast by removal of out-of-focus haze. Z-sequences of optical slices (3D image stacks) are sources for subsequent rendering as anaglyphes, depth-coded maps or 3D movies. TCS is also very well compatible with multi-fluorescence imaging, time-lapse imaging, FLIM, FRAP and FCS measurements – plus a whole world of spectral applications.

Axial Tubule Junctions Control Rapid Calcium Signaling in Atria

The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface.
Super-Resolution
Super-Resolution
May 31, 2017
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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.
Confocal Microscopy
Confocal Microscopy
May 16, 2017
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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.
Confocal Microscopy
Confocal Microscopy
Apr 26, 2017
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Human NK Cell Development Requires CD56-mediated Motility and Formation of the Developmental Synapse

While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34+ precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo.
Confocal Microscopy
Confocal Microscopy
Feb 15, 2017
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Testing the Münch Hypothesis of Long Distance Phloem Transport in Plants

Long distance transport in plants occurs in sieve tubes of the phloem. The pressure flow hypothesis introduced by Ernst Münch in 1930 describes a mechanism of osmotically generated pressure differentials that are supposed to drive the movement of sugars and other solutes in the phloem, but this hypothesis has long faced major challenges. The key issue is whether the conductance of sieve tubes, including sieve plate pores, is sufficient to allow pressure flow. We show that with increasing distance between source and sink, sieve tube conductivity and turgor increases dramatically in Ipomoea nil. Our results provide strong support for the Münch hypothesis, while providing new tools for the investigation of one of the least understood plant tissues.
Fluorescence Microscopy
Fluorescence Microscopy
Feb 14, 2017
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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.
Confocal Microscopy
Confocal Microscopy
Jan 26, 2017
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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.
Multiphoton Microscopy
Multiphoton Microscopy
Dec 14, 2016
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Methods to Calibrate and Scale Axial Distances in Confocal Microscopy as a Function of Refractive Index

Application example of HyVolution Super-Resolution - Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented.
Super-Resolution
Super-Resolution
Dec 09, 2016
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Multispectral Phloem-Mobile Probes: Properties and Applications

Using Arabidopsis (Arabidopsis thaliana) seedlings, we identified a range of small fluorescent probes that entered the translocation stream and were unloaded at the root tip. These probes had absorbance/emission maxima ranging from 367/454 to 546/576 nm and represent a versatile toolbox for studying phloem transport. Of the probes that we tested, naturally occurring fluorescent coumarin glucosides (esculin and fraxin) were phloem loaded and transported in oocytes by the sucrose transporter, AtSUC2. Arabidopsis plants in which AtSUC2 was replaced with barley (Hordeum vulgare) sucrose transporter (HvSUT1), which does not transport esculin in oocytes, failed to load esculin into the phloem.
Fluorescence Microscopy
Fluorescence Microscopy
Nov 29, 2016
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P53- and Mevalonate Pathway–Driven Malignancies Require Arf6 for Metastasis and Drug Resistance

Application example of HvYolution Super-Resolution - Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial–mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program.
Super-Resolution
Super-Resolution
Oct 19, 2016
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Work Efficiently in Developmental Biology with Stereo and Confocal Microscopy: C. elegans

C. Elegans
For scientists, technicians, and teachers working with the worm C. elegans in the research lab or classroom, this report is intended to give useful information to help improve their daly work. The aim is to make the work steps of worm picking, transgenesis, RNA interference, screening, and functional imaging efficient. It also details the various possibilities for equipping a research worm lab or biology classroom/teaching lab explaining worm methods.
Stereo Microscopy
Stereo Microscopy
Sep 19, 2016
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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.
Multiphoton Microscopy
Multiphoton Microscopy
Sep 12, 2016
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