&lt;title&gt;Deconvolution of fluorescence decays and estimation errors&lt;/title&gt;

Narang

Spiecker

et al. 2008

Journal of Biophysics

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670±140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.

Section: Methodsmentioning

confidence: 99%

“…The advantages of the noniterative analysis against the gradient method are its computational speed and the necessity for less data points on the fluorescence decay curve, that is, speedup of data acquisition [ 20 ]. Its major limitation is the instability to signal noise [ 40 ].…”

Section: Methodsmentioning

confidence: 99%

Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H-Based Fluorescence Lifetime Imaging

Narang

Spiecker

et al. 2008

Journal of Biophysics

“…A very stabile and, thus, accurate and fast alternative to the transforms in complex space is based on a non‐iterative linearization technique in real space (time space), initially used to discriminate between bi‐ and tri‐exponential decays in a small time‐interval (94). The stability and speed of this technique and of its advancements has been discussed extensively elsewhere (130).…”

Section: Principle Of Flimmentioning

confidence: 99%

Intravital two‐photon microscopy: focus on speed and time resolved imaging modalities

Andresen

Gunzer

2008

Immunological Reviews

Initially used mainly in the neurosciences, two-photon microscopy has become a powerful tool for the analysis of immunological processes. Here, we describe currently available two-photon microscopy techniques with a focus on novel approaches that allow very high image acquisition rates compared with state-of-the-art systems. This improvement is achieved through a parallelization of the excitation process: multiple beams scan the sample simultaneously, and the fluorescence is collected with sensitive charge-coupled device (CCD)-based line or field detectors. The new technique's performance is compared with conventional single beam laser-scanning systems that detect signals by means of photomultipliers. We also discuss the use of time- and polarization-resolved fluorescence detection, especially fluorescence lifetime imaging (FLIM), which goes beyond simple detection of cells and tissue structures and allows insight into cellular physiology. We focus on the analysis of endogenous fluorophores such as NAD(P)H as a way to analyze the redox status in cells with subcellular resolution. Here, high-speed imaging setups in combination with novel ways of data analysis allow the generation of FLIM data sets almost in real time. The implications of this technology for the analysis of immune reactions and other cellular processes are discussed.

“…The stability and speed of this technique and of its advancements has extensively been discussed elsewhere 50,54 .…”

Section: Flim Data Evaluationmentioning

confidence: 99%

Fluorescence lifetime imaging in biosciences: technologies and applications

Gericke

2008

Front. Phys. China

The biosciences require the development of methods, which allow a non-invasive and rapid investigation of biological systems. In this frame high-end imaging techniques allow an intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e. wide-field, confocal one-photon and two-photon microscopy, respectively, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e. steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at molecular level. The intrinsic disadvantages of steady-state techniques are counteracted by using time-resolved techniques. Among these the fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.