Showing papers on "Photoactivated localization microscopy published in 1998"

Contrast, resolution, pixelation, dynamic range and signal‐to‐noise ratio: fundamental limits to resolution in fluorescence light microscopy

Abstract: In a perfect optical system numerical aperture and wavelength determine resolution. In a real optical system, however, the number of photons collected from a specimen determines the contrast and this limits the resolution. Contrast is affected by the number of picture elements per unit area, the number of photons and the aberrations present in every optical system. The concept of contrast vs. distance functions is used to compare the resolution achievable in confocal and wide-field fluorescence microscopes and the effect of a further reduction of the observable volume. In conclusio: (a) real optical systems will never be able to achieve the theoretical resolution, (b) wide-field fluorescence microscopy will often provide a better resolution than confocal fluorescence microscopy, (c) decreasing the observed volume does not necessarily increase the resolution and (d) using multiple fluorophores can improve the accuracy with which distances are measured. Some numbers for typical situations are provided.

Confocal microscopy of single molecules of the green fluorescent protein

Abstract: Single molecule detection has been extended into life sciences by use of strongly fluorescent labels. The green fluorescent protein (GFP) as a self-fluorescent biomolecule has attracted considerable attention. Here, single molecules of the GFP-mutant Glu222Gln are immobilized in a polyvinylalcohol matrix and detected by confocal fluorescence microscopy. Although this mutant stabilizes one of both conformers of the wild-type GFP, the investigation of its fluorescence dynamics reveals strong signal fluctuations. This fluorescence behaviour is—at least partly—caused by reversible photochemical changes of the protein framework, that can relax into the fluorescent state on different timescales. Thus, this protein appears particularly appropriate for studying the microheterogeneity of the macromolecule GFP on a single molecule level.

Fluorescence correlation microscopy (FCM)-fluorescence correlation spectroscopy (FCS) taken into the cell.

Abstract: Confocal fluorescence correlation spectroscopy (FCS) and other confocal spectroscopic techniques are ideally suited for the analysis of molecular interactions at the subcellular level. However, one requires exact positioning in three dimensions within the cell. Our instrument integrates FCS with high sensitivity digital imaging microscopy and high precision positioning. We present first measurements of intracellular FCS, with specification of the instrumental requirements and methods of data analysis. We propose the term fluorescence correlation microscopy (FCM) for this extended modality of FCS.

Computational and optical methods for improving resolution and signal quality in fluorescence microscopy

Abstract: We present efficient algorithms for image restoration using the maximum a posteriori (MAP) method. Assuming Gaussian or Poisson statistics of the noise and either a Gaussian or an entropy prior distribution for the image, corresponding functionals are formulated and minimized to produce MAP estimations. Efficient algorithms are presented for finding the minimum of these functionals in the presence of non-negativity and support constraints. Performance was tested by using simulated three-dimensional (3D) imaging with a fluorescence confocal laser scanning microscope. Results are compared with those from two existing algorithms for superresolution in fluorescence imaging. An example is given of the restoration of a 3D confocal image of a biological specimen.

Principles and Application of Fluorescence Microscopy

Abstract: This overview discusses the principle of fluorescence along with practical discussions of fluorescent molecular probes, filters and filter sets, multiband filters and multi-dye fluorescence, light sources, objective lenses, image resolution and the point-spread function, fluorescence microscopy of living cells, and immunolabeling.

Nuclear ultrastructure : transmission electron microscopy and image analysis

Abstract: Publisher Summary This chapter provides an overview to both standard and advanced methods for the sample preparation, staining, and structural analysis of nuclear architecture using transmission electron microscopy (TEM). It describes standard TEM techniques or specific three-dimensional reconstruction methodologies. The applications of light microscopy are severalfold. A very powerful application, especially in situations in which in vivo observations are feasible, is the use of light microscopy as a check on sample preparation conditions. In principle, requiring that the fixation and sample preparation conditions used for a TEM project at least preserve the appearance of the sample as observed at lower resolution by light microscopy is a natural and obvious quality check. In practice, this type of control is rarely performed, and many studies of nuclear and chromosome structure have used buffer conditions that produce striking alterations in ultrastructure, obvious even at light microscopy resolutions. The availability of a variety of fluorescent stains specific for various cell organelles and macromolecules, and the recent introduction of the green fluorescent protein (GFP) as a fluorescent epitope tag for the in vivo imaging of specific proteins, facilitates this approach.

Optically sectioned images in wide-field fluorescence microscopy

Abstract: An analysis is presented of the image formation in widefield fluorescence microscopy using standard light The region of support of the resulting optical transfer function is discussed

Life at 10/sup 10/ W/cm/sup 2/: low-damage microscopy in living specimens using multi-photon microscopy

Abstract: Multi-photon excitation microscopy (Denk, Strickler et al. 1990) provides optical sectioning by excitation confinement alone and therefore allows fluorescence imaging of biological samples with minimal photodamage. Generation of fluorescence requires molecular excitation, which for organic chromophores is always accompanied by the possibility of photochemical side reactions. Such reactions can lead to the destruction of the fluorophore itself (photobleaching) or to damage to surrounding biological molecules (photodynamic damage). Photobleaching and photodynamic damage can often be reduced by the removal of oxygen, which is, however, often incompatible with biological viability. Because only molecules in the focal plane are excited within the multi-photon microscope no spatial discrimination is necessary during the detection process leading to efficient utilization of fluorescence photons. This is different from the confocal microscope, which is very wasteful with fluorescence photons and, in particular for thicker samples, uses only a small fraction of the total fluorescence generated.