An improved chamber for direct visualisation of chemotaxis

There has been a growing appreciation over the last decade that chemotaxis plays an important role in cancer migration, invasion and metastasis. Research into the field of cancer cell chemotaxis is still in its infancy and traditional investigative tools have been developed with other cell types and purposes in mind. Direct visualisation chambers are considered the gold standard for investigating the behaviour of cells migrating in a chemotactic gradient. We therefore drew up a list of key attributes that a chemotaxis chamber should have for investigating cancer cell chemotaxis. These include (1) compatibility with thin cover slips for optimal optical properties and to allow use of high numerical aperture (NA) oil immersion objectives; (2) gradients that are relatively stable for at least 24 hours due to the slow migration of cancer cells; (3) gradients of different steepnesses in a single experiment, with defined, consistent directions to avoid the need for complicated analysis; and (4) simple handling and disposability for use with medical samples. Here we describe and characterise the Insall chamber, a novel direct visualisation chamber. We use it to show GFP-lifeact transfected MV3 melanoma cells chemotaxing using a 60x high NA oil immersion objective, which cannot usually be done with other chemotaxis chambers. Linear gradients gave very efficient chemotaxis, contradicting earlier results suggesting that only polynomial gradients were effective. In conclusion, the chamber satisfies our design criteria, most importantly allowing high NA oil immersion microscopy to track chemotaxing cancer cells in detail over 24 hours.     

Supercritical angle fluorescence correlation spectroscopy

We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves.     

Imaging of the cell surface interface using objective coupled widefield surface plasmon microscopy

We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.     

Birefringence of single and bundled microtubules.

We have measured the birefringence of microtubules (MTs) and of MT-based macromolecular assemblies in vitro and in living cells by using the new Pol-Scope. A single microtubule in aqueous suspension and imaged with a numerical aperture of 1.4 had a peak retardance of 0.07 nm. The peak retardance of a small bundle increased linearly with the number of MTs in the bundle. Axonemes (prepared from sea urchin sperm) had a peak retardance 20 times higher than that of single MTs, in accordance with the nine doublets and two singlets arrangement of parallel MTs in the axoneme. Measured filament retardance decreased when the filament was defocused or the numerical aperture of the imaging system was decreased. However, the retardance "area," which we defined as the image retardance integrated along a line perpendicular to the filament axis, proved to be independent of focus and of numerical aperture. These results are in good agreement with a theory that we developed for measuring retardances with imaging optics. Our theoretical concept is based on Wiener's theory of mixed dielectrics, which is well established for nonimaging applications. We extend its use to imaging systems by considering the coherence region defined by the optical set-up. Light scattered from within that region interferes coherently in the image point. The presence of a filament in the coherence region leads to a polarization dependent scattering cross section and to a finite retardance measured in the image point. Similar to resolution measurements, the linear dimension of the coherence region for retardance measurements is on the order lambda/(2 NA), where lambda is the wavelength of light and NA is the numerical aperture of the illumination and imaging lenses.     

Measuring rotations of a few cross-bridges in skeletal muscle

The ability to measure properties of a single cross-bridge in working muscle is important because it avoids averaging the signal from a large number of molecules and because it probes cross-bridges in their native crowded environment. Because the concentration of myosin in muscle is large, observing the kinetics of a single myosin molecule requires that the signal be collected from small volumes. The introduction of small observational volumes defined by diffraction-limited laser beams and confocal detection has made it possible to limit the observational volume to a femtoliter (10(-15) liter). By restraining labeling to 1 fluorophore per 100 myosin molecules, we were able to follow the kinetics of approximately 400 cross-bridges. To reduce this number further, we used two-photon (2P) microscopy. The focal plane in which the laser power density was high enough to produce 2P absorption was thinner than in confocal microscopy. Using 2P microscopy, we were able to observe approximately 200 cross-bridges during contraction. The novel method of confocal total internal reflection (CTIR) provides a method to reduce the observational volume even further, to approximately 1 attoliter (10(-18) liter), and to measure fluorescence with a high signal-to-noise (S/N) ratio. In this method, the observational volume is made shallow by illuminating the sample with an evanescent field produced by total internal reflection (TIR) of the incident laser beam. To guarantee the small lateral dimensions of the observational volume, a confocal aperture is inserted in the conjugate-image plane of the objective. With a 3.5-mum confocal aperture, we achieved a volume of 1.5 attoliter. Association-dissociation of the myosin head was probed with rhodamine attached at cys707 of the heavy chain of myosin. Signal was contributed by one to five fluorescent myosin molecules. Fluorescence decayed in a series of discrete steps, corresponding to bleaching of individual molecules of rhodamine. The S/N ratio was sufficiently large to make statistically significant comparisons from rigor and contracting myofibrils.     

Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) can provide a wealth of information about biological and chemical systems on a broad range of time scales (<1 micros to >1 s). Numerical modeling of the FCS observation volume combined with measurements has revealed, however, that the standard assumption of a three-dimensional Gaussian FCS observation volume is not a valid approximation under many common measurement conditions. As a result, the FCS autocorrelation will contain significant, systematic artifacts that are most severe with confocal optics when using a large detector aperture and aperture-limited illumination. These optical artifacts manifest themselves in the fluorescence correlation as an apparent additional exponential component or diffusing species with significant (>30%) amplitude that can imply extraneous kinetics, shift the measured diffusion time by as much as approximately 80%, and cause the axial ratio to diverge. Artifacts can be minimized or virtually eliminated by using a small confocal detector aperture, underfilled objective back-aperture, or two-photon excitation. However, using a detector aperture that is smaller or larger than the optimal value (approximately 4.5 optical units) greatly reduces both the count rate per molecule and the signal-to-noise ratio. Thus, there is a tradeoff between optimizing signal-to-noise and reducing experimental artifacts in one-photon FCS.     

A custom confocal and two-photon digital laser scanning microscope

We describe a custom one-photon (confocal) and two-photon all-digital (photon counting) laser scanning microscope. The confocal component uses two avalanche photodiodes (APDs) as the fluorescence detector to achieve high sensitivity and to overcome the limited photon counting rate of a single APD ( approximately 5 MHz). The confocal component is approximately nine times more efficient than our commercial confocal microscope (fluorophore fluo 4). Switching from one-photon to two-photon excitation mode (Ti:sapphire laser) is accomplished by moving a single mirror beneath the objective lens. The pulse from the Ti:sapphire laser is 109 fs in duration at the specimen plane, and average power is approximately 5 mW. Two-photon excited fluorescence is detected by a fast photomultiplier tube. With a x63 1.4 NA oil-immersion objective, the resolution of the confocal system is 0.25 microm laterally and 0.52 microm axially. For the two-photon system, the corresponding values are 0.28 and 0.82 microm. The system is advantageous when excitation intensity must be limited, when fluorescence is low, or when thick, scattering specimens are being studied (with two-photon excitation).     

Localization accuracy in single-molecule microscopy

One of the most basic questions in single-molecule microscopy concerns the accuracy with which the location of a single molecule can be determined. Using the Fisher information matrix it is shown that the limit of the localization accuracy for a single molecule is given by, lambda(em)/2pi n(a) square root of gammaAt, where lambda(em), n(a), gamma, A, and t denote the emission wavelength of the single molecule, the numerical aperture of the objective, the efficiency of the optical system, the emission rate of the single molecule and the acquisition time, respectively. Using Monte Carlo simulations it is shown that estimation algorithms can come close to attaining the limit given in the expression. Explicit quantitative results are also provided to show how the limit of the localization accuracy is reduced by factors such as pixelation of the detector and noise sources in the detection system. The results demonstrate what is achievable by single-molecule microscopy and provide guidelines for experimental design.     

3D-Printed high-NA catadioptric thin lens for suppression of XPM background in Stimulated Raman Scattering microscopy

Stimulated Raman Scattering (SRS) is a fast chemical imaging technique with remarkable bioscience applications. Cross Phase Modulation (XPM) is a ubiquitous nonlinear phenomenon that can create spurious background signals that render difficult a high-contrast imaging in SRS measurements. The XPM-induced signal is usually suppressed using high numerical aperture (NA) microscope objectives or condensers to collect the transmitted excitation beam. However, these high NA optics feature short working distances, hence they are not compatible with stage-top incubators, that are necessary to perform live-cell time-lapse experiments in controlled environments. Here, we show a 3D printed high NA compact catadioptric lens that fits inside stage-top incubators and allows the collection of XPM-free SRS signals. The lens delivers SRS images and spectra with a quality comparable to a signal collection with a high-NA microscope objective. We also demonstrate the compatibility of the 3D printed lens with other nonlinear microscopies usually associated with SRS in multimodal microscopes.     

Long-Distance Axial Trapping with Focused Annular Laser Beams

Focusing an annular laser beam can improve the axial trapping efficiency due to the reduction of the scattering force, which enables the use of a lower numerical aperture (NA) objective lens with a long working distance to trap particles in deeper aqueous medium. In this paper, we present an axicon-to-axicon scheme for producing parallel annular beams with the advantages of higher efficiency compared with the obstructed beam approach. The validity of the scheme is verified by the observation of a stable trapping of silica microspheres with relatively low NA microscope objective lenses (NA = 0.6 and 0.45), and the axial trapping depth of 5 mm is demonstrated in experiment.     

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy

Although light microscopy and three-dimensional image analysis have made considerable progress during the last decade, it is still challenging to analyze the genome nano-architecture of specific gene domains in three-dimensional cell nuclei by fluorescence microscopy. Here, we present for the first time chromatin compaction measurements in human lymphocyte cell nuclei for three different, specific gene domains using a novel light microscopic approach called Spatially Modulated Illumination microscopy. Gene domains for p53, p58, and c-myc were labeled by fluorescence in situ hybridization and the sizes of the fluorescence in situ hybridization "spots" were measured. The mean diameters of the gene domains were determined to 103 nm (c-myc), 119 nm (p53), and 123 nm (p58) and did not correlate to the genomic, labeled sequence length. Assuming a spherical domain shape, these values would correspond to volumes of 5.7 x 10(-4) microm(3) (c-myc), 8.9 x 10(-4) microm(3) (p53), and 9.7 x 10(-4) microm(3) (p58). These volumes are approximately 2 orders of magnitude smaller than the diffraction limited illumination or observation volume, respectively, in a confocal laser scanning microscope using a high numerical aperture objective lens. By comparison of the labeled sequence length to the domain size, compaction ratios were estimated to 1:129 (p53), 1:235 (p58), and 1:396 (c-myc). The measurements demonstrate the advantage of the SMI technique for the analysis of gene domain nano-architecture in cell nuclei. The data indicate that chromatin compaction is subjected to a large variability which may be due to different states of genetic activity or reflect the cell cycle state.     

Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection.

We consider the effect of planar dielectric interfaces (e.g., solid/liquid) on the fluorescence emission of nearby probes. First, we derive an integral expression for the electric field radiated by an oscillating electric dipole when it is close to a dielectric interface. The electric field depends on the refractive indices of the interface, the orientation of the dipole, the distance from the dipole to the interface, and the position of observation. We numerically calculate the electric field intensity for a dipole on an interface, as a function of observation position. These results are applicable to fluorescent molecules excited by the evanescent field of a totally internally reflected laser beam and thus very close to a solid/liquid interface. Next, we derive an integral expression for the electric field radiated when a second dielectric interface is also close to the fluorescent molecule. We numerically calculate this intensity as observed through the second interface. These results are useful when the fluorescence is collected by a high-aperture microscope objective. Finally, we define and calculate a "dichroic factor," which describes the efficiency of collection, in the two-interface system, of polarized fluorescence. The limit when the first interface is removed is applicable for any high-aperture collection of polarized or unpolarized fluorescence. The limit when the second interface is removed has application in the collection of fluorescence with any aperture from molecules close to a dielectric interface. The results of this paper are required for the interpretation of order parameter measurements on fluorescent probes in supported phospholipid monolayers (Thompson, N.L., H. M. McConnell, and T. P. Burghardt, 1984, Biophys. J., 46:739-747).     

Cylindrical illumination confocal spectroscopy: rectifying the limitations of confocal single molecule spectroscopy through one-dimensional beam shaping

Cylindrical illumination confocal spectroscopy (CICS) is a new implementation of single molecule detection that can be generically incorporated into any microfluidic system and allows highly quantitative and accurate analysis of single fluorescent molecules. Through theoretical modeling of confocal optics and Monte Carlo simulations, one-dimensional beam shaping is used to create a highly uniform sheet-like observation volume that enables the detection of digital fluorescence bursts while retaining single fluorophore sensitivity. First, we theoretically show that when used to detect single molecules in a microchannel, CICS can be optimized to obtain near 100% mass detection efficiency, <10% relative SD in burst heights, and a high signal/noise ratio. As a result, CICS is far less sensitive to thresholding artifacts than traditional single molecule detection and significantly more accurate at determining both burst rate and burst parameters. CICS is then experimentally implemented, optically characterized, and integrated into separate two microfluidic devices for the analysis of fluorescently stained plasmid DNA and single Cy5 labeled oligonucleotides. CICS rectifies the limitations of traditional confocal spectroscopy-based single molecule detection without the significant operational complications of competing technologies.     

Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens

Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective) and at the axial plane to 1.4nλ/NA² (n = refractive index of the imaging medium, 1.51 for oil immersion), which with visible wavelengths and a 1.4NA oil immersion objective is ∼220 nm and ∼600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT), with its native 50–100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes'' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF), a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes.     

Stereologic estimation of mean nuclear volume in well-differentiated adenocarcinoma and carcinoma in situ of the gallbladder

OBJECTIVE: To investigate mean nuclear volume of cells in well-differentiated adenocarcinomas (20 cases) and carcinoma in situ (20 cases) of the gallbladder by the principle of estimation of the volume of particles with arbitrary shapes. STUDY DESIGN: Hematoxylin and eosin-stained, 4-micron-thick, vertical sections from formalin-fixed, paraffin-embedded tissue blocks were analyzed by using a projection microscope with a 100:1 oil immersion objective (NA 1.3); the final magnification was 2,500:1. The measurements were carried out in 10 microscopic fields for each slide. Mean nuclear volume was obtained by the stereologic method of point-sampled intercepts for vertical sections. RESULTS: Mean nuclear volume in well-differentiated adenocarcinomas (127.67 +/- 46.95 micron 3) was significantly larger than in carcinoma in situ (69.17 +/- 15.74 micron 3) (P < .000001). CONCLUSION: Stereologic estimation of mean nuclear volume may be helpful in the discrimination of malignant and borderline lesions of the gallbladder.     

Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies

Soft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant.     

Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies

Soft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant.     

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC) to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using “off-the-shelf” microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) μm in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.     

Nuclear volume estimation using different sampling, measurement and calculation methods

OBJECTIVE: To study the reliability of volume parameter measured on tissue sections through different sampling, measurement and calculation methods. STUDY DESIGN: The largest nuclear profile image under a 100x, NA 1.30 oil immersion objective of primary spermatocytes and spherical spermatoblasts on 11-micron-thick seminiferous tubule sections and tissue images, under a 20x objective, on 4-micron sections were captured. Their volumes were measured and calculated by the five methods provided by the Technology for Image and Graphics Engineering Research cell image analysis system. RESULTS: The nuclear volumes obtained by nucleator and area equivalent diameter on the largest nuclear profile image were almost the same, including binary images by automated and manual interactive nucleator and grey scale images only by the latter. Nuclear volumes, calculated by random Feret diameter and equivalent diameter of the perimeter, the minimal circumference of the largest nuclear profile binary image, were obviously larger than those of the nucleator and area equivalent diameter. Due to different-sized nuclear slices entrapped in the same section, those nuclear volumes from the seminiferous tubule tissue images were strikingly lower than that of the largest nuclei profile image. The shape factors of primary spermatocytes and spherical spermatoblast nuclei under 100x and 20x objectives were approximately the same. CONCLUSION: The sample preparation, sampling methods and calculation formulas suitable to nuclear form are necessary to obtain reproducible volume parameters.