Anders Liljeborg

Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation

We demonstrate the possibility to increase substantially the number of simultaneously detected fluorophores by utilizing both spectral and lifetime information. Using a two‐detector confocal scanning laser microscope, experiments confirm that four different fluorophores can be detected with good channel separation. The signal‐to‐noise ratio (SNR) of the recorded images is investigated both theoretically and experimentally. It is found that in order to obtain a high SNR fluorophore lifetimes should differ by approximately an order of magnitude.

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.

Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance

We investigate the performance of confocal pH imaging when using phase fluorometry and fluorophores with pH‐dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidally modulated. The lifetime‐dependent phase shift in the fluorescent signal is detected by a lock‐in amplifier, and converted into a pH value through a calibration procedure. A theoretical investigation is made of how the different system parameters will influence the results concerning sensitivity and noise. Experiments carried out with the fluorophore SNAFL‐2 support these theoretical predictions. It is found that, under realistic experimental conditions, we can expect a pH change of 0.1 units to be easily detected in an 8‐bit digital image. However, the pixel‐to‐pixel root mean square noise is often of the order of one pH unit. This comparatively high level of noise has its origin in photon quantum noise. pH measurements on living cells show a systematic deviation from expected values. This discrepancy appears to be the result of fluorophore interaction with various cell constituents, and is the subject of further investigation.

A method to compensate for light attenuation with depth in three‐dimensional DNA image cytometry using a confocal scanning laser microscope

A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three‐dimensional (3‐D) DNA image cytometry is described. The method is based on studying the stack of 2‐D histograms that can be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin‐fixed paraffin‐embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3‐D DNA image cytometry.

Second-order stereology for pores in translucent alumina studied by confocal scanning laser microscopy

The three‐dimensional (3‐D) arrangement of pores in translucent alumina was investigated with a confocal scanning laser microscope (CSLM). By moving the focal plane of the CSLM down into the material, a stack of serial thin optical sections was obtained to produce a 3‐D image of the pores. Computer‐based image analysis was used to obtain the coordinates of the pore centroids. The distance distribution function G(r) and the second‐order functions K(r), L(r), H(r) and g(r) were used to analyse the spatial point pattern of the pore centroids. Estimates of the preceding functions obtained from eight stacks of sections were compared with the corresponding functions for a 3‐D stationary Poisson point process, which served as a reference model for complete spatial randomness. The analysis suggested that the pore centroids were arranged in an aggregated pattern within a range of about 10 μm.

Confocal 3-dimensional DNA image cytometry in thick tissue sections.

We present a three-dimensional confocal DNA image cytometry (3-D CICM) method for analysis of DNA content in 30-40-microns-thick sections of routinely processed paraffin-embedded specimens. A comparison of DNA ploidy profiles obtained by 3-D CICM and conventional DNA image cytometry (ICM) on tissue sections sections showed significantly higher numbers of cells with high DNA content in DNA histograms by 3-D CICM. As estimated by 3-D CICM, the size of nuclei frequently exceeded the thickness of tissue sections used in conventional ICM, which suggested that many nuclei measured by this technique may be incomplete. This artifact was excluded in 3-D CICM by automatic rejection of cut nuclear profiles. This and the favorable ratio of tissue thickness to nuclear size in 3-D CICM permitted the DNA quantitation even in large cells with highly increased DNA ploidy values such as megakaryocytes and Reed-Sternberg cells of Hodgkins disease. Additionally, 3D-CICM allowed evaluation or morphometric parameters and 3-dimensional reconstruction of studied cells.

Computer control for a galvanometer scanner in a confocal scanning laser microscope

A digital control has been developed for a galvanometer scanner used in a confocal scanning laser microscope. The galvanometer scanner utilizes a microprocessor-controlled scan control unit, able to produce repetitive analog waveforms and digital control signals, and to record analog and digital signals. The scan control unit outputs a sawtooth waveform to the galvanometer, records the position signal from the position sensor in the galvanometer, and uses this information to calculate the timing needed to trigger the reading of 1024 x 1024 equally spaced pixels in the specimen. The galvanometer may be statically positioned at an arbitrarily chosen pixel, thereby allowing recording of data from a point within the microscope specimen for an arbitrary length of time. This makes it possible to record spectral information or to measure fluorescent lifetimes at a single point.

Digital Position Encoding Of Galvanometer Scanner In A Laser Microscope

An account is given of a realization of a feedback method to digitize the analog position signal from a moving iron galvanometer. It is employed in a confocal scanning laser microscope for generating digital images. The photometric sampling has to be closely coupled to the position of a mirror that scans a focused laser beam across a microscope specimen. Pictures with low geometric distortion are obtained up to the size 1024 x 1024 pixels.

Simulation of light attenuation within fluorescent microspheres used for liquid fraction separation recorded by a CSLM

In order to separate different proteins, liquid chromatography is often used. The sample is pumped through a column filled with microspheres. The velocity of the proteins are depending on their interaction with the microspheres. The proteins could be labelled with a fluorescent marker and the distribution of the protein within the sphere can be recorded using a CSLM. When collecting optical sections using a CSLM the detected intensity decreases the deeper in the specimen the section is collected. This is due to absorption, scattering and bleaching. For the special case of a single microsphere it is of interest to find out how this combined effect is distributed within the sphere for a certain distribution of the fluorescent stain. When this distribution is known the attenuation can be compensated for. In the simulation the distribution of the stain is supposed to be the result of a diffusion process and all attenuation is supposed to arise from absorption only. The attenuation for a certain volume element (voxel) is supposed to occur from absorption in the voxels above, within the cone formed by the focused excitation light beam. A basic assumption is that the attenuation within each voxel is a fraction of the fluorescent intensity within that same voxel. A simulation program has been written where the parameters of the diffusion process within the microsphere can be controlled. Also the parameters for the attenuation calculation can be set, e.g. the assumed fraction of fluorescent intensity that act as attenuation. 3D datasets can be generated for visualization. Also intensity profiles can be generated along a diameter of the simulated sphere in the depth direction, since the intensity distribution is circularly symmetric in the lateral directions. Some comparisons are made to real microspheres, and the parameters are adjusted for closest resemblance. This adjustment can be done manually but an implementation using non-linear fitting of data is also presented. The simulated diffusion constant, fraction of intensity acting as attenuation, and the maximum intensity are fitted to experimental data from vertical slices of microspheres.

Method for intracellular imaging of ion concentrations, using confocal microscopy and fluorophore lifetimes

There exist a number of fluorescent probes whose lifetimes change in response to ion concentrations (for example H+ and Ca2+) in the surrounding medium. We describe a technique for utilizing this property in a confocal scanning laser microscope. The technique is based on intensity-modulated laser illumination of the specimen, and phase-sensitive lock-in detection of the fluorescent light. In this way we get a lifetime-dependent output signal which, after calibration, can provide information concerning ion concentrations. In the current study we have used a pH sensitive fluorophore, SNAFL-2, to study the performance of this technique. We find that the sensitivity is such that a pH difference of 0.1 units can easily be detected in an 8- bit digital image. Noise measurements show that under realistic conditions we can expect a pixel-to-pixel standard deviation of approximately one to two pH units.