PHOEBE,a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media

Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ～ 1 nm and a lateral (x, y) resolution of ～ 200 nm when used with a high-numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256-pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ～ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (> 0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or a silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM₀₀), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.     

Confocal microscopy of the in-situ crystalline lens

The fine structure of the in-situ rabbit crystalline ocular lens from the ex-vivo rabbit eye was observed with a confocal scanning laser microscope in the scattered light mode. The images were observed through the full thickness of the cornea and aqueous humour to a depth of 50 μm in the anterior ocular lens. The following structures were observed from optical sections of the ocular lens: two concentric regions of the lens capsule, epithelial cells, lens sutures, and surface and interior regions of individual lenticular fibres. The observed lateral resolution of the microscope objective was degraded by imaging across thick (millimetre) structures. This study shows the feasibility of obtaining high-contrast images of transparent objects across 1.7 mm of ocular tissue (cornea and aqueous humour) using confocal light microscopy.     

Near real time in vivo fibre optic confocal microscopy: sub-cellular structure resolved

We have built a fibre optic confocal reflectance microscope capable of imaging biological tissue in near real time. The measured lateral resolution is 3 µm and axial resolution is 6 µm. Images of epithelial cells, excised tissue biopsies, and the human lip in vivo have been obtained at 15 frames s^?1. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. This device has the potential to enable reflected light confocal imaging of internal organs for in situ detection of pathology.     

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.     

A 2D MEMS mirror with sidewall electrodes applied for confocal MACROscope imaging

This paper presents microelectromechanical system micromirrors with sidewall electrodes applied for use as a Confocal MACROscope for biomedical imaging. The MACROscope is a fluorescence and brightfield confocal laser scanning microscope with a very large field of view. In this paper, a microelectromechanical system mirror with sidewall electrodes replaces the galvo-scanner and XYZ-stage to improve the confocal MACROscope design and obtain an image. Two micromirror-based optical configurations are developed and tested to optimize the optical design through scanning angle, field of view and numerical aperture improvement. Meanwhile, the scanning frequency and control waveform of the micromirror are tested. Analysing the scan frequency and waveform becomes a key factor to optimize the micromirror-based confocal MACROscope. When the micromirror is integrated into the MACROscope and works at 40 Hz, the micromirror with open-loop control possesses good repeatability, so that the synchronization among the scanner, XYZ-stage and image acquisition can be realized. A laser scanning microscope system based on the micromirror with 2 μm width torsion bars was built and a 2D image was obtained as well. This work forms the experimental basis for building a practical confocal MACROscope.     

Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens

An imaging technique called orthogonal-plane fluorescence optical sectioning (OPFOS) was developed to image the internal architecture of the cochlea. Expressions for the three-dimensional point spread function and the axial and lateral resolution are derived. Methodologies for tissue preparation and for construction, alignment, calibration and characterization of an OPFOS apparatus are presented. The instrument described produced focused, high-resolution images of optical sections of an intact, excised guineapig cochlea. The lateral and axial resolutions of the images were 10 and 26 μm, respectively, within a 1·5-mm field of view.     

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.     

激光差动共焦透镜中心厚度测量系统的研制

基于高精度光学共焦定位技术研制了一种全新的非接触透镜中心厚度测量系统,该系统利用差动共焦技术的高轴向层析特性和轴向响应曲线的绝对零点对被测透镜的前表面顶点和后表面顶点分别进行精密瞄准定位;同时,利用激光干涉仪获得透镜前、后表面顶点的位置坐标;然后通过光线追迹算法计算透镜中心厚度,进而实现了透镜中心厚度的高精度非接触测量。实验结果表明,该系统测量精度高,测量标准差小于1μm,满足透镜中心厚度测量的精度要求。     

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.     

Three-dimensional imaging in confocal imaging systems with finite sized detectors

We consider the effect of the finite size of the detector on both the lateral and axial resolution of the confocal system. The use of a finite sized detector means that the imaging is no longer truly coherent. We find that the lateral resolution is considerably more sensitive to the detector size than is the axial response. The question of the rejection of flare light is also considered. Experimental results are shown and we find that acceptable extended-focus, auto-focus and height images may be obtained from non truly-confocal systems. We also find that lens apodization has a far greater effect on the axial resolution than the lateral resolution.     

A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch

A difference in refractive index (n) between immersion medium and specimen results in increasing loss of intensity and resolution with increasing focal depth and in an incorrect axial scaling in images of a confocal microscope. Axial thickness measurements of an object on such images are therefore not exact. The present paper describes a simple procedure to determine the correct axial thickness of an object with confocal fluorescence microscopy. We study this procedure for a specimen that has a higher refractive index than the immersion medium and with a thickness up to 100 µm. The measuring method was experimentally tested by comparing the thickness of polymer layers measured on axial images of a confocal microscope in case of a water–polymer mismatch to reference values obtained from an independent technique, i.e. scanning electron microscopy. The case when the specimen has a lower refractive index than the immersion medium is also shown by way of illustration. Measured thickness data of a water layer and an oil layer with the same actual thickness were obtained using an oil-immersion objective lens with confocal microscopy. Good agreement between theory and experiment was found in both cases, consolidating our method.     

彩色共焦系统可调制色散物镜设计

针对彩色共焦距测量系统中测量范围和分辨率的调制问题,采用色散和聚焦功能分离的思路设计了一款色散物镜,其色散功能由纯球面折射透镜组成的色散管镜来实现,聚焦功能则采用商业物镜实现。使用ZEMAX软件对色散管镜的结构、材料及像差进行了设计及优化,仿真结果显示所设计的色散管镜在可见光范围内能获得优于230mm的轴向线性色散,实际加工的成品管镜的轴向线性色散范围可达160mm。实验测量了色散管镜及它结合不同聚焦物镜后的色散特性。实验结果表明,色散管镜结合不同聚焦能力的物镜能够具有高线性度的轴向色散区域;结合4倍和10倍放大倍率的商用物镜,分别获得了1 300μm和225μm的测量范围,其轴向分辨率分别为2μm和0.4μm,实现了测量范围和分辨率的调制。     

X-ray microtomography

A microscope system based on the principles of computerized axial tomography is described for determining the distribution of the X-ray absorption coefficient in a slice from a solid object without cutting sections. An application is given to determining the distribution at a resolution of about 15 μm through a shell of about 0·5 mm diameter.     

A compact Schwarzschild soft X-ray microscope with a laser-produced plasma source

A compact Schwarzschild soft X-ray microscope using a laser-produced plasma soft X-ray source has been developed. The laser-produced plasma source, which is small but of high brilliance, has made it possible to use the soft X-ray microscope in a small laboratory. The microscope is composed of a Schwarzschild objective and a grazing incidence mirror condenser. Image contrast for biological specimens in soft X-ray regions is investigated briefly. It is possible to observe the fine structures of a thin specimen at a wavelength of 15 nm; at this wavelength high-contrast images of biological specimens have been obtained with a single laser shot of pulse width of 8 ns at a resolution of 0·3 μm. The resolution of the system is limited by the detector.     

Confocal scanning light microscopy with high aperture immersion lenses

The imaging characteristics of a confocal scanning light microscope (CSLM) with high aperture, immersion type, lenses (N.A. = 1·3) are investigated. In the confocal arrangement the images of the illumination and detector pinholes are made to coincide in a common point, through which the object is scanned mechanically. Results show that for point objects the theoretically expected improved response by a factor of 1·4 in comparison with standard microscopy can indeed be realized. Low side lobe intensity and absence of glare permits the imaging at high resolution of weak details close to strong features. A further improvement by a factor of 1·25 in point resolution in CSLM is found after apodization with an annular aperture. Due to the scanning approach all possibilities of electronic image processing become available in light microscopy.     

Imaging in the far-red with electronic light microscopy: Requirements and limitations

The acquisition of simultaneous dual confocal images with red and far-red light has both advantages (e.g. lower autofluorescence) and limitations. An understanding of these requisites is necessary to acquire high-quality images and to avoid the misinterpretation of experimental data. The poor detection of far-red light mandates a high optical transfer efficiency for the system, thus the transmittance of the objective lens and its axial and lateral chromatic aberration in the far-red are important factors for consideration. This technical note is an attempt to ‘demystify’ the process of filter set design for confocal microscopy by discussing the considerations that went into the construction of a filter set for use with the reagents cyanine 3.18 (Cy3) and cyanine 5.18 (Cy5), and thus to encourage users to look beyond the multi-purpose designs available commercially. The 568-nm laser line exciting Cy3 is at its emission maximum, which limits the collectable Cy3 fluorescence. High-transmission optical filters with sharp band pass cutoffs are thus desirable for maximum light throughput. Light path mirror efficiency rapidly degrades above 700 nm, but the loss of this portion of the Cy5 emission spectrum is acceptable since the fluorophore is very bright, and these very long wavelengths are also likely to introduce aberration. While resolution is decreased with far-red light, there is also greater penetration and less scattering, and it is thus possible to obtain high-quality images from deeper within the specimen. Although only one make and model of confocal microscope (the Bio-Rad MRC–600) is considered, similar considerations pertain to the design of filter sets for any confocal microscope that accommodates user-installed filters.     

Optical modifications enabling simultaneous confocal imaging with dyes excited by ultraviolet- and visible-wavelength light

Optical modifications to a confocal scanning laser microscope are described which allow simultaneous fluorescence imaging of living specimens excited by ultraviolet (UV)- and visible-wavelength light. Modifications to a Bio-Rad MRC 600 Lasersharp confocal microscope include the introduction of UV-path-specific lenses and a specially designed UV transmitting eyepiece and tube lens. Upon UV excitation these modifications provide similar resolution and field flatness when compared with visible confocal microscopy. The UV-path-specific optics could be adjusted to correct for varying amounts of longitudinal chromatic aberration in commercially available objectives. Eyepiece and tube lenses were chromatically corrected for UV through visible wavelengths to minimize lateral chromatic error. With these modifications, UV-wavelength light may be used to excite ratioing dyes to quantify intracellular ion concentrations, or as an energy source to release caged compounds in a spatially restricted volume, while simultaneously imaging with dyes excited by visible-wavelength light.     

In-vivo observation of cells with a combined high-resolution multiphoton-acoustic scanning microscope

We present a combined multiphoton-acoustic microscope giving collocated access to the local morphological as well as mechanical properties of living cells. Both methods relay on intrinsic contrast mechanisms and dispense with the need of staining. In the acoustic part of the microscope, a gigahertz ultrasound wave is generated by an acoustic lens and the reflected sound energy is detected by the identical lens in a confocal setup. The achieved lateral resolution is in the range of 1 mum. Contrast in the images arises mainly from the local absorption of sound in the cells related to viscose damping. Additionally, acoustic microscopy can access the sound speed as well as the acoustic impedance of the cell membrane and the cell shape, as it is an intrinsic volume scanning technique. The multiphoton image formation bases on the detection of autofluorescence due to endogenous fluorophores. The nonlinearity of two-photon absorption provides submicron lateral and axial resolution without the need of confocal optical detection. In addition, in the near-IR cell damages are drastically reduced in comparison with direct excitation in the visible or UV. The presented setup was aligned with a dedicated procedure to ensure identical image areas. Combined multiphoton/acoustic images of living myoblast cells are discussed focusing on the reliability of the method.     

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.     

Wide-band acousto-optic deflectors for large field of view two-photon microscope

Acousto-optic deflector (AOD) is an attractive scanner for two-photon microscopy because it can provide fast and versatile laser scanning and does not involve any mechanical movements. However, due to the small scan range of available AOD, the field of view (FOV) of the AOD-based microscope is typically smaller than that of the conventional galvanometer-based microscope. Here, we developed a novel wide-band AOD to enlarge the scan angle. Considering the maximum acceptable acoustic attenuation in the acousto-optic crystal, relatively lower operating frequencies and moderate aperture were adopted. The custom AOD was able to provide 60 MHz 3-dB bandwidth and 80% peak diffraction efficiency at 840 nm wavelength. Based on a pair of such AOD, a large FOV two-photon microscope was built with a FOV up to 418.5 μm (40× objective). The spatiotemporal dispersion was compensated simultaneously with a single custom-made prism. By means of dynamic power modulation, the variation of laser intensity within the FOV was reduced below 5%. The lateral and axial resolution of the system were 0.58-2.12 μm and 2.17-3.07 μm, respectively. Pollen grain images acquired by this system were presented to demonstrate the imaging capability at different positions across the entire FOV.