Fluorescence
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The Fundamentals and History of Fluorescence and Quantum Dots
At some point in your research and science career, you will no doubt come across fluorescence microscopy. This ubiquitous technique has transformed the way in which microscopists can image, tag and trace anything from whole organisms to single proteins and beyond. In this article, we will examine what is meant by "fluorescence", the history and basic physics behind its definition, the discovery and application of Green Fluorescent Protein (GFP) and a look at the rapidly expanding field of fluorescent probes including Quantum Dots.Read article -
Introduction to Widefield Microscopy
One of the most basic microscopy techniques is known as ‘Widefield Microscopy’. It is fundamentally any technique in which the entire specimen of interest is exposed to the light source with the resulting image being viewed either by the observer or a camera (which can also be attached to a computer monitor).Read article -
What is Photomanipulation?
The term photomanipulation describes a wide range of techniques that enable the microscopist the transition from passive observer to instigator of events by offering a way of interacting with their sample via targeted illumination. Typically researchers are trying to observe specific processes of interest in order to understand the underlying biological process. Microscopists are often forced to hunt through large populations of cells or acquire hours of time laps footage before they’re able to observe events of interest and in many cases it’s simply not possible to observe certain processes using conventional microscopy techniques alone. Photomanipulation tools enable the microscopist to initiate biological events, precisely adjusting sample labeling, biological activity, local chemical environments and in some instances physically destroy parts of their specimen.Read article -
Photoactivatable, photoconvertible, and photoswitchable Fluorescent Proteins
Fluorescent proteins (FPs) such as GFP, YFP or DsRed are powerful tools to visualize cellular components in living cells. Nevertheless, there are circumstances when classical FPs reach their limits. Watching dedicated, spatially limited protein populations of a certain protein of interest is impossible with common FPs, since they are expressed throughout the entire cell. At this point photoactivatable, photoconvertible and photoswitchable fluorescent proteins enter the stage. The members of this fluorescence toolkit can be activated from a non-fluorescent state, they can change their emission spectrum, or they are even able to be reversibly switched "on and off". With the help of these “optical highlighters”, researchers can track a distinct protein population over time by activating respectively converting their fluorescence with a spatially defined light beam of a given wavelength.Read article -
Work Efficiently in Developmental Biology and Medical Research with Stereo Microscopy: Rodent and Small Animal Surgery
This report provides information which can help improve the routine work of scientists and technicians performing studies involving surgery on small animals and rodents, i.e. mice, rats, hamsters etc., for developmental biology or medical research. The aim is to help make the work steps efficient and cost-effective, where the employment of microscopes is necessary. It also gives useful hints and details on the various microscopes which can be used in a developmental biology or medical research laboratory where small animal or rodent surgery is exploited.Read article -
TIRF Publication List
This monthly updated references list presents current papers using Leica AM TIRF in the major application fields for TIRF microscopy.Read article -
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.Read article -
Webinar: Introduction to Correlative Light and Electron Microscopy (CLEM)
The webinar will provide an overview of the latest advances in Cryo CLEM, which acts as a powerful interface by combining the best of the light and electron microscopy worlds to overcome their independent barriers and determine the location of fluorescent labelled structures within the landscape of an electron micrograph and showcase how Cryo CLEM adds additional value to quantitative 3D imaging and tomography.Read article -
Practical Guide for Excellent GSDIM Super-Resolution Images
Do you know that most protists and bacteria lack in one feature that each of our body cell has? Our cells are touch and communicate with one another. They send and receive a variety of signals that coordinate their behavior to act together as a functional multicellular organism. Exploring the way of cellular communication and the ways how the cell surface interacts to organize tissues and body structures is of great interest. Kees Jalink and his team of scientists at the Netherlands Cancer Institute (NKI) in Amsterdam obtained new scientific insights into the molecular architecture of hemidesmosomes, cytoskeletal components, cell surface receptors and vesicular proteins with the help of Ground-State-Depletion (GSD)/ dSTORM microscopy. In this interview, Kees Jalink comments on their developments in imaging chambers, buffer conditions and image analysis to get the perfect super resolution image.Read article -
Webinar: Introduction to Fluorescence Microscopy
In this seminar we will provide an overview about the latest advances in fluorescence microscopy. You will learn how you can use widefield and confocal microscopes to help you understand life’s questions down to tiny details, at high speed and state-of-the-art image quality both in living and fixed samples.Read article -
Work More Efficiently In Developmental Biology With Stereo Microscopy: Fruit Flies (Drosophila Melanogaster)
For scientists and technicians working with fruit flies, most often genus Drosophila, this report is intended to give useful information to help improve daily laboratory work by making the steps of fly pushing, fluorescent screening, dissection, and documentation/imaging more efficient. It also details various possibilities for properly equipping or stocking a fly lab.Read article -
Measuring the 3D STED-PSF with a new Type of Fluorescent Beads
A new type of fluorescent bead is presented by GATTAquant. These beads, called GATTA-Beads, are characterized by a small diameter (23 nm), high intensity and size uniformity. In combination with state-of the-art STED microscopes such as the Leica TCS SP8 STED 3X and high-end image restoration methods available in the Huygens Software, it is shown that these new beads can be used for accurate STED PSF characterization in 3D. Furthermore, it is shown that the measured 3D STED-PSF can be used to improve image restoration quality in combination with STED deconvolution methods available in the Huygens Software.Read article -
Work Efficiently in Developmental Biology with Stereo and Confocal Microscopy: 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.Read article -
Video Talk by Roger Tsien: Fluorescent Protein Indicators
In this talk, Roger Tsien discusses how fluorescent proteins have been turned into indicators for a wide variety of biological molecules, including pH, ions, redox potential, and signaling molecules like phosphoinositides. The talk also covers reporters used to measure the activity of enzymes like kinases, phosphatases, and proteases. It covers both single proteins whose intensity or wavelength change, as well as reporters using resonance energy transfer (FRET).Read article -
Highly Selective Fluorescent and Colorimetric Probe for Live-cell Monitoring of Sulphide Based on Bioorthogonal Reaction
H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H2S with improved sensitivity and selectivety.Read article -
Webinar: Advances in Neuroscience: New Methods for Correlating Structure and Function
During this webcast, we will present recent advances in targeted cell labelling, tissue clearing, and fluorescence imaging methods for the study of brain function. These exciting methods are helping to accelerate the understanding of how individual cells and complex neural circuits interact both structurally and functionally.Read article -
Introduction to Digital Camera Technology
A significant majority of modern optical microscopy techniques require the use of a digital camera. By working with digital devices researchers can observe specimens on a screen in real time or acquire and store images and quantifiable data. Here we introduce the basic principles behind digital camera technologies commonly encountered in scientific imaging.Read article -
FRAP with TCS SP8 Resonant Scanner
Fast FRAP experiments need a sufficient number of measurement points for meaningful interpretation and fitting analysis. To study very fast translocational processes, the use of a resonant scanner (RS) is preferred. The advantage in using FRAP with the RS is that statistics are much better in experiments that require fast acquisition: If the half time of recovery is about 0.5 sec you may have only about 3 to 4 data points using the conventional scanner, whereas with the resonant scanner you can get about 20 data points.Read article -
Webinar: Dissecting Protein Dynamics in Living Cells by FRAP
This webinar presented by Dr Marco Fritzsche, University of Oxford, and Jennifer Horner, PhD, Leica Microsystems, you will learn about how to use Fluorescence Recovery After Photo-bleaching (FRAP) microscopy to study protein dynamics.Read article -
Video Talk by Roger Tsien: Fluorescent Proteins
Live cell imaging has been revolutionized by the discovery of the green fluorescent protein (GFP). This lecture covers the history of GFP, how it folds and becomes fluorescent, how it has been mutated to produce additional colors (blue, cyan, yellow), and the discovery of red fluorescent proteins from corals. It also covers novel photoswitchable and photoactivatible fluorescent proteins, whose color can be changed by light, and new infrared fluorescent proteins.Read article -
How to Measure FRET
Here, I will expand, including what to measure when doing FRET. There are a number of approaches to FRET quantification: 1. Sensitized Emission – This two-channel imaging technique uses an algorithm that corrects for excitation and emission crosstalk.Read article -
You May Not Know Theodor Förster but You Know His Work: FRET
If you think FRET stands for Fluorescence Resonance Energy Transfer, you are wrong … in good company but wrong. FRET actually stands for Förster Resonance Energy Transfer. Find out why and more about FRET in this article.Read article -
How to Prepare Your Specimen for Immunofluorescence Microscopy
Immunofluorescence (IF) is a powerful method for visualizing intracellular processes, conditions and structures. IF preparations can be analyzed by various microscopy techniques (e.g. CLSM, Epifluorescence, TIRF, GSDIM), depending on the application or the researcher’s interest. Meanwhile, IF has become indispensable for a large number of research groups which have at least access to a simple fluorescence microscope.Read article -
Video Talk by Daniel Axelrod: Total Internal Reflection Fluorescence (TIRF) Microscopy
Total Internal Reflection Fluorescence (TIRF) Microscopy is a technique that only illuminates dye molecules near a surface. In this video, the pioneer of TIRF Microscopy describes what this technique is used for, explains the principles of the evanescent wave, gives many examples of different microscope configurations used in TIRF, and shows how polarized light TIRF can be used to image membrane orientation.Read article -
A Bright Dye for Live-Cell STED Microscopy
The aim of cell biology is to study smallest details on a cellular level preferably in a live cell experiment. By providing fast and direct super-resolution, STED (Stimulated Emission Depletion) microscopy is the perfect tool for studying cellular details in the nanometer range in vivo.Read article -
Nature Methods: Light-sheet Fluorescence Microscopy - Method of the Year 2014
Just about everyone who has examined fluorescent samples under the microscope is aware of the constant struggle to have enough signal to see the labeled structures while also avoiding fluorophore bleaching. What may be less apparent, at least to those who image bright, robust or fixed samples, is how stressful and potentially toxic to living cells and tissues it is to illuminate them with high-intensity light.Read article -
Influencing Factors and Applicability of the Viability EMA-qPCR for a Detection and Quantification of Campylobacter Cells from Water Samples
In recent years, increasing numbers of human campylobacteriosis cases caused by contaminated water have been reported. As the culture-based detection of Campylobacter is time consuming and can yield false-negative results, the suitability of a quantitative real-time PCR method in combination with an ethidium monoazide pretreatment of samples (EMA-qPCR) for the rapid, quantitative detection of viable Campylobacter cells from water samples was investigated.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 -
Super-Resolution Microscopy – Get Your Free e-Book for Download
Until recently, the diffraction of light had placed a fundamental limit on how far biologists could peer into cells with optical microscopes, preventing them from resolving features less than 250 nm in size, missing critical structures within cells. Over the past 20 years scientists have developed several ingenious techniques allowing them to resolve features as small as 20 nm.Read article -
Analysis of Autofluorescence in Polymorphonuclear Neutrophils: A New Tool for Early Infection Diagnosis
Diagnosing bacterial infection (BI) remains a challenge for the attending physician. An ex vivo infection model based on human fixed polymorphonuclear neutrophils (PMNs) gives an autofluorescence signal that differs significantly between stimulated and unstimulated cells. We took advantage of this property for use in an in vivo pneumonia mouse model and in patients hospitalized with bacterial pneumonia.Read article