Widefield fluorescence microscopy with extended resolution

Widefield fluorescence microscopy is seeing dramatic improvements in resolution, reaching today 100 nm in all three dimensions. This gain in resolution is achieved by dispensing with uniform Köhler illumination. Instead, non-uniform excitation light patterns with sinusoidal intensity variations in one, two, or three dimensions are applied combined with powerful image reconstruction techniques. Taking advantage of non-linear fluorophore response to the excitation field, the resolution can be further improved down to several 10 nm. In this review article, we describe the image formation in the microscope and computational reconstruction of the high-resolution dataset when exciting the specimen with a harmonic light pattern conveniently generated by interfering laser beams forming standing waves. We will also discuss extensions to total internal reflection microscopy, non-linear microscopy, and three-dimensional imaging.     

Extended resolution fluorescence microscopy.

Fluorescence microscopy is an essential tool of modern biology, but, like all forms of optical imaging, it is subject to physical limits on its resolving power. In recent years, several exciting techniques have been introduced to exceed these limits, including standing wave microscopy, 4Pi confocal microscopy, I5M and structured illumination microscopy. Several such techniques have been definitively demonstrated for the first time during the past year.     

Rethinking resolution estimation in fluorescence microscopy: from theoretical resolution criteria to super-resolution microscopy

Science China Life Sciences - Resolution is undoubtedly the most important parameter in optical microscopy by providing an estimation on the maximum resolving power of a certain optical microscope....     

Concepts for nanoscale resolution in fluorescence microscopy

Spatio-temporal visualization of cellular structures by fluorescence microscopy has become indispensable in biology. However, the resolution of conventional fluorescence microscopy is limited by diffraction to about 180 nm in the focal plane and to about 500 nm along the optic axis. Recently, concepts have emerged that overcome the diffraction resolution barrier fundamentally. Formed on the basis of reversible saturable optical transitions, these concepts might eventually allow us to investigate hitherto inaccessible details within live cells.     

Ultra-high resolution imaging by fluorescence photoactivation localization microscopy

Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. To control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function, photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable molecules, the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive molecules are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small number of molecules are visible at a given time, their positions can be determined precisely; with only approximately 100 detected photons per molecule, the localization precision can be as much as 10-fold better than the resolution, depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are observed by FPALM of PA-GFP on glass. FPALM images are compared with images of the same molecules by widefield fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and show that the full width at half-maximum of features approximately 86 +/- 4 nm is significantly better than the expected diffraction-limited optical resolution. The number of fluorescent molecules and their brightness distribution have also been determined using FPALM. This new method suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.     

High-speed, miniaturized fluorescence microscopy in freely moving mice

A central goal in biomedicine is to explain organismic behavior in terms of causal cellular processes. However, concurrent observation of mammalian behavior and underlying cellular dynamics has been a longstanding challenge. We describe a miniaturized (1.1 g mass) epifluorescence microscope for cellular-level brain imaging in freely moving mice, and its application to imaging microcirculation and neuronal Ca(2+) dynamics.     

Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap.

Microscopy has become increasingly important for analysis of cells and cell function in recent years. This is due in large part to advances in light microscopy that facilitate quantitative studies and improve imaging of living cells. Analysis of fluorescence signals has often been a key feature in these advances. Such studies involve a number of techniques, including imaging of fluorescently labeled proteins in living cells, single-cell physiological experiments using fluorescent indicator probes, and immunofluorescence localization. The importance of fluorescence microscopy notwithstanding, there are instances in which electron microscopy provides unique information about cell structure and function. Correlative microscopy in which a fluorescence signal is reconciled with a signal from the electron microscope is an additional tool that can provide powerful information for cellular analysis. Here we review two different methodologies for correlative fluorescence and electron microscopy using ultrathin cryosections and the advantages attendant on this approach. (J Histochem Cytochem 49:803-808, 2001)     

Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.     

Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples

Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 x 30 x 75 nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.     

Archaeal DNA replication: spotlight on a rapidly moving field

The replication of DNA is a fundamental step in the cell cycle, which must be coordinated with cell division to ensure that the daughter cells inherit the same genomic material as the parental cell. The recently published complete genome sequences of some archaeal species together with preliminary biochemical studies suggest that the Archaea quite likely duplicate their chromosome by using replication machinery that seems to be a simplified version of the eukaryotic machinery, although their metabolic facets and their cellular morphology are prokaryotic-like. This review is focused on recent progress on the structural and functional analysis of proteins and enzymes involved in the initiation and elongation steps of DNA replication in Archaea. Differences between the genome replication apparatus of the Euryarchaea and the Crenarchaea (the two main phylogenetic divisions of the Archaea domain) are highlighted.     

IQPNNI: moving fast through tree space and stopping in time

An efficient tree reconstruction method (IQPNNI) is introduced to reconstruct a phylogenetic tree based on DNA or amino acid sequence data. Our approach combines various fast algorithms to generate a list of potential candidate trees. The key ingredient is the definition of so-called important quartets (IQs), which allow the computation of an intermediate tree in O(n(2)) time for n sequences. The resulting tree is then further optimized by applying the nearest neighbor interchange (NNI) operation. Subsequently a random fraction of the sequences is deleted from the best tree found so far. The deleted sequences are then re-inserted in the smaller tree using the important quartet puzzling (IQP) algorithm. These steps are repeated several times and the best tree, with respect to the likelihood criterion, is considered as the inferred phylogenetic tree. Moreover, we suggest a rule which indicates when to stop the search. Simulations show that IQPNNI gives a slightly better accuracy than other programs tested. Moreover, we applied the approach to 218 small subunit rRNA sequences and 500 rbcL sequences. We found trees with higher likelihood compared to the results by others. A program to reconstruct DNA or amino acid based phylogenetic trees is available online (http://www.bi.uni-duesseldorf.de/software/iqpnni).     

Kinesin walks the line: single motors observed by atomic force microscopy

Motor proteins of the kinesin family move actively along microtubules to transport cargo within cells. How exactly a single motor proceeds on the 13 narrow lanes or protofilaments of a microtubule has not been visualized directly, and there persists controversy on the relative position of the two kinesin heads in different nucleotide states. We have succeeded in imaging Kinesin-1 dimers immobilized on microtubules with single-head resolution by atomic force microscopy. Moreover, we could catch glimpses of single Kinesin-1 dimers in their motion along microtubules with nanometer resolution. We find in our experiments that frequently both heads of one dimer are microtubule-bound at submicromolar ATP concentrations. Furthermore, we could unambiguously resolve that both heads bind to the same protofilament, instead of straddling two, and remain on this track during processive movement.     

Major signal increase in fluorescence microscopy through dark-state relaxation

We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 micros, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5-25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 micros. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.     

Antibiotic transport in resistant bacteria: synchrotron UV fluorescence microscopy to determine antibiotic accumulation with single cell resolution

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack.     

Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy

The fact that Mycobacterium tuberculosis mobilizes lipid bodies (LB) located in the cytosol during infection process has been proposed for decades. However, the mechanisms and dynamics of mobilization of these lipid droplets within mycobacteria are still not completely characterized. Evidence in favour of this characterization was obtained here using a combined fluorescent microscopy and computational image processing approach. The decrease in lipid storage levels observed under nutrient depletion conditions was correlated with a significant increase in the size of the bacteria. LB fragmentation/condensation cycles were monitored in real time. The exact contribution of lipases in this process was confirmed using the lipase inhibitor tetrahydrolipstatin, which was found to prevent LB degradation and to limit the bacterial cell growth. The method presented here provides a powerful tool for monitoring in vivo lipolysis in mycobacteria and for obtaining new insights on the growth of cells and their entry into the dormant or reactivation phase. It should be particularly useful for studying the effects of chemical inhibitors and activators on cells as well as investigating other metabolic pathways.     

Beyond the diffraction limit: far-field fluorescence imaging with ultrahigh resolution

Fluorescence microscopy is an important and extensively utilised tool for imaging biological systems. However, the image resolution that can be obtained has a limit as defined through the laws of diffraction. Demand for improved resolution has stimulated research into developing methods to image beyond the diffraction limit based on far-field fluorescence microscopy techniques. Rapid progress is being made in this area of science with methods emerging that enable fluorescence imaging in the far-field to possess a resolution well beyond the diffraction limit. This review outlines developments in far-field fluorescence methods which enable ultrahigh resolution imaging and application of these techniques to biology. Future possible trends and directions in far-field fluorescence imaging with ultrahigh resolution are also outlined.