Noise effects and filtering in controlled light exposure microscopy

Phototoxicity and photobleaching are major limitations of fluorescence live-cell microscopy. A straightforward way to limit phototoxicity and photobleaching is reduction of the excitation light dose, but this causes loss of image quality. In confocal fluorescence microscopy, the field of view is illuminated uniformly whereas in controlled light exposure microscopy, illumination is controlled per pixel on the basis of two illumination strategies. The controlled light exposure microscopy foreground strategy discriminates between bright and weak foreground. Bright foreground pixels are illuminated with a reduced light dose resulting in limited excitation of fluorophores and consequently limited phototoxicity and photobleaching. The controlled light exposure microscopy background strategy discriminates between foreground and background. Pixels that are judged to be background are also illuminated with a reduced light dose. The latter illumination strategy may introduce artefacts due to the stochastic character of photon flow. These artefacts are visible as erratic 'darker pixels' in the foreground with a lower pixel value than the neighbouring pixels. This paper describes a special adaptive image processing filter that detects and corrects most of the 'darker pixels'. It opens the possibility to use controlled light exposure microscopy even in high noise (low signal to noise ratio) imaging to further reduce phototoxicity and photobleaching.     

A novel approach for correlative light electron microscopy analysis

Correlative light and electron microscopy (CLEM) is a multimodal technique of increasing utilization in functional, biochemical, and molecular biology. CLEM attempts to combine multidimensional information from the complementary fluorescence light microscopy (FLM) and electron microscopy (EM) techniques to bridge the various resolution gaps. Within this approach the very same cell/structure/event observed at level can be analyzed as well by FLM and EM. Unfortunately, these studies turned out to be extremely time consuming and are not suitable for statistical relevant data. Here, we describe a new CLEM method based on a robust specimen preparation protocol, optimized for cryosections (Tokuyasu method) and on an innovative image processing toolbox for a novel type of multimodal analysis. Main advantages obtained using the proposed CLEM method are: (1) hundred times more cells/structures/events that can be correlated in each single microscopy session; (2) three‐dimensional correlation between FLM and EM, obtained by means of ribbons of serial cryosections and electron tomography microscopy (ETM); (3) high rate of success for each CLEM experiment, obtained implementing protection of samples from physical damage and from loss of fluorescence; (4) compatibility with the classical immunogold and immunofluorescence labeling techniques. This method has been successfully validated for the correlative analysis of Russel Bodies subcellular compartments. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Minimizing light exposure with the programmable array microscope

The exposure of fluorophores to intense illumination in a microscope often results in photobleaching and phototoxicity, thus constituting a major limiting factor in time lapse live cell or single molecule imaging. Laser scanning confocal microscopes are particularly prone to this problem, inasmuch as they require high irradiances to compensate for the inherently low duty cycle of point scanning systems. In the attempt to maintain adequate speed and signal-to-noise ratios, the fluorophores are often driven into saturation, thereby generating a nonlinear response. One approach for reducing photodegradation in the laser scanning confocal microscope is represented by controlled light exposure microscopy, introduced by Manders and colleagues. The strategy is to reduce the illumination intensity in both background areas (devoid of information) as well as in bright foreground regions, for which an adequate signal-to-noise ratio can be achieved with lower excitation levels than those required for the less intense foreground pixels/voxels. Such a variable illumination scheme can also be exploited in widefield microscopes that employ lower irradiance but higher illumination duty cycles. We report here on the adaptation of the controlled light exposure microscopy principle to the programmable array microscope, which achieves optical sectioning by use of a spatial light modulator (SLM) in an image plane as a programmable mask for illumination and conjugate (and nonconjugate) detection. By incorporating the basic controlled light exposure microscopy concept for minimizing exposure, we have obtained a reduction in the rate of photobleaching of up to ~5-fold, while maintaining an image quality comparable to regular imaging with the programmable array microscope.     

Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method

Multiconfocal microscopy gives a good compromise between fast imaging and reasonable resolution. However, the low intensity of live fluorescent emitters is a major limitation to this technique. Aberrations induced by the optical setup, especially the mismatch of the refractive index and the biological sample itself, distort the point spread function and further reduce the amount of detected photons. Altogether, this leads to impaired image quality, preventing accurate analysis of molecular processes in biological samples and imaging deep in the sample. The amount of detected fluorescence can be improved with adaptive optics. Here, we used a compact adaptive optics module (adaptive optics box for sectioning optical microscopy), which was specifically designed for spinning disk confocal microscopy. The module overcomes undesired anomalies by correcting for most of the aberrations in confocal imaging. Existing aberration detection methods require prior illumination, which bleaches the sample. To avoid multiple exposures of the sample, we established an experimental model describing the depth dependence of major aberrations. This model allows us to correct for those aberrations when performing a z‐stack, gradually increasing the amplitude of the correction with depth. It does not require illumination of the sample for aberration detection, thus minimizing photobleaching and phototoxicity. With this model, we improved both signal‐to‐background ratio and image contrast. Here, we present comparative studies on a variety of biological samples.     

Predictive-focus illumination for reducing photodamage in live-cell microscopy

Due to photobleaching and phototoxicity induced by high-intensity excitation light, the number of fluorescence images that can be obtained in live cells is always limited. This limitation becomes particularly prominent in multidimensional recordings when multiple Z-planes are captured at every time point. Here we present a simple technique, termed predictive-focus illumination (PFI), which helps to minimize cells' exposure to light by decreasing the number of Z-planes that need to be captured in live-cell 3D time-lapse recordings. PFI utilizes computer tracking to predict positions of objects of interest (OOIs) and restricts image acquisition to small dynamic Z-regions centred on each OOI. Importantly, PFI does not require hardware modifications and it can be easily implemented on standard wide-field and spinning-disc confocal microscopes.     

Mapping the distribution of emissive molecules in human ocular lipofuscin granules with near-field scanning optical microscopy

Several high resolution imaging techniques are utilized to probe the structure of human ocular lipofuscin granules. Atomic force microscopy reveals typical granule sizes to be about one micrometre in diameter and hundreds of nanometres in height, in agreement with previous electron microscopy results. For issues concerning the role of lipofuscin in age-related macular degeneration, recent attention has focused on the orange-emitting fluorophore, A2E. Confocal microscopy measurements are presented which reveal the presence of a highly emissive component in the granules, consistent with the presence of A2E. It is shown, however, that the interpretation of these results is complicated by the lack of structural details about the particles. To address these issues, near-field scanning optical microscopy (NSOM) measurements are presented which measure both the lipofuscin fluorescence and topography, simultaneously. These measurements reveal distinct structure in the fluorescence image which do not necessarily correlate with the topography of the granules. Moreover, direct comparison between the NSOM fluorescence and topography measurements suggests that A2E is not the major component in lipofuscin. These measurements illustrate the unique capabilities of NSOM for probing into the microstructure of lipofuscin and uncovering new insights into its phototoxicity.     

Microscopic oxygen imaging based on fluorescein bleaching efficiency measurements

Photobleaching of the fluorophore fluorescein in an aqueous solution is dependent on the oxygen concentration. Therefore, the time‐dependent bleaching behavior can be used to measure of dissolved oxygen concentrations. The method can be combined with epi‐fluorescence microscopy. The molecular states of the fluorophore can be expressed by a three‐state energy model. This leads to a set of differential equations which describe the photobleaching behavior of fluorescein. The numerical solution of these equations shows that in a conventional wide‐field fluorescence microscope, the fluorescence of fluorescein will fade out faster at low than at high oxygen concentration. Further simulation showed that a simple ratio function of different time‐points during a fluorescence decay recorded during photobleaching could be used to describe oxygen concentrations in an aqueous solution. By careful choice of dye concentration and excitation light intensity the sensitivity in the oxygen concentration range of interest can be optimized. In the simulations, the estimation of oxygen concentration by the ratio function was very little affected by the pH value in the range of pH 6.5–8.5. Filming the fluorescence decay by a charge‐coupled‐device (ccd) camera mounted on a fluorescence microscope allowed a pixelwise estimation of the ratio function in a microscopic image. Use of a microsensor and oxygen‐consuming bacteria in a sample chamber enabled the calibration of the system for quantification of absolute oxygen concentrations. The method was demonstrated on nitrifying biofilms growing on snail and mussel shells, showing clear effects of metabolic activity on oxygen concentrations. Microsc. Res. Tech. 77:341–347, 2014. © 2014 Wiley Periodicals, Inc.     

Correlative light and electron microscopy for the analysis of cell division

Correlative light and electron microscopy (CLEM) has recently gained increasing attention, because it enables the acquisition of dynamic as well as ultrastructural information about subcellular processes. It is the power of combining the two imaging modalities that gives additional information as compared to using the imaging techniques separately. Here, we briefly summarize two CLEM approaches for the analysis of cells in mitosis and cytokinesis.     

Rapid single‐molecule imaging in cyclic olefin copolymer channels

Rapid preparation of high quality capture surfaces is a major challenge for surface‐based single‐molecule protein binding assays. Here we introduce a simple method to activate microfluidic chambers made from cyclic olefin copolymer for single‐molecule imaging with total internal reflection fluorescence microscopy. We describe a surface coating protocol and demonstrate single‐molecule imaging in off‐the‐shelf microfluidic parts that can be activated for binding assays within a few minutes. As the first example, biotinylated protein directly captured on the neutravidin‐coated surface was detected using fluorescently labeled antibody. We then showed detection of a fusion construct containing green fluorescence protein and verified its single fluorophore behavior by observing stepwise photobleaching events. Finally, a target protein was identified in the crude cell lysate using antibody–sandwich complex formation. In all experiments, controls were completed to ensure that nonspecific binding to the surface was minimal. Based on our results, we conclude that the simple surface preparation described in this paper enables single‐molecule imaging assays without time‐consuming coating procedures. Microsc. Res. Tech. 78:309–316, 2015. © 2015 Wiley Periodicals, Inc.     

Multicolour analysis and local image correlation in confocal microscopy

Multiparameter fluorescence microscopy is often used to identify cell types and subcellular organelles according to their differential labelling. For thick objects, the quantitative comparison of different multiply labelled specimens requires the three-dimensional (3-D) sampling capacity of confocal laser scanning microscopy, which can be used to generate pseudocolour images. To analyse such 3-D data sets, we have created pixel fluorogram representations, which are estimates of the joint probability densities linking multiple fluorescence distributions. Such pixel fluorograms also provide a powerful means of analysing image acquisition noise, fluorescence cross-talk, fluorescence photobleaching and cell movements. To identify true fluorescence co-localization, we have developed a novel approach based on local image correlation maps. These maps discriminate the coincident fluorescence distributions from the superimposition of noncorrelated fluorescence profiles on a local basis, by correcting for contrast and local variations in background intensity in each fluorescence channel. We believe that the pixel fluorograms are best suited to the quality control of multifluorescence image acquisition. The local image correlation methods are more appropriate for identifying co-localized structures at the cellular or subcellular level. The thresholding of these correlation maps can further be used to recognize and classify biological structures according to multifluorescence attributes.     

Real-time computation of subdiffraction-resolution fluorescence images

In the recent past, single-molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction-resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high-precision fluorophore localization and subsequent visualization of found fluorophore positions in a super-resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non-maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real-time data processing is possible and that super-resolution imaging with organic fluorophores of cellular structures with ∼20 nm optical resolution can be completed in less than 10 s.     

Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1

Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.     

Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells

FLAP is a new method for localized photo‐labelling and subsequent tracking of specific molecules within living cells. It is simple in principle, easy to implement and has a wide potential application. The molecule to be located carries two fluorophores: one to be photobleached and the other to act as a reference label. Unlike the related methods of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), the use of a reference fluorophore permits the distribution of the photo‐labelled molecules themselves to be tracked by simple image differencing. In effect, FLAP is therefore comparable with methods of photoactivation. Its chief advantage over the method of caged fluorescent probes is that it can be used to track chimaeric fluorescent proteins directly expressed by the cells. Although methods are being developed to track fluorescent proteins by direct photoactivation, these still have serious drawbacks. In order to demonstrate FLAP, we have used nuclear microinjection of cDNA fusion constructs of β‐actin with yellow (YFP) and cyan (CFP) fluorescent proteins to follow both the fast relocation dynamics of monomeric (globular) G‐actin and the much slower dynamics of filamentous F‐actin simultaneously in living cells.     

Ultrafast photon counting applied to resonant scanning STED microscopy

To take full advantage of fast resonant scanning in super‐resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue‐to‐digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (～50 × 50 μm) with fast scanning that reduces photobleaching, and advances the time‐gated continuous wave STED technology to the usage of resonant scanning with hardware‐based time‐gating. The assembled system provides superb signal‐to‐noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant‐scanning continuous wave STED microscopy with online time‐gated detection.     

Analysis of heterogeneous fluorescence photobleaching by video kinetics imaging: The method of cumulants

The method of cumulants has been applied to digital video fluorescence microscopy. The method is used to reconstruct the distribution of fluorescent molecules before the initiation of fluorescence photobleaching, and to characterize heterogeneous photobleaching by imaging one or more of the cumulants of the bleaching decay rate. Using the pipelined pixel processor of the image analysis system for the bulk of the calculations, rather than the general-purpose host-computer CPU, the video kinetics imaging can be performed in near real-time. The method is applied to chick embryo myotubes labelled with fluorescein-conjugated α-bungarotoxin. The pre-bleach fluorescence distribution is derived, and the image of fluorescein fluorescence is separated from glutaraldehyde-induced autofluorescence on the basis of the spatially resolved average photobleaching decay rate.     

Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new,efficient upconverting fluorophore

Three-dimensional confocal imaging of polymer samples was achieved by the use of two-photon excited fluorescence in both positive and negative contrast modes. The fluorophore was a new and highly efficient two-photon induced upconverter, resulting in improved signal strength at low pumping power. Because of the relatively long wavelength of the excitation source (798 nm from a mode-locked Ti:Sap-phire laser), this technique shows a larger penetration depth into the samples than provided by conventional single-photon fluorescence confocal microscopy. Single-photon and two-photon images of the same area of each sample show significant differences. The results suggest the possibility of using two-photon confocal microscopy, in conjunction with highly efficient fluorophores, as a tool to study the surface, interface, and fracture in material science applications.     

Fibre‐optical microendoscopy

Microendoscopy has been an essential tool in exploring micro/nano mechanisms in vivo due to high‐quality imaging performance, compact size and flexible movement. The investigations into optical fibres, micro‐scanners and miniature lens have boosted efficiencies of remote light delivery to sample site and signal collection. Given the light interaction with materials in the fluorescence imaging regime, this paper reviews two classes of compact microendoscopy based on a single fibre: linear optical microendoscopy and nonlinear optical microendoscopy. Due to the fact that fluorescence occurs only in the focal volume, nonlinear optical microendoscopy can provide stronger optical sectioning ability than linear optical microendoscopy, and is a good candidate for deep tissue imaging. Moreover, one‐photon excited fluorescence microendoscopy as the linear optical microendoscopy suffers from severe photobleaching owing to the linear dependence of photobleaching rate on excitation laser power. On the contrary, nonlinear optical microendoscopy, including two‐photon excited fluorescence microendoscopy and second harmonic generation microendoscopy, has the capability to minimize or avoid the photobleaching effect at a high excitation power and generate high image contrast. The combination of various nonlinear signals gained by the nonlinear optical microendoscopy provides a comprehensive insight into biophenomena in internal organs. Fibre‐optical microendoscopy overcomes physical limitations of traditional microscopy and opens up a new path to achieve early cancer diagnosis and microsurgery in a minimally invasive and localized manner.     

Fluorescence resonance energy transfer and anisotropy reveals both hetero- and homo-energy transfer in the pleckstrin homology-domain and the parathyroid hormone-receptor

We present a method and an apparatus of polarized fluorescence resonance energy transfer (FRET) and anisotropy imaging microscopy done in parallel for improved interpretation of the photophysical interactions. We demonstrate this apparatus to better determine the protein-protein interactions in the pleckstrin homology domain and the conformational changes in the Parathyroid Hormone Receptor, a G-protein coupled receptor, both fused to the cyan and yellow fluorescent proteins for either inter- or intramolecular FRET. In both cases, the expression levels of proteins and also background autofluorescence played a significant role in the depolarization values measured in association with FRET. The system has the sensitivity and low-noise capability of single-fluorophore detection. Using counting procedures from single-molecule methods, control experiments were performed to determine number densities of green fluorescence protein variants CFP and YFP where homo resonance energy transfer can occur. Depolarization values were also determined for flavins, a common molecule of cellular background autofluorescence. From the anisotropy measurements of donor and acceptor, the latter when directly excited or when excited by energy transfer, we find that our instrumentation and method also characterizes crucial effects from homotransfer, polarization specific photobleaching and background molecules.     

Optimizing imaging parameters for the separation of multiple labels in a fluorescence image

A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image. Equations are derived that allow the calculation of the signal‐to‐noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels. We then ask how such imaging parameters have to be chosen for optimal fluorophore separation. We optimize the signal‐to‐noise ratio or optimize a newly defined ‘figure of merit’, which is a measure of efficiency in the use of emitted photons. The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information. A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected. The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching. Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.