Nils R.D. Aslund

Confocal imaging for 3-D digital microscopy

Optical serial sectioning based on the depth-discriminating ability of confocal laser scanning can be combined with digital image processing to realize fast and easy-to-use 3-D microscopy. A great advantage as compared with traditional methods, e.g., using a microtome, is that the specimen is left undamaged. An account is given of an instrument designed for this purpose and of feasibility studies that have been carried out to assess the usefulness of the method in fluorescence microscopy.

Simultaneous confocal recording of multiple fluorescent labels with improved channel separation

Confocal microscopes are often used to study specimens labelled with fluorophores. A commonly used method for simultaneous recording of the distribution of multiple fluorophores is to divide the fluorescent light emitted by the specimen into different wavelength regions using dichroic and bandpass filters. These different wavelength regions are then distributed to multiple detectors. However, the broad and overlapping spectra of commonly used fluorophores often result in considerable crosstalk between channels. A new technique, intensity‐modulated multiple‐beam scanning (IMS) microfluorometry, can be used to reduce this cross‐talk substantially.

Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique

Abstract A new technique for simultaneous recording of multiple fluorophores has been implemented in a confocal scanning laser microscope. Dual excitation wavelengths, intensity-modulated at different frequencies and two lock-in amplifiers tuned to the corresponding frequencies are used. In this way two fluorophores can be independently, albeit simultaneously, excited. This technique, Intensity-modulated Multiple-beam Scanning (IMS) microfluorometry, has the potential to virtually eliminate the cross-talk between fluorophores that often occurs when recording multiple-labelled specimens. Furthermore, it will offer simultaneous information about both the excitation and emission spectra of the fluorophores used. Also, variations in decay time over the image area can be studied independently and simultaneously for two fluorophores.

Simultaneous lifetime imaging of two fluorophores using a confocal laser microscope

We demonstrate the possibility to study, simultaneously, the variations over the image area of the lifetimes of two different fluorophores, in a confocal laser microscope. In this way information conveyed by the lifetimes can be extracted, in particular complementary information from two different fluorophores. The fluorophores are excited by laser light of two different wavelengths, which are modulated at different frequencies using electro-optical modulators. By frequency-selective detection, using two lock-in amplifiers, it is possible to efficiently separate signals that emanate from each of the fluorophores. Further, by using 2- phase lock-in amplifiers the phase of each of the separated signals is determined. Since the phase shift of the emitted light relative to the exciting light depends on the lifetime of the fluorophore, the technique allows mapping of the lifetime. Two images are obtained, one for each fluorophore. The method can be extended to supply information concerning the excitation cross sections at the two laser wavelengths used.

Using intensity-modulated scanning beams in combination with lock-in detection for recording multiple-labeled fluorescent specimens in confocal laser microscopy

We demonstrate how the new Intensity-modulated Multiple-beam Scanning technique can be used in improve simultaneous recording of multiple fluorophores in confocal scanning laser microscopes.

Compensating for depth-dependent light attenuation at 3-D imaging with a confocal microscope

An account has been given previously of a 3-D image processing method to compensate for depth dependent light attenuation in images from a confocal microscope, working in the epifluorescence mode. A basic assumption is that there are regions of the specimen that are homogeneous in the sense that the attenuation is constant within the region. It is shown that a stack of 2-D histograms of adjacent images usually shows distinguishable features through which homogeneous regions of this kind can be traced. It is also shown how the attenuation factor of the region is obtained. Its inverse is the correction factor applicable to the region. The region may be extracted from the stack to be dealt with separately. The method has been further developed by introducing a dynamic display procedure. It makes both the search and the extraction more efficient. Further, techniques have been implemented to perform the compensation automatically by changing the PM tube voltage during the recording using computer control. In the present paper an account is given on these improvements. A review is also given of some fundamentals of the method and of an application of the method.

Mirror deflection control for a confocal scanning laser microscope employing a time-modulated laser and a linear diode array

A mirror deflection device for a CSLM has been developed. It performs repetitive scanning according to a preset waveform which can be chosen arbitrarily. It can also be used to perform stationary positioning at arbitrarily chosen points. A digital memory, comprising dual banks, is used to allow switching from one actuating waveform to another. The movement of the mirror is recorded very accurately. A burst of sequential pulse from a diode laser is deflected by the mirror and recorded by means of a linear diode array. The target pattern is analyzed digitally. The objective is to implement a control strategy whereby a new actuating waveform can be derived in order to correct any deviation between the desired waveform and the recorded one. Some results obtained with the device are reported. Foreseen applications encompass spectral analysis of selected regions and kinetic studies where a trade-off between speed and number of image points is necessary.

Recording of dual-labeled specimens using frequency-multiplexed confocal imaging and intensity-modulated two-wavelength excitation

We demonstrate the possibility to use intensity-modulated excitation and frequency multiplexing in combination with lock-in detection to make multi-parameter measurements with a confocal scanning laser microscope. This approach can be used, for example, when studying dual-labelled fluorescent specimens. Frequency-multiplexed confocal imaging has the potential to reduce a main problem in connection with detection of multiple-labelled specimens, namely cross-talk between the recorded signals from the two fluorophores. In addition, it has the advantage compared with the traditional method that the fluorescent spectra are utilized much more efficiently, thereby greatly improving signal quality.

Three-dimensional image processing method to compensate for depth-dependent light attenuation in images from a confocal microscope

When looking into the depth of a semitransparent specimen, using a confocal laser microscope working in the epifluorescence mode, it is often observed that the recorded images are darker the deeper the optical sections are located in the specimen. One reason for this is that light is absorbed in the specimen on its way to and from the section. A manual method to compensate for this darkening is to vary the electronic amplification at the recording. The appropriate amplification depends not only on the depth but also on the specimen, its shape and density, etc. Methods to replace the manual adjustments with computer methods, applied to stacks of uncompensated images recorded at different equidistant depths, have been suggested. A basic assumption is then that there are regions of the specimen that are homogeneous enough to serve as reference regions for the compensation. A key problem is to detect these regions. An interactive method to trace homogeneous regions in a stack of recorded images is described. It is also shown how image segmentation can be performed to extract such regions.