A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images

A programmable array microscope (PAM) incorporates a spatial light modulator (SLM) placed in the primary image plane of a widefield microscope, where it is used to define patterns of illumination and/or detection. We describe the characteristics of a special type of PAM collecting two images simultaneously. The conjugate image (I_c) is formed by light originating from the object plane and returning along the optical path of the illumination light. The non‐conjugate image (I_nc) receives light from only those regions of the SLM that are not used for illuminating the sample. The dual‐signal PAM provides much more time‐efficient excitation than the confocal laser scanning microscope (CLSM) and greater utilization of the available emission light. It has superior noise characteristics in comparison to single‐sided instruments. The axial responses of the system under a variety of conditions were measured and the behaviour of the novel I_nc image characterized. As in systems in which only I_c images are collected (Nipkow‐disc microscopes, and previously characterized PAMs), the axial response to thin fluorescent films showed a sharpening of the axial response as the unit cell of the repetitive patterns decreased in size. The dual‐signal PAM can be adapted to a wide range of data analysis and collection strategies. We investigated systematically the effects of patterns and unit cell dimensions on the axial response. Sufficiently sparse patterns lead to an I_c image formed by the superposition of the many parallel beams, each of which is equivalent to the single scanning spot of a CLSM. The sectioning capabilities of the system, as given by its axial responses, were similar for a given scan pattern and for processed pseudorandom sequence (PRS) scans with the same size of the unit cell. For the PRS scans, optical sectioning was achieved by a subtraction of an I_nc image or, alternatively, a scaled widefield image from the I_c image. Based on the comparative noise levels of the two methods, the non‐conjugate subtraction was significantly superior. A point spread function for I_c and I_nc was simulated and properties of the optical transfer functions (OTFs) were compared. Simulations of the OTF in non‐conjugate imaging did not suffer from the missing cone problem, enabling a high quality deconvolution of the non‐conjugate side alone. We also investigated the properties of images obtained by subjecting the I_c and I_nc data to a combined maximum likelihood deconvolution.     

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.     

Theory of confocal fluorescence imaging in the programmable array microscope (PAM)

The programmable array microscope (PAM) uses a spatial light modulator (SLM) to generate an arbitrary pattern of conjugate illumination and detection elements. The SLM dissects the fluorescent light imaged by the objective into a focal conjugate image, I_c, formed by the ‘in-focus’ light, and a nonconjugate image, I_nc, formed by the ‘out-of-focus’ light. We discuss two different schemes for confocal imaging using the PAM. In the first, a grid of points is shifted to scan the complete image. The second, faster approach, uses a short tiled pseudorandom sequence of two-dimensional patterns. In the first case, I_c is analogous to a confocal image and I_nc to a conventional image minus I_c. In the second case I_c and I_nc are the sum and the difference, respectively, of a conventional and a confocal image. The pseudorandom sequence approach requires post-processing to retrieve the confocal part, but generates significantly higher signal levels for an equivalent integration time.     

Fibre-optic two-photon scanning fluorescence microscopy

Two geometries of a novel two‐photon fluorescence microscope incorporating single‐mode fibre optics for the delivery of ultrashort‐pulsed illumination to a remote sample are characterized. First, a 785 nm single‐mode optical fibre is implemented in a scanning microscope, which demonstrates that an improvement in axial resolution is achieved due to the non‐linear response of the fibre to intense ultrashort‐pulsed light. Second, a 785 nm single‐mode optical fibre coupler is adapted, in which case spectral broadening and blue shifting of the ultrashort‐pulsed laser beam caused by the non‐linear response of the fibre to ultrashort‐pulsed illumination are experimentally characterized. An investigation into the impact of temporal broadening of the ultrashort‐pulsed beam on the systems is also considered. The coupling efficiency of both geometries for various illumination wavelengths is also presented. The introduction of the fibre coupler to the system has significant advantages, including an improved optical sectioning effect and a reduction in the number of bulk optical components resulting in a low‐cost, compact instrument. Sets of three‐dimensional images of fluorescent polymer microspheres and biological material confirm these features.     

Fluorescence recovery after photobleaching and photoconversion in multiple arbitrary regions of interest using a programmable array microscope

Photomanipulation (photobleaching, photoactivation, or photoconversion) is an essential tool in fluorescence microscopy. Fluorescence recovery after photobleaching (FRAP) is commonly used for the determination of lateral diffusion constants of membrane proteins, and can be conveniently implemented in confocal laser scanning microscopy (CLSM). Such determinations provide important information on molecular dynamics in live cells. However, the CLSM platform is inherently limited for FRAP because of its inflexible raster (spot) scanning format. We have implemented FRAP and photoactivation protocols using structured illumination and detection in a programmable array microscope (PAM). The patterns are arbitrary in number and shape, dynamic and adjustable to and by the sample characteristics. We have used multispot PAM–FRAP to measure the lateral diffusion of the erbB3 (HER3) receptor tyrosine kinase labeled by fusion with mCitrine on untreated cells and after treatment with reagents that perturb the cytoskeleton or plasma membrane or activate coexpressed erbB1 (HER1, the EGF receptor EGFR). We also show the versatility of the PAM for photoactivation in arbitrary regions of interest, in cells expressing erbB3 fused with the photoconvertible fluorescent protein dronpa. dronpa. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Fluorescence saturation in confocal microscopy

The effects of fluorescence saturation on imaging in confocal microscopy have been studied. To include saturation it was necessary to deviate from the widely assumed linear relationship between the fluorescence and the illumination intensity. The lateral response for a point-like object, as well as the optical sectioning power, decreases depending on the degree of saturation. For very high illumination intensities the response for a saturated point object approached that of a conventional fluorescence microscope in which the fluorescence was not saturated. The decrease in the axial confocal response has been confirmed qualitatively by experiment.     

Optical sectioning microscope with a binary hologram based beam scanning

We describe the development of a beam scanning microscope that can perform optical sectioning based on the principle of confocal microscopy. The scanning is performed by a laser beam diffracted from a dynamic binary hologram implemented using a liquid crystal spatial light modulator. Using the proposed scanning mechanism, unlike the conventional confocal microscopes, scanning over a two-dimensional area of the sample can be obtained without the use of a pair of galvo mirror scanners. The proposed microscope has a number of advantages, such as superior frame to frame repeatability, simpler optical arrangement, increased pixel dwell time relative to the time between two pixels, illumination of only the sample points without pulsing the laser, and absolute control over the amplitude and phase of the illumination beam on a pixel to pixel basis. The proposed microscope can be particularly useful for applications requiring very long exposure time or very large working distance objective lenses. In this paper we present experimental implementation of the setup using a nematic liquid crystal spatial light modulator and proof-of-concept experimental results.     

Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment

We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross‐talk between adjacent imaging channels. We demonstrate that a time‐multiplexed non‐linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single‐photon confocal system. The background becomes irrelevant for thin (< 15 µm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength. Theoretical and experimental axial responses of practically implemented microscopes are given.     

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.     

Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy

Monomolecular films of polymerized dimethyl-bis[pentacosadiinoic-oxyethyl] ammonium bromide (EDIPAB) provide one- and two-photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3-D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi-confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB-layers can be used as substrates for the cultivation of cells.     

Single-pinhole confocal imaging of sub-resolution sparse objects using experimental point spread function and image restoration

Sparse fluorescent pointlike subresolution objects have been imaged using a diffraction limited single-pinhole confocal fluorescence microscope. A Maximum likelihood image restoration algorithm has been used in conjunction with a measure of the experimental point spread function for improving the three-dimensional imaging of subresolution sparse objects. The experimental point-spread-function profiles have been improved by a factor of 1.95 in lateral direction and 3.75 in axial direction resulting in full-width half maximum (FWHM) values of 91 +/- 11 nm and 160 +/- 26 nm. This amounts to 1. 43 and 2.15 in optical units, respectively. The lateral and axial FWHM of the sparse pointlike subresolution objects is about 5 and 3 times smaller than the wavelength. This result points to the attractive possibility of utilising a compact confocal architecture for localising punctuate fluorescent objects having subresolution dimensions. The key resides in the utilisation of the measured point spread function coupled to an appropriate image restoration approach, and, of course, in the stability of the confocal system being used.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

Optical sectioning in fluorescence microscopy

We review the origins of optical sectioning in fluorescence microscopy in terms of the structure of the illumination used to generate the fluorescence within the specimen. We note that the conventional microscope using essentially uniform illumination does not exhibit optical sectioning whereas the confocal microscope using point (many spatial frequencies) illumination does. We show that the optical sectioning strength of a confocal microscope is not optimal and discuss the advantages of using a single spatial frequency for the structure of the illumination and the detection. In this case the optical sectioning strength is shown to be up to 25% narrower than in the ideal confocal case.     

Direct-view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration

We present a comparison between theoretical and experimental results for the axial response to a plane mirror specimen of the direct-view microscope employing multiple-pinhole arrays. The effects of pinhole size, pinhole spacing and array geometry are investigated in detail with a view to (i) achieving good optical sectioning characteristics and (ii) maximizing the amount of light available for imaging. The implications of our results for practical systems as regards pinhole-array design and fabrication are also discussed.     

Basic building units and properties of a fluorescence single plane illumination microscope

The critical issue of all fluorescence microscopes is the efficient use of the fluorophores, i.e., to detect as many photons from the excited fluorophores as possible, as well as to excite only the fluorophores that are in focus. This issue is addressed in EMBL's implementation of a light sheet based microscope [single plane illumination microscope (SPIM)], which illuminates only the fluorophores in the focal plane of the detection objective lens. The light sheet is a beam that is collimated in one and focused in the other direction. Since no fluorophores are excited outside the detectors' focal plane, the method also provides intrinsic optical sectioning. The total number of observable time points can be improved by several orders of magnitude when compared to a confocal fluorescence microscope. The actual improvement factor depends on the number of planes acquired and required to achieve a certain signal to noise ratio. A SPIM consists of five basic units, which address (1) light detection, (2) illumination of the specimen, (3) generation of an appropriate beam of light, (4) translation and rotation of the specimen, and finally (5) control of different mechanical and electronic parts, data collection, and postprocessing of the data. We first describe the basic building units of EMBL's SPIM and its most relevant properties. We then cover the basic principles underlying this instrument and its unique properties such as the efficient usage of the fluorophores, the reduced photo toxic effects, the true optical sectioning capability, and the excellent axial resolution. We also discuss how an isotropic resolution can be achieved. The optical setup, the control hardware, and the control scheme are explained in detail. We also describe some less obvious refinements of the basic setup that result in an improved performance. The properties of the instrument are demonstrated by images of biological samples that were imaged with one of EMBL's SPIMs.     

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.     

Time-domain whole-field fluorescence lifetime imaging with optical sectioning

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.     

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.     

Axial Phase-Darkfield-Contrast (APDC), a new technique for variable optical contrasting in light microscopy

Axial phase-darkfield-contrast (APDC) has been developed as an illumination technique in light microscopy which promises significant improvements and a higher variability in imaging of several transparent 'problem specimens'. With this method, a phase contrast image is optically superimposed on an axial darkfield image so that a partial image based on the principal zeroth order maximum (phase contrast) interferes with an image, which is based on the secondary maxima (axial darkfield). The background brightness and character of the resulting image can be continuously modulated from a phase contrast-dominated to a darkfield-dominated character. In order to achieve this illumination mode, normal objectives for phase contrast have to be fitted with an additional central light stopper needed for axial (central) darkfield illumination. In corresponding condenser light masks, a small perforation has to be added in the centre of the phase contrast providing light annulus. These light modulating elements are properly aligned when the central perforation is congruent with the objective's light stop and the light annulus is conjugate with the phase ring. The breadth of the condenser light annulus and thus the intensity of the phase contrast partial image can be regulated with the aperture diaphragm. Additional contrast effects can be achieved when both illuminating light components are filtered at different colours. In this technique, the axial resolution (depth of field) is significantly enhanced and the specimen's three-dimensional appearance is accentuated with improved clarity as well as fine details at the given resolution limit. Typical artefacts associated with phase contrast and darkfield illumination are reduced in our methods.     

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.