Showing papers on "Photoactivated localization microscopy published in 2001"

Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes

Abstract: We report on the generation of various hole-centered beams in the focal region of a lens and investigate their effectiveness to break the diffraction barrier in fluorescence microscopy by stimulated emission. Patterning of the phase of the stimulating beam across the entrance pupil of the objective lens produces point-spread-functions with twofold, fourfold, and circular symmetry, which narrow down the focal spot to 65--100 nm. Comparison with high-resolution confocal images exhibits a resolution much beyond the diffraction barrier. Particles that are only 65-nm apart are resolved with focused light.

Cell biology beyond the diffraction limit: near-field scanning optical microscopy.

Abstract: Throughout the years, fluorescence microscopy has proven to be an extremely versatile tool for cell biologists to study live cells. Its high sensitivity and non-invasiveness, together with the ever-growing spectrum of sophisticated fluorescent indicators, ensure that it will continue to have a prominent role in the future. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution - similar to 250 urn - dictated by the laws of diffraction. The challenge to break this diffraction limit has led to the development of several novel imaging techniques. o­ne of them, near-field scanning optical microscopy (NSOM), allows fluorescence imaging at a resolution of o­nly a few tens of nanometers and, because of the extremely small near-field excitation volume, reduces background fluorescence from the cytoplasm to the extent that single-molecule detection sensitivity becomes within reach. NSOM allows detection of individual fluorescent proteins as part of multimolecular complexes o­n the surface of fixed cells, and similar results should be achievable under physiological conditions in the near future

Methods in cellular imaging

Abstract: PART 1: BASICS OF FLUORESCENCE, FLUROPHORES, MICROSCOPY AND DETECTORS 1. Basics of Fluorescence 2. Flurophores and Their Labelling Procedures for Monitoring Various Biological Signals 3. Detectors for Fluorescence Microscopy 4. Basics of a Light Microscopy Imaging System and Its Application in Biology 5. Laser Scanning Confocal Microscopy Applied to Living Cells and Tissues 6. Functional Imaging of Mitochondria Within Cells 7. Diffusion Measurements by Fluorescence Recovery After Photobleaching 8. Processing Microscope-Acquired Images for Use In Multimedia, Print, and the World Wide Web PART 2: MULTIPHOTON EXCITATION FLUROSCENCE MICROSCOPY 9. Basic Principles of Multiphoton Excitation Microscopy 10. Building a Two-Photon Microscope Using a Laser Scanned Confocal Architecture 11. Two-Photon Microscopy in Highly Scattering Tissue 12. Multiphoton Laser Scanning Microscopy and Dynamic Imaging in Embryos 13. In vivo Diffusion Measurements Using Multiphoton Excitation Fluorescence Photobleaching Recovery and Fluorescence Correlation Spectroscopy 14. Cellular Response to Laser Radiation in Fluorescence Microscopes PART 3: FLUORESCENCE RESONANCE ENERGY TRANSFER AND LIFETIME IMAGING MICROSCOPY 15. Measurement of Fluorescence Resonance Energy Transfer in the Optical Microscope 16. Frequency-Domain Fluorescence Lifetime Imaging Microscopy: A Window on the Biochemical Landscape of the Cell 17. Wide-Field, Confocal, Two-Photon, and Lifetime Resonance Energy Transfer Imaging Microscopy 18. One- and Two-Photon Confocal Fluorescence Lifetime Imaging and Its Applications 19. Biological Applications of Time-Resolved, Pump-Probe Fluorescence Microscopy and Spectroscopy in the Frequency Domain PART 4: OTHER ADVANCED METHODS IN CELLULAR IMAGING 20. Spectral Microscopy for Quantitative Cell and Tissue Imaging 21. Total Internal Reflection Fluorescence Microscopy 22. Laser Traps in Cell Biology and Biophysics 23. Bioluminescence Imaging of Gene Expression in Living Cells and Tissues 24. Imaging Living Cells and Mapping Their Surface Molecules with the Atomic Force Microscope INDEX

Fluorescence resonance energy transfer microscopy: a mini review.

Abstract: Fluorescence resonance energy transfer (FRET) microscopy is a better method than the x-ray diffraction, nuclear magnetic resonance, or electron microscopy for studying the structure and localization of proteins under physiological conditions. In this paper, we describe four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBPalpha) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides two-dimensional spatial distribution of steady-state protein-protein interactions. The two-photon imaging technique provides a better FRET signal (less bleedthrough and photobleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. The fluorescence lifetime methods allow us to monitor FRET signals at the moment of the protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution. This paper will provide a brief review of the above-mentioned FRET techniques.

Correlative Fluorescence and Electron Microscopy on Ultrathin Cryosections: Bridging the Resolution Gap

Abstract: SUMMARY Microscopy has become increasingly important for analysis of cells and cell function in recent years. This is due in large part to advances in light microscopy that facilitate quantitative studies and improve imaging of living cells. Analysis of fluorescence signals has often been a key feature in these advances. Such studies involve a number of techniques, including imaging of fluorescently labeled proteins in living cells, single-cell physiological experiments using fluorescent indicator probes, and immunofluorescence localization. The importance of fluorescence microscopy notwithstanding, there are instances in which electron microscopy provides unique information about cell structure and function. Correlative microscopy in which a fluorescence signal is reconciled with a signal from the electron microscope is an additional tool that can provide powerful information for cellular analysis. Here we review two different methodologies for correlative fluorescence and electron microscopy using ultrathin cryosections and the advantages attendant

Two-photon Fluorescence Light Microscopy

Abstract: Two-photon fluorescence microscopy allows three-dimensional imaging of biological specimens in vivo. Compared with confocal microscopy, it offers the advantages of deeper tissue penetration and less photodamage but has the disadvantage of slightly lower resolution. Keywords: two-photon; microscopy; tissue optics; fluorescence

Multiple-objective microscopy with three-dimensional resolution near 100 nm and a long working distance.

Abstract: The resolution of microscopes is limited by the sizes of their point-spread functions. The invention of confocal, theta, and 4Pi microscopes has permitted the classic Abbe limit to be exceeded. We propose the use of a combination of 4Pi and theta microscopy to decrease resolution by using four illumination objectives and two detection objectives. Using middle numerical aperture, long-working-distance objectives yielded a resolution near 100 nm in the three dimensions, which opens the possibility of exploring large volumes with a high resolution.

Transmitted light fluorescence microscopy revisited.

Abstract: From its introduction in 1967 by Ploem (1), reflected light fluorescence microscopy, commonly called “epi-fluorescence,” has enjoyed wide acceptance. Its optical path is relatively simple: full-spectrum light passing through an excitation filter is reflected by the dichromatic mirror into the objective lens to illuminate the sample; the excited sample emits fluorescent light, which is re-collected by the objective lens and passed through the emission filter to the camera. The recent development of biosensors based on genetically encoded variants of green fluorescent protein (GFP), coupled with advances in digital, multi-modes, epi-fluorescence microscopy, has introduced new powerful tools for observing protein dynamics and proteinprotein interactions at high spatial and temporal resolution within living cells. However, there are some disadvantages inherent in epifluorescence microscopy: a) mechanical switching of filter cubes to

Multi-Wavelength Epi-Illumination in Fluorescence Microscopy

Abstract: Fluorescence is a process where a substance after having absorbed light (photons) emitts a radiation the wavelength (colour) of which is longer than that of the absorbed light, and where this emission stops immediately after cessation of the excitation. This phenomenon is the basic element of fluorescence microscopy and its application. Besides the "classical” excitation of fluorescence in a light microscope it is possible today to obtain the same emission effect via the modern technology of confocal laser scanning microscopy by an excitation with two or multiple photons having longer wavengths than those of the emitted ones. Fluorescence occurs either as autofluorescenc of biological and/or inorganic structures or as so called secondary fluorescence after a treatment of the specimen with special dyes (fluorochromes, fluorescent markers). To perform fluorescence in a microscope the following requirements have to be met: powerful energy sources (highpressure mercury arc lamp, halogen lamp, etc.), adequate transmission filter systems (filterblocks) selecting the excitation light and the emitted radiation perfectly, and, last but not least, optical parts and outfits suitable for fluorescence, i.e. collector lenses, illuminators, beamsplitters, objectives, tube lenses and eyepieces. Compared with the situation of today, fluorescence microscopy was for the first time applied with the use of transmitted light and darkfield microscopy. This was due to its limited rangers of application in those days. But with its increasing importance to histology, cytology, molecular biology and immuno-diagnosis the demand for a fundamental improvement of illumination and observation techniques came up more and more. This was the hour of birth of incident light fluorescence microscopy. During its course of nearly 40 years of application and development this technique became one of the basic tools for routine and research work in biology, medicine, science and industry. The progress in incident light fluorescence microscopy was particularly determined by the research work of Ploem and his function as trend setter and also by the forward strategy of Leitz resp. Leica Wetzlar in developing the optical instruments required. This course of development is described chronologically in the present contrbution. A synoptical table of fluorescence microscopy techniques gives relevant information about microscopes, objectives, fluorochromes, light sources and filter systems concerning the fields of application and the methods referring to this.

Biological Applications of Time-Resolved, Pump-Probe Fluorescence Microscopy and Spectroscopy in the Frequency Domain

Abstract: In biological applications of optical microscopy, technical developments often lead to novel imaging modalities with significant applications. For example, the development of confocal microscopy led to microscopic imaging with enhanced contrast (see Chapter 5), and the more recent development of two-photon fluorescence microscopy revolutionized fluorescence microscopy by providing an imaging modality capable of high image contrast, reduced photodamage, and exciting possibilities in controlling localized photochemical reactions in three dimensions (see Chapters 9-13).

Simultaneous Optical Coherence and Two-Photon Fluorescence Microscopy

Abstract: We demonstrate simultaneous non-fluorescence and fluorescence imaging with sub-micron resolution in tissues by combining optical coherence tomography (OCT) and two-photon-excited fluorescence (TPEF) microscopy