Immersion oil for high-resolution live-cell imaging at 37 degrees C: optical and physical characteristics.

The use of normal immersion oil, developed for 23 degrees C, at 37 degrees C greatly compromises both axial resolution and signal intensity. We developed and characterized an immersion oil for optimal performance in live-cell imaging at 37 degrees C. We quantify the improvements in resolution and intensity obtained when using the new oil instead of its standard 23 degrees C counterparts.

Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and SIPchart-based calibration procedures.

The fluorescence intensity image of an axially integrated through-focus series of a thin standardized uniform fluorescent layer can be used for image intensity correction and calibration in sectioning microscopy. This intensity image is in fact available from the earlier introduced Sectioned Imaging Property (SIP) charts (Brakenhoff et al., 2005). It is shown that the integrated intensity of a z-stack from a biological sample, imaged under identical conditions as the layer, can be calibrated in terms of fluorescence layer units of the calibration layer. The imaging after such calibration becomes, as a first approximation, independent of the microscope system and imaging conditions. This is demonstrated on axially integrated images of standard fluorescent beads and standard BPAE Fluorocells. Corrections on the microscope imaging conditions include shading effects, imaging with different magnifications and objectives, and using different microscope systems. It is also shown that with the present approach the actual underlying three-dimensional (3D) fluorescence data set itself can be corrected for variations in point spread function (PSF) imaging efficiency over the imaging data cube. Realizing such calibration between imaging conditions or systems requires basically only the 2D fluorescer molecule density of the reference layers and the section distances with which the layer data are collected.

Analysis of the influence of spherical aberration from focusing through a dielectric slab in quantitative nonlinear optical susceptibility measurements using third-harmonic generation.

The third-order nonlinear susceptibility (chi(3)) can be measured quantitatively using third-harmonic generation (THG) from two different interfaces. For the first time it is demonstrated both in experiments and theory that the magnitude of the THG signals from the two interfaces is not only determined by material properties (refractive index and chi(3)), but also by optical aberrations. It is found that this method of chi(3) determination can be applied without additional correction factors only for focusing conditions with a numerical aperture (NA)

Imaging colloidal particle induced topological defects in a nematic liquid crystal using third harmonic generation microscopy.

The nature of the third-harmonic generation (THG) process in a nematic liquid crystal is investigated for the case of tightly focused, low intensity, laser beams. Colloidal particle induced topological defects in a liquid crystal are visualized in three-dimensions using the dependence of the THG signal on both changes in non-linear susceptibility and the orientation of the liquid crystal director relative to the incident laser polarization state. We have found that the interpretation of THG images in a liquid crystal is complicated not only by the change in polarisation of the electric field as it propagates through the medium but also by anisotropic refractive index mismatch induced aberrations.

Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts.

Thin, uniformly fluorescing reference layers can be used to characterize the imaging conditions in confocal, or more general, sectioning microscopy. Through-focus datasets of such layers obtained by standard microscope routines provide the basis for the approach. A set of parameters derived from these datasets is developed for defining a number of relevant sectioned imaging properties. The main characteristics of a particular imaging situation can then be summarized in a Sectioned Imaging Property-chart or SIPchart. We propose the use of such charts for the characterization of imaging properties in confocal and multiphoton microscopy. As such, they can be the basis for comparison of sectioned imaging condition characteristics, quality control, maintenance or reproduction of sectioned imaging conditions and other applications. Such charts could prove useful in documenting the more relevant properties of the instrumentation used in microscopy studies. The method carries the potential to provide the basis for a general characterization of sectioned imaging conditions as the layers employed can be characterized and fabricated to standard specifications. A limited number of such thin, uniformly fluorescing layers is available from our group for this purpose. Extension of the method to multiphoton microscopy is discussed.

Image calibration in fluorescence microscopy.

A fluorescence image calibration method is presented based on the use of standardized uniformly fluorescing reference layers. It is demonstrated to be effective for the correction of non-uniform imaging characteristics across the image (shading correction) as well as for relating fluorescence intensities between images taken with different microscopes or imaging conditions. The variation of the illumination intensity over the image can be determined on the basis of the uniform bleaching characteristics of the layers. This permits correction for the latter and makes bleach-rate-related imaging practical. The significant potential of these layers for calibration in quantitative fluorescence microscopy is illustrated with a series of applications. As the illumination and imaging properties of a microscope can be evaluated separately, the methods presented are also valuable for general microscope testing and characterization.

Four-dimensional imaging of chromatin dynamics during the assembly of the interphase nucleus.

Large-scale chromatin organization is likely to play an important role in epigenetic control of gene expression. This implies that after mitosis the correct chromatin organization must be re-established in the nuclei of the two daughter cells. Here we analyze the dynamic behavior of chromatin during the transition from late anaphase to G1 in dividing HeLa cells, which express green fluorescent protein-tagged histone H2B. Time-lapse confocal microscopy was used to image the movement and the decondensation of chromatin as cell division progresses. Typically, time series of over 100 three-dimensional images (4D images) were collected, spanning a time period of up to three hours. Special care was taken to avoid photodamage, since cell cycle progression is exquisitely sensitive to photochemical damage. Quantitative analysis of the 4D images revealed that during the anaphase to G1 transition the movement of chromatin domains relative to other chromatin is remarkably limited. Chromatin dynamics can best be described as a radial expansion of the cluster of chromosomes that is present in late anaphase. We find that decondensation occurs in two phases. First a rapid decondensation by about a factor of two, followed by a slower phase in which part of the chromatin does not decondense any further, whereas the remaining chromatin decondenses further about two fold.

Optical far-field microscopy of single molecules with 3.4 nm lateral resolution.

Optical far-field imaging of single molecules in a frozen solution at 1.2 K with a lateral resolution of 3.4 nm is reported. The mechanical stability of the fluorescence microscope, especially of the low-temperature insert, allows for the localization of fluorescing molecules with a reproducibility of better than 5 nm within observation times up to 10 min. For observation times of 9 h the reproducibility of the lateral position is limited to about 20 nm due to mechanical drift. Lateral position and orientation of 314 single molecules, present within the confocal detection volume of approximately 10 microm(3), are obtained. The possibility to correct for mechanical drift by monitoring the position of a spatial reference in the sample is demonstrated.

Image cytometric method for quantifying the relative amount of DNA in bacterial nucleoids using Escherichia coli.

An image cytometric method for quantifying integrated fluorescence was developed to measure the relative DNA contents of bacterial nucleoids. Image analysis was performed with newly developed macros in combination with the program Object-Image, all downloadable from http://simon.bio.uva.nl/object-image.html. Four aspects of the method were investigated. (i) Good linearity was found over a ten-fold range of fluorescence intensity in a test with a calibration kit of fluorescent latex spheres. (ii) The accuracy of the method was tested with a narrowly distributed Escherichia coli population, which was obtained by growing cells into stationary phase. The width of the image cytometric distribution was approximately 6%, in good agreement with results obtained by flow cytometry. (iii) The error contribution of manual focusing could be kept below 2%, although a strong dependency between integrated fluorescence and focus position was observed. (iv) The results were verified with a flow cytometer, which gave similar distributions for the DNA contents per cell expressed in chromosome equivalents (4.8 fg of DNA). We used the presented method to evaluate whether the DNA conformation had any effect on the total fluorescence of bacterial nucleoids. Experiments using nucleoids with the same amount of DNA in either a dispersed or a compact conformation showed no significant difference in integrated fluorescence, indicating that it is possible to determine the DNA content per nucleoid independently of the actual organization of the DNA.

Probing microscopic chemical environments with high-intensity chirped pulses.

By varying the chirp of high-intensity pulses, we can use the chirp-condition-dependent fluorescence yield to distinguish among different molecules or the same molecule in different microenvironments. As an example of the latter we show that SNAFL-2, a well-known pH-sensitive dye, shows large modulation in fluorescence yield in response to both variation in acidity and variation in chirp condition. Future application of this technique as a novel contrast mechanism within fluorescence microscopy is discussed.

Optical saturation measurements of fluorophores in solution with pulsed femtosecond excitation and two-dimensional CCD camera detection.

We present a new method for the measurement of saturation of the optical transition of fluorescent molecules in solution, which is based on detection with a CCD camera of a two-dimensional projection of the three-dimensional, spatially nonuniform fluorescence intensity distribution as generated in a bulk solution of the fluorophore by excitation with focused femtosecond optical pulses. Essential to the method is (a) a combination of information from a measurement in saturation and one not in saturation and (b) for the measurement in saturation, the simultaneous observation of both saturated and nonsaturated regions of the fluorescence intensity distribution. The experimental setup is straightforward and good agreement is found between the theory and the experimental data.

Apodization and the point-spread autocorrelation function.

A novel function, the point-spread autocorrelation function (PSAF), which is closely related to the point-spread function, of a high numerical aperture microscope objective is introduced. The function is both experimentally measured and theoretically modeled for various apodization conditions. These include varying the effective numerical aperture of the objective, applying annuli of different size, and illuminating the objective with a spatially nonuniform intensity distribution. An excellent agreement between experimental data and theoretical modeling is obtained without the use of any fitting parameters. The PSAF technique is sensitive to the various apodization conditions, affecting both the width of the PSAF signal and the amplitude of the sidelobes. A potential use of the technique is the measurement of the effective numerical aperture and the optimization of the illumination conditions in complex microscopical systems.

The mechanism of red cell (dis)aggregation investigated by means of direct cell manipulation using multiple optical trapping.

We used multiple optical trapping to study the mechanism of red cell (dis)aggregation. Two sets of optical 'tweezers' were used to bring two red blood cells together to form a two-cell aggregate and then to pull them apart, to study the interaction between the cells. We found that cross-bridging occurred in normal reversible aggregation as we observed binding and the occurrence of small tethers between opposite cell membranes. Furthermore, the cells could only be parted by sliding them side by side with a maximum velocity in the order of microm/s indicating accumulation of the cross-bridges.

Analysis of efficiency of two-photon versus single-photon absorption of fluorescence generation in biological objects.

Starting from basic absorption cross-section values and breakdown limitations on possible field strengths it is shown that the total exposure required for equivalent fluorescence generation by single-photon absorption (SPA) is at least an order of magnitude lower than by two-photon absorption (TPA). The difference is such that TPA may in fact in many cases offer no advantage over SPA with respect to the damage induced during fluorescence imaging of biological materials.

Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system.

The bilateral imaging approach known from confocal applications operating in the line mode was used to realize real-time two-photon imaging. It is shown that the sectioning inherent to two-photon imaging could be improved by the introduction of a confocal line aperture in the imaging path. Using a high-power, low-repetition-rate amplified Ti:sapphire system, various biological objects were visualized including live boar sperm.

Improved axial resolution by point-spread autocorrelation function imaging.

Based on an interferometric spatial autocorrelation of two shifted point-spread functions combined with confocal detection, we demonstrate that the point-spread autocorrelation function technique provides improved axial resolution compared with conventional confocal imaging. The principle of the technique is demonstrated for a one-sided f luorescing step object.

A new method to study shape recovery of red blood cells using multiple optical trapping.

In this new method for studying the shape recovery of deformed red blood cells, three optical traps ("optical tweezers") induce a parachute-shaped red cell deformation, which is comparable to the deformation in small capillaries. The shape recovery is recorded, and a relaxation time is obtained for each individual red blood cell. The sensitivity of this technique for the detection of differences in relaxation times is demonstrated on subpopulations of density-separated red blood cells: "young" cells have shorter (162 ms) and "old" cells have longer (353 ms) relaxation times compared with the total population (271 ms). The relaxation time is remarkably shorter (114 ms) when the plasma surrounding the cells is replaced by a phosphate-buffered saline solution. The main advantages of this technique are the relatively short measuring and preparation time and the physiological type of deformation and shape recovery in which all relevant cell properties play a role. Therefore, especially when automated further, the technique may be a powerful tool for the study of (sub)populations of pathological red blood cells.

Isolation of single yeast cells by optical trapping.

Individual yeast cells can be successfully isolated and recultured on plates with a new isolation method making use of optical trapping with infrared laser light. The cells can be selected on morphological criteria by high resolution microscopy. The isolation device is constructed from two coverslips separated by spacers, in which selected cells are transferred to a plastic capillary, using the optical trap. To test the procedure, selection experiments were done with a mixture of two Saccharomyces cerevisiae strains, distinguishable both in fluorescence microscopy and on agar plates. These experiments showed that only selected cells were isolated, and close to 100% of the isolated stationary-phase cells formed colonies on agar plates, indicating a high recovery. A lower recovery was obtained with exponential-phase cells, possibly because of a higher sensitivity to laser irradiation. Applications for this method may include the isolation of mutants with altered morphology and the isolation of subpopulations of yeast cultures, for their separate investigation or for the initiation of pure cultures.