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.     

Quantitative determination of the reduction of phototoxicity and photobleaching by controlled light exposure microscopy

Phototoxicity and photobleaching are major limitations in live-cell fluorescence microscopy. They are caused by fluorophores in an excited singlet or triplet state that generate singlet oxygen and other reactive oxygen species. The principle of controlled light exposure microscopy (CLEM) is based on non-uniform illumination of the field of view to reduce the number of excited fluorophore molecules. This approach reduces phototoxicity and photobleaching 2- to 10-fold without deteriorating image quality. Reduction of phototoxicity and photobleaching depends on the fluorophore distribution in the studied object, the optical properties of the microscope and settings of CLEM electronics. Here, we introduce the CLEM factor as a quantitative measure of reduction in phototoxicity and photobleaching. Finally, we give a guideline to optimize the effect of CLEM without compromising image quality.     

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.     

MRT letter: high speed scanning has the potential to increase fluorescence yield and to reduce photobleaching

True confocal microscopy requires point-shaped illumination and detection. To generate an image, a diffraction limited spot is moved over the sample. Single spot scanning has suffered in the past from low image rates; a solution is the employment of very fast scanning devices (resonant scanners) for x-movement. In the process of introducing resonant scanning devices, it was found that both signal yield is improved and bleaching is decreased-in contrary to the assumed performance. This article will show by a simple and well understood model a straightforward explanation for the potential increase of signal yield and decrease in photobleaching. The time that is ruling the dose-rate effects is the effective time; a fluorochrome is illuminated. This time depends on the diameter of the spot that is moved over the sample and the speed at which the spot moves. In essence, the scan process causes a pulsed illumination of the fluorochromes. Various schemes of pulsed illumination are simulated with a fluorescence model. The model includes a dark state, where fluorochromes will exit the fluorescence process and slowly decay back into the ground state. Upon splitting a single dose into two pulses separated by a dark time-reflecting an increased scan speed-the amount of fluorescence emission is increased and bleaching is reduced. These results show a potential increase of fluorescence and a lower photobleaching upon higher scan speed. As illumination during the bleach-phase in a FRAP-experiment is similar to a light pulse, the findings also suggest to critically consider the very beginning of fluorescence recovery in terms of triplet relaxation process that potentially could falsify the measurements.     

Method of calibration of a fluorescence microscope for quantitative studies

Confocal microscopy is based on measurement of intensity of fluorescence originating from a limited volume in the imaged specimen. The intensity is quantized in absolute (albeit arbitrary) units, producing a digital 3D micrograph. Thus, one may obtain quantitative information on local concentration of biomolecules in cells and tissues. This approach requires estimation of precision of light measurement (limited by noise) and conversion of the digital intensity units to absolute values of concentration (or number) of molecules of interest. To meet the first prerequisite we propose a technique for measurement of signal and noise. This method involves registration of a time series of images of any stationary microscope specimen. The analysis is a multistep process, which separates monotonic, periodic and random components of pixel intensity change. This approach permits simultaneous determination of dark and photonic components of noise. Consequently, confidence interval (total noise estimation) is obtained for every level of signal. The algorithm can also be applied to detect mechanical instability of a microscope and instability of illumination source. The presented technique is combined with a simple intensity standard to provide conversion of relative intensity units into their absolute counterparts (the second prerequisite of quantitative imaging). Moreover, photobleaching kinetics of the standard is used to estimate the power of light delivered to a microscope specimen. Thus, the proposed method provides in one step an absolute intensity calibration, estimate of precision and sensitivity of a microscope system.     

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.     

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.     

The effects of spherical aberration on multiphoton fluorescence excitation microscopy

Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descanned emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples.     

Evaluating the performance of fluorescence microscopes

A simple means of evaluating the performance of fluorescence microscopes is described. The proposed test gives an overall figure of merit that takes into account all of the important instrumental parameters that determine image quality. The essence of the test is to use a specimen whose photobleaching rate is a measure of the illumination time–intensity integral. When this time–intensity integral is controlled, the signal-to-noise ratio in the image of small subresolution objects becomes a system-independent measure of light throughput, resolution and intrinsic noise.     

A new algorithm to reduce noise in microscopy images implemented with a simple program in python

All microscopical images contain noise, increasing when (e.g., transmission electron microscope or light microscope) approaching the resolution limit. Many methods are available to reduce noise. One of the most commonly used is image averaging. We propose here to use the mode of pixel values. Simple Python programs process a given number of images, recorded consecutively from the same subject. The programs calculate the mode of the pixel values in a given position (a, b). The result is a new image containing in (a, b) the mode of the values. Therefore, the final pixel value corresponds to that read in at least two of the pixels in position (a, b). The application of the program on a set of images obtained by applying salt and pepper noise and GIMP hurl noise with 10-90% standard deviation showed that the mode performs better than averaging with three-eight images. The data suggest that the mode would be more efficient (in the sense of a lower number of recorded images to process to reduce noise below a given limit) for lower number of total noisy pixels and high standard deviation (as impulse noise and salt and pepper noise), while averaging would be more efficient when the number of varying pixels is high, and the standard deviation is low, as in many cases of Gaussian noise affected images. The two methods may be used serially.     

Artefacts in restored images due to intensity loss in three-dimensional fluorescence microscopy

Computational algorithms for three-dimensional deconvolution have proven successful in reducing blurring and improving the resolution of fluorescence microscopic images. However, discrepancies between the imaging conditions and the models on which such deconvolution algorithms are based may lead to artefacts and/or distortions in the images restored by application of the algorithms. In this paper, artefacts associated with a decrease of fluorescence intensity with time or slice in three-dimensional wide-field images are demonstrated using simulated images. Loss of intensity, whether due to photobleaching or other factors, leads to artefacts in the form of bands or stripes in the restored images. An empirical method for correcting the intensity losses in wide-field images has been implemented and used to correct biological images. This method is based on fitting a decreasing function to the slice intensity curve computed by summing all pixel values in each slice. The fitted curve is then used for the calculation of correction factors for each slice.     

Wavelength considerations in confocal microscopy of botanical specimens

We have used a multiple-laser confocal microscope with lines at 325, 442, 488, 514 and 633 nm to investigate optical sectioning of botanical specimens over a wide range of wavelengths. The 442-nm line allowed efficient excitation of Chromomycin A3, with minimal background autofluorescence, to visualize GC-rich heterochromatin as an aid to chromosome identification. Sequential excitation with 442- and 488-nm light enabled ratio imaging of cytosolic pH using BCECF. The red HeNe laser penetrated deep into intact plant tissues, being less prone to scattering than shorter blue lines, and was also used to image fluorescent samples in reflection, prior to fluorescence measurements, to reduce photobleaching. Chromatic corrections are more important in confocal microscope optics than in conventional microscopy. Measured focus differences between blue, green and red wavelengths, for commonly used objectives, were up to half the optical section thickness for both our multi-laser system and a multi-line single-laser instrument. This limited high-resolution sectioning at visible wavelengths caused a loss in signal. For ultraviolet excitation the focus shift was much larger and had to be corrected by pre-focusing the illumination. With this system we have imaged DAPI-stained nuclei, callose in pollen tubes using Aniline Blue and the calcium probe Indo-1.     

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.     

A statistical approach for intensity loss compensation of confocal microscopy images

In this paper, a probabilistic technique for compensation of intensity loss in confocal microscopy images is presented. For single-colour-labelled specimen, confocal microscopy images are modelled as a mixture of two Gaussian probability distribution functions, one representing the background and another corresponding to the foreground. Images are segmented into foreground and background by applying Expectation Maximization algorithm to the mixture. Final intensity compensation is carried out by scaling and shifting the original intensities with the help of parameters estimated for the foreground. Since foreground is separated to calculate the compensation parameters, the method is effective even when image structure changes from frame to frame. As intensity decay function is not used, complexity associated with estimation of the intensity decay function parameters is eliminated. In addition, images can be compensated out of order, as only information from the reference image is required for the compensation of any image. These properties make our method an ideal tool for intensity compensation of confocal microscopy images that suffer intensity loss due to absorption/scattering of light as well as photobleaching and the image can change structure from optical/temporal section-to-section due to changes in the depth of specimen or due to a live specimen. The proposed method was tested with a number of confocal microscopy image stacks and results are presented to demonstrate the effectiveness of the method.     

Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy

Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.     

基于模糊形态筛和四元数的水下彩色图像增强

水下彩色图像增强是水下视觉领域的一个重要问题.其中,照明引起的非均匀亮度往往严重影响了对水下图像增强的效果.首先运用彩色模糊形态筛改善非均匀照明情况,然后通过围绕水体色彩的四元数几何旋转,将水下背景像素赋予灰度或低饱和度色彩,而目标像素色彩保持不变,从而扩大前景目标与背景的差别.实验结果对比表明,新的彩色水下图像增强算法修正了水下图像的色彩不平衡,更适于处理水下图像的亮度不均匀情况.     

Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spread bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64°, both the dorsal and ventral membranes were illuminated, while images excited above 66° illuminated only focal contacts. Above 74° the fluorescence of focal contacts was dominated by background noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 ± 13 nm. Focal contact areas estimated by TIRFM (1·3 ± 0·7 μm²) agreed closely with areas observed by immunofluorescence staining of vinculin (1·5 ± 0·3 μm²).     

Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors

Multiphoton excitation was originally projected to improve live cell fluorescence imaging by minimizing photobleaching effects outside the focal plane, yet reports suggest that photobleaching within the focal plane is actually worse than with one photon excitation. We confirm that when imaging enhanced green fluorescent protein, photobleaching is indeed more acute within the multiphoton excitation volume, so that whilst fluorescence increases as predicted with the square of the excitation power, photobleaching rates increase with a higher order relationship. Crucially however, multiphoton excitation also affords unique opportunities for substantial improvements to fluorescence detection. By using a Pockels cell to minimize exposure of the specimen together with multiple nondescanned detectors we show quantitatively that for any particular bleach rate multiphoton excitation produces significantly more signal than one photon excitation confocal microscopy in high resolution Z‐axis sectioning of thin samples. Both modifications are readily implemented on a commercial multiphoton microscope system.     

Determination of green fluorescent protein by ultraviolet-excited gel imaging

The green fluorescent protein has gained interest in bioanalytical applications due to its visible fluorescence. As the usage of green fluorescent protein increases, more appropriate fluorescence instrumentation is required. Most fluorescence instrumentation uses ultraviolet light as the excitation source for the determination of green fluorescent protein. However, ultraviolet radiation may damage biological molecules and affect the quantitative analysis. In this study, the effects of the ultraviolet radiation period and the mass of green fluorescent protein on the fluorescence determination were characterized using gel imaging. The ultraviolet illumination period affected the green fluorescent protein fluorescence intensity. The fluorescence increased with the ultraviolet illumination time from 30–90 s. However, the fluorescence intensity decreased when the excitation period was longer, probably due to photobleaching. The photobleaching decreased when a higher concentration of enhanced green fluorescent protein was employed. This gel imaging study has provided a better understanding of the optimum conditions for the determination of green fluorescent protein.     

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.