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.     

A new high-aperture glycerol immersion objective lens and its application to 3D-fluorescence microscopy

High‐resolution light microscopy of glycerol‐mounted biological specimens is performed almost exclusively with oil immersion lenses. The reason is that the index of refraction of the oil and the cover slip of ~1.51 is close to that of ~1.45 of the glycerol mountant, so that refractive index mismatch‐induced spherical aberrations are tolerable to some extent. Here we report the application of novel cover glass‐corrected glycerol immersion lenses of high numerical aperture (NA) and the avoidance of these aberrations. The new lenses feature a semi‐aperture angle of 68.5°, which is slightly larger than that of the diffraction‐limited 1.4 NA oil immersion lenses. The glycerol lenses are corrected for a quartz cover glass of 220 µm thickness and for a 80% glycerol‐water immersion solution. Featuring an aberration correction collar, the lens can adapt to glycerol concentrations ranging between 72% and 88%, to slight variations of the temperature, and to the cover glass thickness. As the refractive index mismatch‐induced aberrations are particularly important to quantitative confocal fluorescence microscopy, we investigated the axial sectioning ability and the axial chromatic aberrations in such a microscope as well as the image brightness as a function of the penetration depth. Whereas there is a significant decrease in image brightness associated with oil immersion, this decrease is absent with the glycerol immersion system. In addition, we show directly the compression of the optic axis in the case of oil immersion and its absence in the glycerol system. The unique advantages of these new lenses in high‐resolution microscopy with two coherently used opposing lenses, such as 4 Pi‐microscopy, are discussed.     

Near-field nano-ellipsometer for ultrathin film characterization

We describe a near‐field ellipsometer for accurate characterization of ultrathin dielectric films. Optical tunnelling mimics the absorption in metallic films, enabling accurate measurement of the refractive index of ultrathin dielectric film. A regression model shows that a refractive index resolution of 0.001 for films as thin as 1 nm is possible. A solid‐immersion nano‐ellipsometer that incorporates this near‐field ellipsometric technique with a solid‐immersion lens is constructed to demonstrate the viability of this technique. Such a nano‐ellipsometer can accurately characterize thin films ranging in thickness from subnanometre to micrometres with potential transverse resolution of the order of 100 nm.     

Blind deconvolution of 3D fluorescence microscopy using depth‐variant asymmetric PSF

The 3D wide‐field fluorescence microscopy suffers from depth‐variant asymmetric blur. The depth‐variance and axial asymmetry are due to refractive index mismatch between the immersion and the specimen layer. The radial asymmetry is due to lens imperfections and local refractive index inhomogeneities in the specimen. To obtain the PSF that has these characteristics, there were PSF premeasurement trials. However, they are useless since imaging conditions such as camera position and refractive index of the specimen are changed between the premeasurement and actual imaging. In this article, we focus on removing unknown depth‐variant asymmetric blur in such an optical system under the assumption of refractive index homogeneities in the specimen. We propose finding few parameters in the mathematical PSF model from observed images in which the PSF model has a depth‐variant asymmetric shape. After generating an initial PSF from the analysis of intensities in the observed image, the parameters are estimated based on a maximum likelihood estimator. Using the estimated PSF, we implement an accelerated GEM algorithm for image deconvolution. Deconvolution result shows the superiority of our algorithm in terms of accuracy, which quantitatively evaluated by FWHM, relative contrast, standard deviation values of intensity peaks and FWHM. Microsc. Res. Tech. 79:480–494, 2016. © 2016 Wiley Periodicals, Inc.     

Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index

Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive‐index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high‐end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented. With these methods, we measured axial scaling factors as a function of refractive‐index mismatch for high‐aperture confocal microscopy imaging. We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account. There was however a strong deviation with the theoretical predictions using (high‐angle) geometrical optics, which predict much lower scaling factors. As an illustration, we measured the PSF of a correctly calibrated point‐scanning confocal microscope and showed that a nearly index‐matched, micron‐sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles.     

Analysis of spherical aberration of a water immersion objective: application to specimens with refractive indices 1.33–1.40

The method of using immersion medium to correct spherical aberration for water immersion objectives when the samples are not water is investigated. Spherical aberration is measured by an interferometer converted from a confocal microscope for samples with different refractive indices. When the proper refractive index of the immersion medium and thickness of cover slip are selected, the measured spherical aberration approaches zero. A theoretical model can be used for prediction of the immersion medium to correct spherical aberration for various samples. Using the thinnest available cover slip (100 μm), the zero spherical aberration condition can be applied to samples with refractive index as high as 1.40. Confocal images in the condition of almost no spherical aberration are included to demonstrate the improvement of axial resolution due to this correction.     

3D deconvolution of spherically aberrated images using commercial software

Refractive index mismatch between the specimen and the objective immersion oil results in spherical aberration, which causes distortion and spreading of the point spread function, as well as incorrect readings of the axial coordinates. These effects have to be taken into account when performing three-dimensional restoration of wide-field fluorescence images. By using objects with well-defined geometry (fluorescently stained Escherichia coli or actin filaments) separated from a cover slip by a layer of oil with known refractive index, we investigated the accuracy of three-dimensional shape restoration by the commercial programs Huygens and Autoquant. Aberration correction available in the software dramatically reduced the axial blur compared to deconvolution that ignored the refractive index mismatch. At the same time, it failed to completely recover the cylindrical symmetry of bacteria or of actin fibres, which showed up to a three to five times larger width along the optical axis compared to the lateral plane. The quality of restoration was only moderately sensitive to the exact values of the specimen refractive index but in some cases improved significantly by assuming a reduced NA of the objective. Because image restoration depends on the knowledge of the vertical scale, we also performed detailed measurements of the axial scaling factor and concluded (in agreement with some previous authors) that scaling is adequately described by the simple paraxial formula, even when high-NA oil-immersion objectives are used.     

Volume measurements in three-dimensional microscopy

A refractive index mismatch between the oil immersion and the microscopic object can lead to a severe over-estimation of the object's size. The cause of this effect is explained and a simple calibration method to compensate for its occurrence is presented. A practical example is discussed. The analysis applies to both conventional three-dimensional, and confocal microscopy.     

High‐resolution wide‐field microscopy with adaptive optics for spherical aberration correction and motionless focusing

Live imaging in cell biology requires three‐dimensional data acquisition with the best resolution and signal‐to‐noise ratio possible. Depth aberrations are a major source of image degradation in three‐dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of the mismatch between the sample refractive index and the immersion medium index. We have built a wide‐field fluorescence microscope that incorporates a large‐throw deformable mirror to simultaneously focus and correct for depth aberration in three‐dimensional imaging. Imaging fluorescent beads in water and glycerol with an oil immersion lens we demonstrate a corrected point spread function and a 2‐fold improvement in signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.     

2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy

The use of high numerical aperture immersion lenses in optical microscopy is compromised by spherical aberrations induced by the refractive index mismatch between the immersion system and the embedding medium of the sample. Especially when imaging >10 micro m deep into the specimen, the refractive index mismatch results in a noticeable loss of image brightness and resolution. A solution to this problem is to adapt the index of the embedding medium to that of the immersion system. Unfortunately, not many mounting media are known that are both index tunable as well as compatible with fluorescence imaging. Here we introduce a nontoxic embedding medium, 2,2'-thiodiethanol (TDE), which, by being miscible with water at any ratio, allows fine adjustment of the average refractive index of the sample ranging from that of water (1.33) to that of immersion oil (1.52). TDE thus enables high resolution imaging deep inside fixed specimens with objective lenses of the highest available aperture angles and has the potential to render glycerol embedding redundant. The refractive index changes due to larger cellular structures, such as nuclei, are largely compensated. Additionally, as an antioxidant, TDE preserves the fluorescence quantum yield of most of the fluorophores. We present the optical and chemical properties of this new medium as well as its application to a variety of differently stained cells and cellular substructures.     

Refractive-index-induced aberrations in two-photon confocal fluorescence microscopy

The effect of refractive index mismatch on the image quality in two-photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high-aperture objectives when the image plane is moved deeper into the sample. When exciting at 740 nm and recording the fluorescence around 460 nm in a glycerol-mounted sample using a lens of a numerical aperture of 1·4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 μm. In an aqueous sample, the same decrease is observed at a depth of 3 μm. By reducing the numerical aperture to 1·0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermat's principle. Advice is given concerning how the effects can be taken into account in practice.     

Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin

In this work, we compared the performance of objectives with similar numerical aperture of 0.75 but different immersion media of air, water, glycerin, and oil in the imaging of human skin epithelium and dermis. In general, we found that the oil immersion objective recorded the strongest intensity at the same mechanical depth. We also characterized the focal shifts and found that with decreasing refractive index, the focal shift becomes increasingly more negative (for both the epithelium and dermis). In imaging the dermis, we estimated the image resolution at the depths of 18.8 and 30.2 microm, and found that the image resolution were comparable at these depths under the four types of immersion conditions. Our results demonstrate that by changing the immersion media, the main microscopic imaging effects are the recorded axial intensities and the focal shifts. The effects on the image resolution are negligible.     

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.     

Effects of specimen refractive index on confocal imaging

The aberrations introduced when focusing within a specimen with a refractive index equal to that of water using an oil-immersion objective are investigated theoretically. The peak intensity in the confocal point spread function drops by a factor of two for focusing less than 10 μm into the specimen. The effects on scaling of dimensions in the resulting images are discussed. The image exhibits an axial stretching by a factor of about 1.12.     

Improved correction of axial geometrical distortion in index-mismatched fluorescent confocal microscopic images using high-aperture objective lenses

Extracting quantitative data from microscopic volume images is straightforward when the refractive indices of the immersion medium and the mounting medium are equal. The readings of the position of the specimen stage can be directly used to measure depth and width. Imperfectly matched immersion and mounting media result in axial geometrical distortion. Linear correction of the axial distortion using the paraxial estimate of the axial scaling factor yields results that may differ as much as 4% from the actual values. From calculations based on a theoretical expression of the 3‐D point‐spread function in the focal region of a high‐aperture microscope focussing into a mismatched mounting medium, we derived axial scaling factors that result in quantitative results accurate to better than 1%. From a non‐linear correction procedure, an improved formula for the paraxial estimate of the axial scaling factor is derived.     

Optical characteristics of thin film coating and measurement of its thickness

Theoretical analysis of the optical characteristics of thin film coatings was carried out. The relations of refractive index, phase change and film thickness were studied. As a result, a new optical method of measuring thickness of thin metallic coatings was developed. Further, the technique can also be used to find the refractive index of the thin film and the phase change of the light beam owing to being reflected from the coating. It is shown that the new method is very accurate for measuring the thin coating thickness. A specimen coating was tested with a polarised laser beam and the measured phase change falls between the reasonable range of 0 and π.     

Modern techniques in reflectance measurements

In the field of quantitative investigation of microscopic images reflectance measurements with the aid of photoelectric equipment play an important role. Reflectances can be used for the identification of various components in a polished specimen, for chemical analysis and for the determination of the refractive index of a specimen as well as for a determination of the various absorption parameters of reflecting solid material. The parameters involved are: reflectances at given wavelengths, spectral reflectance curves in the visible, UV-, and near IR-spectral range, reflectances of optically anisotropic surfaces at varying orientations of the vibration plane of linearly polarized light, reflectances in air and in an immersion liquid (mainly in standard immersion oil). From spectral curves, quantitative expressions of colour can be derived. Due to improvements in reflectance standard materials and technical design of apparatus the measurements and calculations can be carried out very conveniently, quickly and precisely.     

The attachment of mineralised tissues to coverslips for observing dynamic events by confocal microscopy

In confocal scanning optical fluorescence microscopy, using high-aperture oil immersion lenses, the best images are obtained for focus planes immediately under the coverslip, or under a continuation of the coverslip with a medium with the same, high refractive index. Therefore placing the coverslip with a layer of oil underneath it is an advantage if the sample will allow it. If experimental manipulations that can displace the coverslip are involved, —for example, attempts to make dynamic observation of fluid flow—the coverslip is best cemented to the sample with a high-index material. This report describes the use of adhesive systems developed for restorative dentistry to achieve a durable attachment of dental tissues to microscope coverslips. The technique described in this paper has been used for monitoring real-time fluid movement in dentine. The samples were examined with a high-frame-rate confocal microscope (a tandem scanning microscope). The adhesive technology also could be utilised in the microscopic preparation of other porous translucent materials.     

The role of specimen-induced spherical aberration in confocal microscopy

We present an overview of recent theories for describing specimen-induced spherical aberration in confocal microscopy. One of these theories is used to compute numerically the role of spherical aberration in general confocal, and especially in biological confocal, microscopy for a variety of three-layer specimen structures. In particular, we study the effect of specimen-induced spherical aberration on the maximum value of the overall confocal point spread function, the accompanying focal shift and the size of the optical probe in both fluorescence and brightfield confocal microscopy.     

Spatial and temporal changes of subchondral bone proceed to articular cartilage degeneration in rats subjected to knee immobilization

This study was aimed to investigate the spatial and temporal changes of subchondral bone and its overlying articular cartilage in rats following knee immobilization. A total of 36 male Wistar rats (11–13 months old) were assigned randomly and evenly into 3 groups. For each group, knee joints in 6 rats were immobilized unilaterally for 1, 4, or 8 weeks, respectively, while the remaining rats were allowed free activity and served as external control groups. For each animal, femurs at both sides were dissected after sacrificed. The distal part of femur was examined by micro‐CT. Subsequently, femoral condyles were collected for further histological observation and analysis. For articular cartilage, significant changes were observed only at 4 and 8 weeks of immobilization. The thickness of articular cartilage and chondrocytes numbers decreased with time. However, significant changes in subchondral bone were defined by micro‐CT following immobilization in a time‐dependent manner. Immobilization led to a thinner and more porous subchondral bone plate, as well as a reduction in trabecular thickness and separation with a more rod‐like architecture. Changes in subchondral bone occurred earlier than in articular cartilage. More importantly, immobilization‐induced changes in subchondral bone may contribute, at least partially, to changes in its overlying articular cartilage. Microsc. Res. Tech. 79:209–218, 2016. © 2016 Wiley Periodicals, Inc.