Practical Confocal Microscopy and the Evaluation of System Performance (original) (raw)
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Advanced microscopy: Laser scanning confocal microscopy
Methods in Molecular Biology, 2011
Fluorescence microscopy is an important and fundamental tool for biomedical research. Optical microscopy is almost non-invasive and allows highly spatially resolved images of organisms, cells, macromolecular complexes, and biomolecules to be obtained. Generally speaking, the architecture of the observed structures is not significantly modified and the environmental conditions can be kept very close to physiological reality. The development of fluorescence microscopy was revolutionized with the invention of laser scanning confocal microscopy (LSCM). With its unique three-dimensional representation and analysis capabilities, this technology gives us a more real view of the world.
Confocal laser scanning microscopy
Bios, Oxford; Springer, New York; Springer, Singapore, 1997
Confocal microscopy is a new science. While the idea for a confocal microscope was first patented by Minsky in 1957, and the first purely analogue mechanical confocal microscope was designed and produced by Eggar and Petran a decade later, it was not until the late seventies, with the advent of affordable computers and lasers, and the development of digital image processing software, that the first single-beam confocal laser scanning microscopes were developed in a number of laboratories and applied to biological and materials specimens. For biologists, a crucial turning point came in 1985 with the publication of six papers from four separate laboratories independently demonstrating the power of the confocal fluorescence microscope to eliminate out-of-focus blur, and thus to obtain three-dimensional (3D) data from intact biological specimens by non-invasive optical sectioning. In a remarkably rapid development, in which both of us were involved, the first commercial confocal laser scanning fluorescence microscopy systems were produced within 2 years of these publications by a small Oxfordshire company which has now become Bio-Rad Microscience Ltd. A number of other companies, including all the major microscope manufacturers worldwide, were swift to produce their own confocal instruments of widely varying designs and capabilities. During the last decade the availability of confocal laser scanning microscopes of ever-increasing power and sophistication has revolutionized the science of microscopy as applied to cell and develop- mental biology, physiology, cytogenetics, diagnostic pathology, and the material sciences. In particular, the ability to obtain a time-series of three-dimensional images from a living specimen, with temporal and spatial resolutions as good as or superior to video microscopy, has opened up new avenues of investigations previously impossible to contemplate. Over the last decade, several excellent books and reviews on confocal microscopy have been published, but there has been a noticeable gap in the availability of a small handbook that introduces the interested student or research worker to this important microscopical technique, and that illustrates how it might benefit their own research. It is this gap that we hope this Handbook, the latest in the Royal Microscopical Society Microscopy Handbooks series, will fill. Starting from first principles, this Handbook explains to the reader what a confocal microscope is, how it is constructed and used what its benefits. are, and. why i.ts imaging performance is superior to'that of a conventional optical mIcroscope. It discusses multiparameter confocal fluorescence microscopy, describes digital image processing and animation of 3D confocal images, illustrates applications of confocal microscopy in both the biomedical and the materials sciences and concludes with a discussion of future developments in this new area of microscopy. Richly illustrated with colour micrographs and diagrams chosen for their clarity and didactic quality, this book also contains an up-to-date bibliography of the most informative publications on confocal microscopy, a catalogue of World Wide Web sites of relevance, and a listing of the names and addresses of confocal microscope and fluorescence filter manufacturers, image processing software vendors, and reagent suppliers. We hope that you will find it useful.
How the Confocal Laser Scanning Microscope entered Biological Research
Biology of the Cell, 2003
A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge is presented. The rapid uptake of this technology is explained by the wide use of fluorescence in the 80s. The key innovations were the scanning of the light beam over the specimen rather than vice-versa and a high magnification at the level of the detector, allowing the use of a macroscopic iris. These were followed by an achromatic all-reflective relay system, a non-confocal transmission detector and novel software for control and basic image processing. This design was commercialized successfully and has been produced and developed over 17 years, surviving challenges from alternative technologies, including solid-state scanning systems. Lessons are pointed out from the unusual nature of the original funding and research environment. Attention is drawn to the slow adoption of the instrument in diagnostic medicine, despite promising applications.
The Journal of Cell Biology, 1987
Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.
LASER SCANNING CONFOCAL MICROSCOPY
Laser scanning confocal microscopy has become an invaluable tool for a wide range of investigations in the biological and medical sciences for imaging thin optical sections in living and fixed specimens ranging in thickness up to 100 micrometers. Modern instruments are equipped with 3-5 laser systems controlled by high-speed acousto-optic tunable filters (AOTFs), which allow very precise regulation of wavelength and excitation intensity. Coupled with photomultipliers that have high quantum efficiency in the near-ultraviolet, visible and near-infrared spectral regions, these microscopes are capable of examining fluorescence emission ranging from 400 to 750 nanometers. Instruments equipped with spectral imaging detection systems further refine the technique by enabling the examination and resolution of fluorophores with overlapping spectra as well as providing the ability to compensate for autofluorescence. Recent advances in fluorophore design have led to improved synthetic and naturally occurring molecular probes, including fluorescent proteins and quantum dots, which exhibit a high level of photostability and target specificity.
Resolution in the ApoTome and the confocal laser scanning microscope: comparison
Journal of Biomedical Optics, 2009
The essential feature of the confocal laser scanning microscope ͑cLSM͒ is the generation of optical sections by the removal of out-of-focus light. About ten years ago, structured illumination microscopy ͑SIM͒ was introduced as an alternative method for obtaining optical sections from biological specimens. Here we compare the resolution of the ApoTome ͑commercial SIM by Zeiss͒ to that achieved by a cLSM ͑Zeiss LSM 510͒. If fluorescent beads are used as test objects, then the ApoTome will achieve a lower axial resolution than the cLSM. In contrast to that, its lateral resolution scores slightly better. If subresolution homogeneous fluorescent layers are used as test objects, then the ApoTome will achieve a higher axial resolution than the cLSM. The ApoTome's axial resolution is homogeneous over the field-of-view while that of the cLSM changes markedly. Finally, the anisotropy of the ApoTome's resolution was found to be negligible for standard applications while its capability to resolve fine structures within stained tissue slices is limited to one or two cell layers and thus worse than in the cLSM.
Evaluation of confocal microscopy system performance
Methods in molecular biology (Clifton, N.J.), 2006
The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. When tests are made to evaluate the performance of a CLSM, the usual subjective assessment is accomplished by using a histological test slide to create a "pretty picture." Without the use of functional tests, many of the machines could be working at suboptimal performance levels, delivering suboptimum performance and possibly misleading data. To replace the subjectivity in evaluating a confocal microscope, tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, scanning stability, photomultiplier tube quality, overall machine stability, and system noise. These tests will help serve as a guide for other investigators to ensure that their machines are working correctly to provide data that are accurate with the necessary resolution, sensitivity, and precision. Util...
Power and limits of laser scanning confocal microscopy
Biology of the Cell, 1994
In confocal microscopy, the object is illuminated and observed so as to rid the resulting image of the light from out-offocus planes. Imaging may be performed in the reflective or in the fluorescence mode. Confocal microscopy allows accurate and nondestructive optical sectioning in a plane perpendicular or parallel to the optical axis of the microscope. Further digital three-dimensional treatments of the data may be performed so as to visualize the specimen from a variety of angles. Several examples illustrating each of these possibilities are given. Three-dimensional reconstitution of nuclear components using a cubic representation and a raytracing based method are also given. Instrumental and experimental factors can introduce some bias into the acquisition of the 3-D data set: self-shadowing effects of thick specimens, spherical aberrations due to the sub-optimum use of the objective lenses and photobleaching processes. This last phenomenon is the one that most heavily hampers the quantitative analysis needed for 3-D reconstruction. We delineate each of these problems and indicate to what extent they can be solved. Some tips are given for the practice of confocal microscope and image recovery: how to determine empirically the thickness of the optical slices, how to deal with extreme contrasts in an image, how to prevent artificial flattening of the specimens. Finally, future prospects in the field are outlined. Particular mention of the use of pulsed lasers is made as they may be an alternative to UV-lasers and a possible means to attenuate photodamage to biological specimens. confocal microscopy / fluorescence microscopy / reflectance microscopy / photobleaching / three-dimensional imaging 13th I n t e r n a t i o n a l C o n g r e s s on E l e c t r o n M i c r o s c o p y