Polarization-preserving confocal microscope for optical experiments in a dilution refrigerator with high magnetic field

We present the design and operation of a fiber-based cryogenic confocal microscope. It is designed as a compact cold-finger that fits inside the bore of a superconducting magnet, and which is a modular unit that can be easily swapped between use in a dilution refrigerator and other cryostats. We aimed at application in quantum optical experiments with electron spins in semiconductors and the design has been optimized for driving with and detection of optical fields with well-defined polarizations. This was implemented with optical access via a polarization maintaining fiber together with Voigt geometry at the cold finger, which circumvents Faraday rotations in the optical components in high magnetic fields. Our unit is versatile for use in experiments that measure photoluminescence, reflection, or transmission, as we demonstrate with a quantum optical experiment with an ensemble of donor-bound electrons in a thin GaAs film.     

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.     

Resolution and optical sectioning in the confocal microscope

In this short review, we present a self-contained discussion of the image formation properties of the fluorescent confocal microscope. The optical sectioning or depth discrimination property is discussed in detail and new analytic formulae are presented, which relate the optical sectioning strength to the wavelength, numerical aperture and pinhole aperture size in a particularly simple fashion.     

Optical-fibre scanning near-field optical microscope for cryogenic operation

We have developed a new type of scanning near-field optical microscope (SNOM) utilizing optical fibres. The probe tip is controlled by shear force feedback with a fibre interferometer and signal light is collected directly by a multimode fibre. These features make the SNOM head more compact and less sensitive to vibration. Further advantages of this new type of SNOM are that it obviates the need for optical windows in the cryostat and offers easy optical alignment.     

Optical-fibre scanning near-field optical microscope for cryogenic operation

We have developed a new type of scanning near-field optical microscope (SNOM) utilizing optical fibres. The probe tip is controlled by shear force feedback with a fibre interferometer and signal light is collected directly by a multimode fibre. These features make the SNOM head more compact and less sensitive to vibration. Further advantages of this new type of SNOM are that it obviates the need for optical windows in the cryostat and offers easy optical alignment.     

A method for characterizing longitudinal chromatic aberration of microscope objectives using a confocal optical system

We describe a novel method of characterizing the longitudinal chromatic aberration of microscope objectives by recording a series of axial responses as a function of wavelength as a plane reflector is scanned through the focal region of a confocal microscope. Measurements are presented for a variety of objectives with differing degrees of correction. The use of the chromatic focal shift to measure surface profiles is also discussed.     

A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K

We developed a cryostat suitable for a laser scanning confocal microscope which allows for a short working distance and thus the usage of an objective with a high numerical aperture ensuring high collection efficiency. The in situ preparation of a thin layer of amorphous water is realized in a part of the cryostat, a Dewar vessel, which is put onto a custom-made, liquid-nitrogen immersed spin-coater. First tests on the setup are performed on a perylenemonoimide/polymethyl methacrylate model system using a standard oil objective and a dry objective at ambient temperature as well as a dry objective at liquid nitrogen temperature (77 K). Fluorescence resonance energy transfer (FRET) measurements on doubly labeled, freeze-quenched polyproline chains show the applicability of the new method on biomolecules. The alternating laser excitation (ALEX) is modified to a line-scanning process (slow ALEX) to optimize the sorting of the labeled molecules. Photophysics and photochemistry at liquid nitrogen temperature are investigated.     

A low temperature scanning tunneling microscope for electronic and force spectroscopy

In this article, we describe and test a novel way to extend a low temperature scanning tunneling microscope with the capability to measure forces. The tuning fork that we use for this is optimized to have a high quality factor and frequency resolution. Moreover, as this technique is fully compatible with the use of bulk tips, it is possible to combine the force measurements with the use of superconductive or magnetic tips, advantageous for electronic spectroscopy. It also allows us to calibrate both the amplitude and the spring constant of the tuning fork easily, in situ and with high precision.     

A low-wavenumber-extended confocal Raman microscope with very high laser excitation line discrimination

We describe the simple modification of a confocal Raman imaging microscope to incorporate two ultra-narrow holographic notch filters. The modified microscope rejects the laser excitation line (Rayleigh peak) by a discrimination factor of ～10(11) and allows simultaneous measurements of Stokes/anti-Stokes Raman shifts as close as ～10/20 cm(-1) to the Rayleigh line. The extremely high rejection ratio of the Rayleigh peak results in its intensity becoming comparable to typical Raman scattering signals. This is essential for micro-Raman spectroscopy and imaging in the low-wavenumber region. We illustrate the resulting performance with measurements on silicon/silica, sapphire, sulfur, L-cystine, as well as on single-walled carbon nanotubes (SWNTs). We find that both aggregated (bulk) and individual (deposited on substrate) SWNTs demonstrate strong and broad characteristic Raman features below ～100 cm(-1)-in a region which has remained essentially unexplored in measurements of bulk SWNT samples and which has so far been inaccessible for Raman spectroscopy of individual SWNTs.     

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.     

数字共焦显微仪序列光学切片自动采集方法研究

提出一种基于图像处理的数字共焦显微仪序列光学切片自动采集方法。方法分两个步骤:第一步是对细胞进行自动聚焦;第二步是细胞的起始点自动调焦定位及进行光学切片。方法采用清晰度评价函数作为判别标准,将灰度差分算法与拉普拉斯函数法相结合,通过计算机实现两个步骤的自适应调焦控制,进而实现光学切片的自动采集。实验结果表明,方法有较高的自动聚焦和细胞起始点定位的速度和准确性,对三维球形细胞序列光学切片图像采集,简单易行,效果良好。     

A confocal fibre scanning microscope for magnetic domain imaging

A compact confocal single-mode optical fibre scanning microscope for imaging magnetic domain structures, based on the polar magneto-optic Kerr effect, has been developed. The images obtained correspond to those obtained using single mirror scanning but this design offers a more compact structure and can be made more immune from system depolarization which makes two-axis mirror scanning difficult to implement when very small changes in polarization need to be detected.     

A white light confocal microscope for spectrally resolved multidimensional imaging

Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.     

A microfluidic vibrating wire viscometer for operation at high pressure and high temperature

This article discusses a microfluidic vibrating wire viscometer with an internal volume of a few microliters. Accuracy of order ±10% for viscosities ranging from 0.1 to 100 cP is demonstrated for temperatures (10 °C-175 °C) and pressures (10-24,000 psi) suitable for oilfield use by prior calibration in air and toluene. Comparison between multiple data sets indicates that a large fraction of the discrepancy between literature values is systematic, indicating that future refinements may be possible with better interpretation. Confinement effects are evaluated and are not found to play a significant role, which is surprising since the ratio (6.6) of the fluid channel width to the wire diameter is very low.     

Detectability: A new criterion for evaluation of the confocal microscope

We introduce a new criterion to explore the performance, with respect to signal-noise ratio for fluorescent imaging, in the confocal microscope. This criterion, which we term detectability, has three forms: transverse, axial, and three-dimensional. It describes the ease with which a small object can be detected in transverse images, axial images, and three-dimensional images.     

En bloc optical sectioning of resin-embedded specimens using a confocal laser scanning microscope

Reconstruction of 3D structures of specimens embedded for light or electron microscopy is usually achieved by cutting serial sections through the tissues, then assembling the images from each section to reconstruct the original structure or feature. This is both time-consuming and destructive, and may lead to areas of particular interest being missed. This paper describes a method of examining specimens which have been fixed in glutaraldehyde and embedded in epoxy resin, by utilising the autofluorescence preserved or enhanced by aldehyde fixation, and by using a confocal laser scanning microscope to section optically such specimens in the block down to a depth of about 200 μm. In this way, the accurate estimation of the depth of particular features could be used to facilitate subsequent sectioning at the light microscope or electron microscope level for more detailed studies, and 3D images of tissues/structures within the block could be easily prepared if required.     

A scanning tunnelling microscope for operation in air

In this paper a scanning tunnelling microscope for operation in air is described with emphasis on the coarse and fine sample positioning mechanism, piezoelectric scanner and vibration isolation. Line scan (amplitude modulation), top view (brightness modulation) and combined shaded topographical images of the surface of highly orientated pyrolitic graphite are shown.     

A standard video-rate confocal laser-scanning reflection and fluorescence microscope

A confocal laser-scanning microscope (CLSM) differs from a conventional microscope by affording an extreme depth discrimination, as well as a slightly improved resolution. These features afford improved imaging, and make possible new imaging techniques. The CLSM developed at TNO has standard video-rate imaging, and is capable of working in reflection and in fluorescence mode simultaneously. Nonconfocally the laser-scanning microscope can also be used in transmission mode. In addition to the evident advantages of a fast system when searching objects or studying living objects, the time needed to produce an image of extended depth of focus and high resolution is very short. Furthermore, the high-speed averaging of many images at low laser-power levels, and the short dwelling time of the focused laser beam (60 ns) obviate quenching effects in fluorescence microscopy and prevent damage to the object. In this article the TNO-CLSM system is outlined. The most important specifications are summarized, and some representative micrographs obtained with the system are shown. Furthermore, the performance of the system is illustrated by some experimental results.     

A compact sub-Kelvin ultrahigh vacuum scanning tunneling microscope with high energy resolution and high stability

We designed a scanning tunneling microscope working at sub-Kelvin temperatures in ultrahigh vacuum (UHV) in order to study the magnetic properties on the nanoscale. An entirely homebuilt three-stage cryostat is used to cool down the microscope head. The first stage is cooled with liquid nitrogen, the second stage with liquid (4)He. The third stage uses a closed-cycle Joule-Thomson refrigerator of a cooling power of 1 mW. A base temperature of 930 mK at the microscope head was achieved using expansion of (4)He, which can be reduced to ≈400 mK when using (3)He. The cryostat has a low liquid helium consumption of only 38 ml/h and standing times of up to 280 h. The fast cooling down of the samples (3 h) guarantees high sample throughput. Test experiments with a superconducting tip show a high energy resolution of 0.3 meV when performing scanning tunneling spectroscopy. The vertical stability of the tunnel junction is well below 1 pm (peak to peak) and the electric noise floor of tunneling current is about 6fA/√Hz. Atomic resolution with a tunneling current of 1 pA and 1 mV was achieved on Au(111). The lateral drift of the microscope at stable temperature is below 20 pm/h. A superconducting spilt-coil magnet allows to apply an out-of-plane magnetic field of up to 3 T at the sample surface. The flux vortices of a Nb(110) sample were clearly resolved in a map of differential conductance at 1.1 K and a magnetic field of 0.21 T. The setup is designed for in situ preparation of tip and samples under UHV condition.