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.     

激光共焦高速扫描显微成像的高帧速重构算法

针对激光共焦扫描显微镜的往复式逐行扫描成像方式带来的帧图像数据分割难的问题,在分析系统扫描方式、振镜的实际运动方式与理论运动方式差异的基础上,利用相邻两帧图像相似性大的特点,提出了一套完整的高帧速重构算法。该算法通过连续帧特征区域差分的方式实现了一维信号序列的自适应分割,即实现了对一维信号序列进行动态排列及分割成二维阵列图像数据,从而重构出多帧高精度图像。实验表明,该算法的成像误差低于1.6%,适用于成像速度高达300帧/s的激光共焦扫描显微成像。     

Development of a standing-wave fluorescence microscope with high nodal plane flatness

This article reports about the development and application of a standing-wave fluorescence microscope (SWFM) with high nodal plane flatness. As opposed to the uniform excitation field in conventional fluorescence microscopes an SWFM uses a standing-wave pattern of laser light. This pattern consists of alternating planar nodes and antinodes. By shifting it along the axis of the microscope a set of different fluorescent structures can be distinguished. Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of structures which would appear axially unresolved in a conventional or confocal fluorescence microscope. An SWFM is most powerful when the axial extension of the specimen is comparable to the wavelength of light. Otherwise several planes are illuminated simultaneously and their separation is hardly feasible. The objective of this work was to develop a new SWFM instrument which allows standing-wave fluorescence microscopy with controlled high nodal plane flatness. Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and processing. Another design goal was to build a compact, easy-to-use instrument to foster a more widespread use of this new technique. The instrument developed uses a green-emitting helium–neon laser as the light source, a piezoelectric movable beamsplitter to generate two mutually coherent laser beams of variable relative phase and two single-mode fibres to transmit these beams to the microscope. Each beam is passed on to the specimen by a planoconvex lens and an objective lens. The only reflective surface whose residual curvature could cause wavefront deformations is a dichroic beamsplitter. Nodal plane flatness is controlled via interference fringes by a procedure which is similar to the interferometric test of optical surfaces. The performance of the instrument was tested using dried and fluorescently labelled cardiac muscle cells of rats. The SWFM enabled the distinction of layers of stress fibres whose axial separation was just a fraction of a wavelength. Layers at such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could therefore not be distinguished by these two methods. To obtain futher information from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Krishnamurthi et al. (Proc. SPIE, 2655, 1996, 18–25). To minimize specimen-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the refractive indices could be checked via the method presented here for the measurement of nodal plane flatness. For this purpose the fluorescent layer of latex beads would simply be replaced by the specimen. A combination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit the full potential of standing-wave fluorescence microscopy.     

Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope

High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.     

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.     

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.     

Optimizing the performance of confocal point scanning laser microscopes over the full field of view

To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view three simple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent material and a rectangular grid with a readily defined centre. Using these specimens the adjustment of any combination of confocal scanning laser visualization system and light microscope can be examined throughout the field of view. The effects of misalignment of the various subcomponents of a confocal scanning laser microscope on both the axial spread function of a plane and the shading pattern over the image field are described. Finally, where the design of the confocal optics permits, the three specimens can be used to facilitate the alignment of the various components to the optimal level achievable.     

New imaging modes for lenslet-array tandem scanning microscopes

The tandem scanning microscope permits confocal images to be obtained in real time and viewed directly by eye. The light budget of these instruments may be increased from a few percent to a few tens of percent by incorporating an array of microlenses so as to increase the amount of illumination light that reaches the specimen. These instruments are configured for fluorescence imaging together with laser illumination. We describe how the versatility of the instrument may be enhanced to permit the use of incoherent light sources as well as extending the imaging modes to include bright‐field reflection.     

An optical spectrometer for a confocal scanning laser microscope

An optical spectrometer for the visible range has been developed for the confocal scanning laser microscope (CSLM) Phoibos 1000. The spectrometer records information from a single point or a user-defined region within the microscope specimen. A prism disperses the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit cools the diode array, thereby reducing the detector dark current to a level, which allows integration times of up to 60 s. The spectral resolving power, λ/Δλ, ranges from 400 at λ = 375 nm to 100 at λ = 700 nm. Since the entrance aperture of the spectrometer has the same diameter as the detector aperture of the CSLM, the three-dimensional spatial resolution for spectrometer readings is equivalent to that of conventional confocal scanning, that is, down to 0.2 μm lateral and 0.8 μm axial resolution with an N.A.=1.3 objective.     

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.     

直接测距型无扫描激光主动成像验证系统

为提高成像效率,研究了基于雪崩光电二极管(APD)阵列和分束照明的直接测距型无扫描激光主动成像系统。系统采用脉冲激光器作为光源,通过测量激光脉冲飞行时间来获取目标距离信息;基于达曼光栅进行分束照明以提高能量利用率。另外,光学系统采用收发共孔径结构,保证了目标处激光脚点和APD阵列各像元的几何对准。与参考APD所采用固定阈值时刻鉴别法不同,系统采用固定阈值与恒比定时相结合的时刻鉴别法处理回波APD阵列的输出信号以适应外光路参数的变化。各通道采用专用计时芯片TDC-GP22测量启停脉冲之间的时间间隔,实现了45ps的计时分辨率。最后,将19m和31m处放置的两个角反射器作为合作目标进行了成像实验,并给出了所获取的距离图像。结果表明:两个目标的平均距离分别为19.28m和31.54m,测距误差分别为0.28m和0.54m,显示提出的设计方案切实可行。     

Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering

This paper addresses the problem of intensity correction of fluorescent confocal laser scanning microscope images. Confocal laser scanning microscope images are frequently used in medicine for obtaining 3D information about specimen structures by imaging a set of 2D cross sections and performing 3D volume reconstruction afterwards. However, 2D images acquired from fluorescent confocal laser scanning microscope images demonstrate significant intensity heterogeneity, for example, due to photo‐bleaching and fluorescent attenuation in depth. We developed an intensity heterogeneity correction technique that (a) adjusts the intensity heterogeneity of 2D images, (b) preserves fine structural details and (c) enhances image contrast, by performing spatially adaptive mean‐weight filtering. Our solution is obtained by formulating an optimization problem, followed by filter design and automated selection of filtering parameters. The proposed filtering method is experimentally compared with several existing techniques by using four quality metrics: contrast, intensity heterogeneity (entropy) in a low frequency domain, intensity distortion in a high frequency domain and saturation. Based on our experiments and the four quality metrics, the developed mean‐weight filtering outperforms other intensity correction methods by at least a factor of 1.5 when applied to fluorescent confocal laser scanning microscope images.     

Autofluorescence of living cells

We have investigated the autofluorescence of viable mammalian cells (DU-145 and V79) with a confocal laser scanning microscope equipped with a UV laser. Our aim was to investigate the autofluorescence dependence on different treatments in mitochondria and lysosomes by using different reagents and to improve the confocal laser scanning microscope image quality by deconvolution. The following conclusions were drawn from the results: (1) not all of the autofluorescence comes from mitochondria; (2) one can significantly affect the signal which comes from the mitochondria; (3) the other organelles involved are probably lysosomes; (4) it is harder to affect the autofluorescence signal from the lysosomes than that from the mitochondria, and (5) deconvoluted autofluorescence images provide better information than undeconvoluted ones.     

High efficiency beam splitter for multifocal multiphoton microscopy

In this article we present the development of a multibeam two-photon laser scanning microscope. A new type of beam splitter to create the multitude of laser beams is described. This type of beam splitter has higher transmission and generates more uniform beams than can be achieved with the microlens approach used by other groups. No crosstalk exists between the different foci due to small temporal delays between the individual beams. The importance of dispersion compensation to obtain maximum efficiency of the microscope is discussed. With optimum compensation the fluorescence signal was raised by a factor of 14. Different modes of detecting the fluorescence signals and their effect on imaging speed and resolution are discussed.     

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.     

An optical sectioning programmable array microscope implemented with a digital micromirror device

The defining feature of a programmable array microscope (PAM) is the presence of a spatial light modulator in the image plane. A spatial light modulator used singly or as a matched pair for both illumination and detection can be used to generate an optical section. Under most conditions, the basic optical properties of an optically sectioning PAM are similar to those of rotating Nipkow discs. The method of pattern generation, however, is fundamentally different and allows arbitrary illumination patterns to be generated under programmable control, and sectioning strategies to be changed rapidly in response to specific experimental conditions. We report the features of a PAM incorporating a digital micromirror device, including the axial sectioning response to fluorescent thin films and the imaging of biological specimens. Three axial sectioning strategies were compared: line scans, dot lattice scans and pseudo-random sequence scans. The three strategies varied widely in light throughput, sectioning strength and robustness when used on real biological samples. The axial response to thin fluorescent films demonstrated a consistent decrease in the full width at half maximum (FWHM), accompanied by an increase in offset, as the unit cells defining the patterns grew smaller. Experimental axial response curves represent the sum of the response from a given point of illumination and cross-talk from neighbouring points. Cross-talk is minimized in the plane of best focus and when measured together with the single point response produces a decrease in FWHM. In patterns having constant throughput, there appears to be tradeoff between the FWHM and the size of the offset. The PAM was compared to a confocal laser scanning microscope using biological samples. The PAM demonstrated higher signal levels and dynamic range despite a shorter acquisition time. It also revealed more structures in x - z sections and less intensity drop-off with scanning depth.     

A confocal beam scanning white-light microscope

We report on a confocal beam scanning microscope utilizing a continuous Xe short-arc lamp operating in the visible spectrum with unprecedented radiance. Measurements of lateral and vertical resolution will be presented and compared with those of an equivalent scanning laser microscope. Resolution of the white-light microscope is equivalent to that of the scanning laser microscope. White-light microscope images positively stand out from those of the scanning laser microscope by their lack of artefacts caused by interference.     

A line scanning confocal fluorescent microscope using a CMOS rolling shutter as an adjustable aperture

Traditional confocal microscopy uses a physical aperture barrier to prevent out-of-focus light from reaching the detector. The physical nature of a conventional aperture limits control over the system confocality. We describe a new line scanning confocal microscope that eliminates a need for a physical aperture by employing a software-controllable rolling shutter on a CMOS camera. A confocal image is obtained by synchronizing motion of the rolling shutter and the laser line scanning over a sample. Confocal resolution of this microscope is adjustable in real time and independently established for each fluorescence channel by changing the rolling shutter width. This technology has been implemented in the IN Cell Analyzer 6000 system by GE Healthcare.     

Use of backscattered electron detector arrays for forming backscattered electron images in the scanning electron microscope

The backscattered electron (BSE) signal in the scanning electron microscope (SEM) can be used in two different ways. The first is to give a BSE image from an area that is defined by the scanning of the electron beam (EB) over the surface of the specimen. The second is to use an array of small BSE detectors to give an electron backscattering pattern (EBSP) with crystallographic information from a single point. It is also possible to utilize the EBSP detector and computer-control system to give an image from an area on the specimen--for example, to show the orientations of the grains in a polycrystalline sample ("grain orientation imaging"). Some further possibilities based on some other ways for analyzing the output from an EBSP detector array, are described.     

A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope

We report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic‐scale knife edge in the form of a simple transmission electron microscopy grid attached to a glass microscope slide, and a light‐collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, a beam profile in the form of an error function can be obtained and by repeating this with the knife edge at different axial positions relative to the beam waist, the divergence and astigmatism of the postobjective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multiphoton laser scanning microscopy. Our knife‐edge method has several advantages over alternative knife‐edge methods used in microscopy including that the knife edge is easy to prepare, that the beam can be characterized also directly under a cover slip, as necessary to reduce spherical aberrations for objectives designed to be used with a cover slip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited.