Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation

We demonstrate the possibility to increase substantially the number of simultaneously detected fluorophores by utilizing both spectral and lifetime information. Using a two-detector confocal scanning laser microscope, experiments confirm that four different fluorophores can be detected with good channel separation. The signal-to-noise ratio (SNR) of the recorded images is investigated both theoretically and experimentally. It is found that in order to obtain a high SNR fluorophore lifetimes should differ by approximately an order of magnitude.     

Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique

We demonstrate the simultaneous recording of confocal lifetime images of multiple fluorophores. The confocal microscope used in the study combines intensity-modulated laser illumination, lock-in detection and spectral separation of the fluorescent light. A theoretical investigation is presented that describes how the signal-to-noise ratio (SNR) depends on various factors such as modulation frequency, degree of modulation and number of detected photons. Theory predicts that, compared with ordinary intensity images, lifetime images will have a SNR that is, at best, approximately four times lower. Experimental results are presented that confirm this prediction.     

A confocal laser microscope scanner for digital recording of optical serial sections

A confocal laser microscope scanner developed at our institute is described. Since an ordinary microscope is used, it is easy to view the specimen prior to scanning. Confocal imaging is obtained by laser spot illumination, and by focusing the reflected or fluorescent light from the specimen onto a pinhole aperture in front of the detector (a photomultiplier tube). Two rotating mirrors are used to scan the laser beam in a raster pattern. The scanner is controlled by a microprocessor which coordinates scanning, data display, and data transfer to a host computer equipped with an array processor. Digital images with up to 1024 × 1024 pixels and 256 grey levels can be recorded. The optical sectioning property of confocal scanning is used to record thin (～ 1 μm) sections of a specimen without the need for mechanical sectioning. By using computer-control to adjust the focus of the microscope, a stack of consecutive sections can be automatically recorded. A computer is then used to display the 3-D structure of the specimen. It is also possible to obtain quantitative information, both geometric and photometric. In addition to confocal laser scanning, it is easy to perform non-confocal laser scanning, or to use conventional microscopic illumination techniques for (non-confocal) scanning. The design has proved reliable and stable, requiring very few adjustments and realignments. Results obtained with this scanner are reported, and some limitations of the technique are discussed.     

Detection of doubly stained fluorescent specimens using confocal microscopy

Studies of doubly stained specimens were performed with a confocal scanning microscope. The instrument used provides the possibility of making separate detections of the fluorescent dyes. The optimal choice of excitation wavelengths and optical filters are discussed. The fluorphores that were used are Lucifer Yellow, Texas Red, fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate.     

Imaging enzyme activity with polarization-sensitive confocal fluorescence microscopy

We describe a technique for imaging enzyme activity through steady‐state fluorescence anisotropy measurements on a per‐pixel basis with a confocal microscope. With this method, enzyme activity is reported by changes in the fluorescence anisotropy of a fluorescently labelled substrate. Enzymatic cleavage of the substrate yields smaller labelled fragments that tumble more readily than the intact substrate and therefore yield a lower anisotropy. Anisotropy is recovered to an accuracy of 7% or better on and off the optical axis to depths of 210 µm using objective numerical apertures as high as 0.75. Enzyme imaging experiments were performed with Bodipy‐FL‐labelled bovine serum albumin (BSA) attached to sepharose beads as a substrate for trypsin and proteinase K. Anisotropy images acquired up to 1 h after enzyme addition revealed more rapid digestion of BSA with proteinase K than with trypsin, but in both cases anisotropy decreased by at least five‐fold. Fluorescence lifetime and time‐resolved anisotropy decay measurements were made on the construct in fluid solution to reveal the effects of enzyme activity. The Bodipy‐FL lifetime increased from 1.34 ns for the construct without enzyme to 5.98 ns after 1 h in the presence of proteinase K. Anisotropy decays yielded average rotational correlation times of 1.13 ns before enzymatic action and 0.27 ns after enzymatic action, consistent with the presence of smaller Bodipy‐containing protein fragments. These results suggest wide applicability of the technique in biological systems when used in conjunction with appropriately designed constructs.     

Reduction of cross-talk between fluorescent labels in scanning laser microscopy

By using dual detectors in combination with a dichroic filter, it is possible to record simultaneously the distribution of two fluorescent labels in a specimen. It is often difficult, however, to obtain a good separation, i.e. each detector will generally detect light from more than one fluorophore. In such cases it is desirable to find image-processing methods to improve the separation. A simple method is to form a linear combination of the recorded images. In this paper we investigate the necessary prerequisites for this method to be successful, and we also investigate to what extent these are fulfilled in some practical cases. In this context the spectral properties of the fluorophores turn out to be of crucial importance. Even when the necessary prerequisites are not strictly fulfilled, a considerable improvement in image quality can, nevertheless, be obtained.     

Scanning and detection techniques used in a confocal scanning laser microscope

A two-mirror scanning mechanism for confocal microscopy is described. No optical components, in addition to the scanning mirrors, are used. Design criteria and performance of the scanner are discussed. The photometric linearity of a detector unit incorporating a photomultiplier tube is reported, and a dual detector unit with tunable split wavelength is described.     

Two-colour two-photon confocal microscopy with isotropic three-dimensional resolution and parallel excitation

We demonstrate a novel design of two-colour two-photon fluorescence microscope in which isotropic three-dimensional imaging resolution and high scanning speed can be achieved simultaneously. In our scheme, a three-dimensional optical lattice constructed by multi-beam interference is used for two-colour two-photon fluorescence excitation. Our simulation results show that a resolution of 113.5 nm can be achieved in both transverse and axial directions with two pump pulses at the wavelengths of 400 and 800 nm, respectively; meanwhile, imaging speed can be greatly improved compared with that of traditional two-photon scanning fluorescence microscopes.     

Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation

In this paper, we experimentally demonstrated a two-channel frequency division multiplexing confocal fluorescence microscopy system using a UV laser as the excitation source. In our two-channel frequency division multiplexing confocal fluorescence system, the incoming laser beam was divided into two beams and each beam was modulated with an individual carrier frequency. These two laser beams were then spatially combined with a small angle and focused into two diffraction-limited spots on the targeted cell (rat neural cell) surface to generate fluorescent signal. As a result, the fluorescent signals from two spots of the rat neural cell surface can be demodulated and distinguished during data processing. Furthermore, a quantitative analysis on the cross-talk among different frequencies was provided as well. The experimental results confirm that the two-channel frequency division multiplexing confocal fluorescence technology can not only maintain the high spatial resolution, but also realize the multiple points detection simultaneously with high temporal resolution (within millisecond level range), which benefits the dynamic studies of living biological cells.     

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.     

Measurement and restoration of the point spread function of fluorescence confocal microscopy

The point spread function of an objective lens of a fluorescence confocal microscope was directly measured by imaging fluorescent beads. We analysed how the measurement of the point spread function was influenced by the diameter of the fluorescent beads and how the restoration technique with a deconvolution algorithm improved the measuring performance. Numerical and experimental results are presented for a typical point spread function and a zero‐centred point spread function.     

Measurement of distance with the nanoscale precise imaging by rapid beam oscillation method

We discuss here the principles of a novel optical method in which the scanning of a laser spot around a fluorescent object is used to determine its shape, orientation, and fluorophore distribution. The scanning pattern is adapted to the shape of the object according to a feedback principle based on intensity modulation measurements. The modulation of the intensity with respect to the angular coordinate is used to keep the orbit centered on the object. The modulation induced by rapid oscillations of the orbit radius is used to measure the local distance from the surface with nanometer precision. We provide a model to describe the fundamental relationship between modulation and distance and discuss the range of validity of several approximate expressions. According to this model, the distance can be measured with a precision dependent on the steepness of the point spread function and the total number of detected photons. To test our findings, we performed experiments with one or two channels on fluorescent spheres of known size and characterized the modulation function of our microscope setup. We conclude that the method can be used to measure distances in the range 10–200 nm between two surfaces labeled with two different probes. Microsc. Res. Tech. 75:1253–1264, 2012. © 2012 Wiley Periodicals, Inc.     

Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index

The effect of refractive-index mismatch, as encountered in the observation of biological specimens, on the image acquisition process in confocal fluorescence microscopy is investigated theoretically. The analysis takes the vectorial properties of light into account and is valid for high numerical apertures. Quantitative predictions on the decrease of resolution, intensity drop and shift of focus are given for practical situations. When observing with a numerical aperture of 1·3 (oil immersion) and an excitation wavelength of 514 nm the centre of the focus shifts 1·7 μm per 10 μm of axial displacement in an aqueous medium, thus yielding an image that is scaled by a factor of 1·2 in the axial direction. Furthermore, it can be expected that for a fluorescent plane 20 μm deep inside an aqueous medium the peak intensity is 40% less than for a plane which is 10 μm deep. In addition, the axial resolution is decreased by a factor of 1·4. The theory was experimentally verified for test samples with different refractive indices.     

The point-spread function of a confocal microscope: its measurement and use in deconvolution of 3-D data

We have measured the point-spread function (PSF) for an MRC-500 confocal scanning laser microscope using subresolution fluorescent beads. PSFs were measured for two lenses of high numerical aperture—the Zeiss plan-neofluar 63 × water immersion and Leitz plan-apo 63 × oil immersion—at three different sizes of the confocal detector aperture. The measured PSFs are fairly symmetrical, both radially and axially. In particular there is considerably less axial asymmetry than has been demonstrated in measurements of conventional (non-confocal) PSFs. Measurements of the peak width at half-maximum peak height for the minimum detector aperture gave approximately 0·23 and 0·8 μm for the radial and axial resolution respectively (4·6 and 15·9 in dimensionless optical units). This increased to 0·38 and 1·5 μm (7·5 and 29·8 in dimensionless units) for the largest detector aperture examined. The resulting optical transfer functions (OTFs) were used in an iterative, constrained deconvolution procedure to process three-dimensional confocal data sets from a biological specimen—pea root cells labelled in situ with a fluorescent probe to ribosomal genes. The deconvolution significantly improved the clarity and contrast of the data. Furthermore, the loss in resolution produced by increasing the size of the detector aperture could be restored by the deconvolution procedure. Therefore for many biological specimens which are only weakly fluorescent it may be preferable to open the detector aperture to increase the strength of the detected signal, and thus the signal-to-noise ratio, and then to restore the resolution by deconvolution.     

Robust approaches to quantitative ratiometric FRET imaging of CFP/YFP fluorophores under confocal microscopy

Ratiometric quantification of CFP/YFP FRET enables live-cell time-series detection of molecular interactions, without the need for acceptor photobleaching or specialized equipment for determining fluorescence lifetime. Although popular in widefield applications, its implementation on a confocal microscope, which would enable sub-cellular resolution, has met with limited success. Here, we characterize sources of optical variability (unique to the confocal context) that diminish the accuracy and reproducibility of ratiometric FRET determination and devise practical remedies. Remarkably, we find that the most popular configuration, which pairs an oil objective with a small pinhole aperture, results in intractable variability that could not be adequately corrected through any calibration procedure. By quantitatively comparing several imaging configurations and calibration procedures, we find that significant improvements can be achieved by combining a water objective and increased pinhole aperture with a uniform-dye calibration procedure. The combination of these methods permitted remarkably consistent quantification of sub-cellular FRET in live cells. Notably, this methodology can be readily implemented on a standard confocal instrument, and the dye calibration procedure yields a time savings over traditional live-cell calibration methods. In all, identification of key technical challenges and practical compensating solutions promise robust sub-cellular ratiometric FRET imaging under confocal microscopy.     

Microfractography of granitic rocks under confocal scanning laser microscopy

Scanning laser microscopy, in the confocal mode (CSLM) has been applied to a granitic rock to characterize its fissure space. The technique provides a unique three-dimensional picture of the rock microfractography. CSLM is unique in observing fine details of the fractographic network (connectivity, tortuosity, etc.), its geometry and its relation to other rock-forming components. The fractographic images with standard fluorescence microscopy are compared with those obtained with CSLM. The examples presented emphasize the advantages of CSLM: three-dimensional visualization of the microfractographic network, crack connectivity, automatic evaluation of direction and slope of fissures. These studies are related to the migration of radionuclides in the geosphere. The relations between potentially water-conducting open fissures, and the rock-forming minerals provide a means of modelling the ‘radionuclide retardation mechanism’, a security factor in their definitive storage in rock masses.     

Systematic evaluation of FRAP experiments performed in a confocal laser scanning microscope

The diffusion coefficient as well as the dimensionality of the diffusion process can be determined by straightforward and facile data analysis, when fluorescence recovery after photobleaching (FRAP) is measured as a function of time and space by means of confocal laser scanning microscopy. Experiments representing one-dimensional diffusion from a plane source or two-dimensional diffusion from a line source are readily realized. In the data analysis, the deviations of the actual initial conditions from ideal models are consistently taken into account, so that no calibration measurements are needed. The method is applied to FRAP experiments on solutions of Rhodamine B in glycerol and aqueous suspensions of polymethyl methacrylate microspheres.     

Conventional, confocal and two-photon fluorescence microscopy investigations of polymer-supported oxygen sensors

Luminescence‐based, polymer‐supported oxygen sensors, particularly those based on platinum group complexes, continue to be of analytical importance. Commercial applications range from the macroscopic (e.g. aerodynamic investigations in wind tunnels, monitoring of oxygen concentration during fermentation, and measurement of biological oxygen demand) to the microscopic (e.g. imaging of oxygen in blood, tissue, cells and other biological samples). Problems hindering the design of improved oxygen sensors include non‐linear Stern–Volmer calibration plots and the multi‐exponentiality of measured lifetime decays, both of which are attributed primarily to heterogeneity of the sensor molecule in the polymer support matrix. Conventional, confocal and two‐photon fluorescence microscopy have proven to be invaluable tools with which the microscale heterogeneity and response of luminescence‐based oxygen sensors can be investigated and compared to the macroscopic response. Results obtained for three ruthenium(II) α‐diimine complexes in polydimethylsiloxane polymer supports indicate the presence of unquenched microcrystals within the polymer matrix that probably degrade oxygen quenching sensitivity and linearity of the Stern–Volmer quenching plot. Two‐photon fluorescence microscopy proved most useful for imaging microcrystals within sensor films, and conventional microscopy allowed direct comparison between microscopic and macroscopic sensor response. The implications of the results in the rational design and mass production of luminescence‐based oxygen sensors are significant.     

Methods for large data volumes from confocal scanning laser microscopy of lung

Confocal scanning laser microscopy makes it possible to obtain series of optical sections in precise registration. Certain studies of lung parenchyma, however, require both the fine resolution obtainable with high-numerical-aperture (NA) objectives and the extensive fields of view that usually would be achieved only with low-NA objectives. This article presents a technique that resolves this conflict by using a sequence of operations: (i) to correct intensity variations on individual sections due to non-uniform illumination/detection characteristics of the microscope; (ii) to correct intensity variations between successive sections in a series due to, for example, depth-related absorption or step changes in detector sensitivity; (iii) to adjust adjacent, overlapping stacks of sections to a common intensity level; and (iv) to fuse a group of such overlapping stacks into a single series of larger sections. This resulting stack may contain, for example, a complete cross-section of an alveolar ductal unit about 500 μm or more in diameter at about 1-μm pixel resolution.