Ralf Wolleschensky

Rapid three-dimensional imaging and analysis of the beating embryonic heart reveals functional changes during development.

We report an accurate method for studying the functional dynamics of the beating embryonic zebrafish heart. The fast cardiac contraction rate and the high velocity of blood cells have made it difficult to study cellular and subcellular events relating to heart function in vivo. We have devised a dynamic three‐dimensional acquisition, reconstruction, and analysis procedure by combining (1) a newly developed confocal slit‐scanning microscope, (2) novel strategies for collecting and synchronizing cyclic image sequences to build volumes with high temporal and spatial resolution over the entire depth of the beating heart, and (3) data analysis and reduction protocols for the systematic extraction of quantitative information to describe phenotype and function. We have used this approach to characterize blood flow and heart efficiency by imaging fluorescent protein‐expressing blood and endocardial cells as the heart develops from a tube to a multichambered organ. The methods are sufficiently robust to image tissues within the heart at cellular resolution over a wide range of ages, even when motion patterns are only quasiperiodic. These tools are generalizable to imaging and analyzing other cyclically moving structures at microscopic scales. Developmental Dynamics 235:2940–2948, 2006.

High-speed confocal fluorescence imaging with a novel line scanning microscope.

Research in the life sciences increasingly involves the investigation of fast dynamic processes at the cellular and subcellular level. It requires tools to image complex systems with high temporal resolution in three-dimensional space. For this task, we introduce the concept of a fast fluorescence line scanner providing image acquisition speeds in excess of 100 frames per second at 512 x 512 pixels. Because the system preserves the capability for optical sectioning of confocal systems, it allows us to observe processes with three-dimensional resolution. We describe the principle of operation, the optical characteristics of the microscope, and cover several applications in particular from the field of cell and developmental biology. A commercial system based on the line scanning concept has been realized by Carl Zeiss (LSM 5 LIVE).

Biphoton focusing for two-photon excitation

We study two-photon excitation using biphotons generated via the process of spontaneous parametric down conversion in a nonlinear crystal. We show that the focusing of these biphotons yields an excitation distribution that is the same as the distribution of one-photon excitation at the pump wavelength. We also demonstrate that biphoton excitation in the image region yields a distribution whose axial width is approximately that of the crystal thickness and whose transverse width is that of the pump at the input to the crystal.

Sensitive imaging of spectrally overlapping flourochromes using the LSM 510 META

Multi-color fluorescence microscopy has become a popular way to discriminate between multiple proteins, organelles or functions in a single cell or animal and can be used to approximate the physical relationships between individual proteins within the cell, for instance, by using Fluorescence Resonance Energy Transfer (FRET). However, as researchers attempt to gain more information from single samples by using multiple dyes or fluorescent proteins (FPs), spectral overlap between emission signals can obscure the data. Signal separation using glass filters is often impractical for many dye combinations. In cases where there is extensive overlap between fluorochromes, separation is often physically impossible or can only be achieved by sacrificing signal intensity. Here we test the performance of a new, integrated laser scanning system for multispectral imaging, the Zeiss LSM 510 META. This system consists of a sensitive multispectral imager and online linear unmixing functions integrated into the system software. Below we describe the design of the META device and show results from tests of the linear unmixing experiments using fluorochromes with overlapping emission spectra. These studies show that it is possible to expand the number of dyes used in multicolor applications.

High-speed scanning confocal microscope for the life sciences

Research in the Life Sciences increasingly involves the investigation of fast dynamic processes at the cellular and sub-cellular level. It requires tools to image complex systems with high temporal resolution in three-dimensional space. For this task we introduce a fast fluorescence line scanner with image acquisition speeds in excess of 100 frames per second at 512x512 pixels and with a more than 10- fold increased sensitivity compared to point scanning confocal systems. Since the system preserves the capability for optical sectioning of confocal systems it allows to observe processes in three dimensions. We describe the principle of operation, the optical characteristics of the microscope and cover several applications in particular from the field of developmental biology.

Imaging of Vitrified Biological Specimens by Confocal Cryo-Fluorescence Microscopy and Cryo-FIB/SEM Tomography

The investigation of vitrified biological specimens (i.e. samples that are plunge or high-pressure-frozen) enables the visualization of cellular ultrastructure in a near-native fully hydrated state, unadulterated by harmful preparation methods. Here, we focus on two recent cryo-imaging modalities and discuss their impact on cryo-correlative workflows. First, we present confocal cryo-fluorescence microscopy, utilizing a novel confocal detector scheme with improved signal-to-noise ratio (SNR) and resolution. Second, we show volume imaging of multicellular specimens by cryo-focused ion beam scanning electron microscopy (FIB/SEM) .

SIM and PALM: high-resolution microscopy methods and their consequences for cell biology

The diffraction limit in traditional fluorescence microscopy (approximately 200 and 600 nanometers in lateral and axial directions, respectively) has restricted the applications in bio-medical research. However, over the last 10 years various techniques have emerged to overcome this limit. Each of these techniques has its own characteristics that influence its application in biology. This paper will show how two of the techniques, Structured Illumination Microscopy (SIM) and PhotoActivated Localization Microscopy (PALM), complement each other in imaging of biological samples beyond the resolution of classical widefield fluorescence microscopy. As a reference the properties of two well known standard imaging techniques in this field, confocal Laser Scanning Microscopy (LSM) and Total Internal Reflection (TIRF) microscopy, are compared to the properties of the two high resolution techniques. Combined SIM/PALM imaging allows the extremely accurate localization of individual molecules within the context of various fluorescent structures already resolved in 3D with a resolution of up to 100nm using SIM. Such a combined system provides the biologist with an unprecedented view of the sub-cellular organization of life.

Need for Standardization of Fluorescence Measurements from the Instrument Manufacturer's View

Characterization of fluorescence imaging systems from the manufacturers view creates several challenges. What are the key parameters for which characterization is appropriate? How can the standardization procedures developed for use during manufacture be applied during installation and application? With so many instrument variables, how can procedures be developed that give precise diagnostic information? These are not simply questions of “standardized tests”. There are also issues of finding shared confidence in the tests amongst the different users of the systems. Ideally such tests should also allow objective comparison of the performance of systems of different design or from different manufacturers.