Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells

FLAP is a new method for localized photo‐labelling and subsequent tracking of specific molecules within living cells. It is simple in principle, easy to implement and has a wide potential application. The molecule to be located carries two fluorophores: one to be photobleached and the other to act as a reference label. Unlike the related methods of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), the use of a reference fluorophore permits the distribution of the photo‐labelled molecules themselves to be tracked by simple image differencing. In effect, FLAP is therefore comparable with methods of photoactivation. Its chief advantage over the method of caged fluorescent probes is that it can be used to track chimaeric fluorescent proteins directly expressed by the cells. Although methods are being developed to track fluorescent proteins by direct photoactivation, these still have serious drawbacks. In order to demonstrate FLAP, we have used nuclear microinjection of cDNA fusion constructs of β‐actin with yellow (YFP) and cyan (CFP) fluorescent proteins to follow both the fast relocation dynamics of monomeric (globular) G‐actin and the much slower dynamics of filamentous F‐actin simultaneously in living cells.     

Contactless determination of nuclear compressibility using 3D image- and model-based analysis of drug-induced cellular deformation

Mechanical properties of the chromatin-bearing nucleus in normal and pathological cells are of general interest for epigenetics and medicine. Conventional techniques for quantitative measurements of material properties of cellular matter are based on application of controlled forces onto the cellular or nuclear boundary and do not allow probing intracellular structures that are not directly accessible for physical contact inside the living cell. In this work, we present a novel approach for contactless determination of the nuclear compressibility (i.e. the Poisson's ratio ν) in living cells by means of image- and model-based analysis of drug-induced cell deformation. The Poisson's ratio of the HeLa cell nucleus is determined from time-series of 3D images as a parameter of constitutive model that minimizes the dissimilarity between the numerically predicted and experimentally observed images.     

Video-rate confocal reflection microscopy of neoplastic cells: Rate of intracellular movement and peripheral motility characteristic of neoplastic cell line (RSK4) with high degree of growth independence in vitro

Video-rate laser confocal interference reflection microscopy was used to demonstrate rapid motion of intracellular organelles and features at the cell periphery in a fully transformed neoplastic cell line, RSK4, and in four other neoplastic cell populations. In the RSK4 cells, vibrational and trafficking movements of intracellular particles at a rate greater than 25 Hz and ranging down to 5 Hz were recorded. Rapidly moving processes changed to ruffles, then microspikes, and previously undetectable ephemeral intercellular contacts were seen. Dynamic cyclical changes were revealed in the sizes of the podosomal close contacts of the transformed cells. The visibility of such features and the temporal and spatial resolution are improved over earlier methods. The fact that fast cellular and intracellular movements can be detected with this microscopic technique offers new possibilities in attempting to recognise differences between unimpaired living cells, and it may prove useful in the identification of malignant cells.     

Four-dimensional factor analysis of confocal image sequences (4D-FAMIS) to detect and characterize low copy numbers of human papillomavirus DNA by FISH in HeLa and SiHa cells

Visualization and localization of specific DNA sequences were performed by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), and four-dimensional factor analysis of biomedical image sequences (4D-FAMIS). HeLa and SiHa cells containing, respectively 20–50 and 1–2 copies per cell of human papillomavirus (HPV) DNA type 18 and 16 integrated in cellular DNA were used as models. HPV-DNA was identified using DNA probes containing the whole genome of HPV-DNA type 18 or 16, and DNA–DNA hybrids were revealed by alkaline phosphatase and Fast Red. Cell nuclei were counterstained with thiazole orange (TO) or TOTO-iodide. 4D image sequences were obtained using successive dynamic or spectral sequences of images on different optical sections from CLSM. The location of fluorescent signals within the preparations was determined by FAMIS. This original method summarizes image sequences into a reduced number of images called factor images, and curves called factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. We distinguished between Fast Red and nucleus stainings in HPV-DNA hybridization signals by taking into account differences in their extinction velocities (fluorescence decay rate) or spectral patterns, and in their focus (depth emission profiles). In HeLa cells, factor images showed that Fast-Red-stained targets could be distinguished from nucleus stainings, and were located on different focal planes of the nuclei. In SiHa cells, 4D-FAMIS determined as few as 1–2 copies per cell of HPV-DNA type 16 located in continuous focal planes. Therefore, 4D-FAMIS, together with CLSM, made the detection and characterization of low copy numbers of genes in whole cells possible.