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Journal articles on the topic 'Confocal fluorescence microscopy'

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Published: 4 June 2021

Last updated: 24 January 2023

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1

Zhibin Wang, Zhibin Wang, Guohua Shi Guohua Shi, and Yudong Zhang Yudong Zhang. "Adaptive aberration correction in confocal scanning fluorescence microscopy." Chinese Optics Letters 12, s1 (2014): S11103–311105. http://dx.doi.org/10.3788/col201412.s11103.

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2

Volkov, I. A., N. V. Frigo, L. F. Znamenskaya, and O. R. Katunina. "Application of Confocal Laser Scanning Microscopy in Biology and Medicine." Vestnik dermatologii i venerologii 90, no. 1 (February 24, 2014): 17–24. http://dx.doi.org/10.25208/0042-4609-2014-90-1-17-24.

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Fluorescence confocal laser scanning microscopy and reflectance confocal laser scanning microscopy are up-to-date highend study methods. Confocal microscopy is used in cell biology and medicine. By using confocal microscopy, it is possible to study bioplasts and localization of protein molecules and other compounds relative to cell or tissue structures, and to monitor dynamic cell processes. Confocal microscopes enable layer-by-layer scanning of test items to create demonstrable 3D models. As compared to usual fluorescent microscopes, confocal microscopes are characterized by a higher contrast ratio and image definition.

3

Wright, S. J., J. S. Walker, H. Schatten, C. Simerly, J. J. McCarthy, and G. Schatten. "Confocal fluorescence microscopy with the tandem scanning light microscope." Journal of Cell Science 94, no. 4 (December 1, 1989): 617–24. http://dx.doi.org/10.1242/jcs.94.4.617.

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Applications of the tandem scanning confocal microscope (TSM) to fluorescence microscopy and its ability to resolve fluorescent biological structures are described. The TSM, in conjunction with a cooled charge-coupled device (cooled CCD) and conventional epifluorescence light source and filter sets, provided high-resolution, confocal data, so that different fluorescent cellular components were distinguished in three dimensions within the same cell. One of the unique features of the TSM is the ability to image fluorochromes excited by ultraviolet light (e.g. Hoechst, DAPI) in addition to fluorescein and rhodamine. Since the illumination is dim, photobleaching is insignificant and prolonged viewing of living specimens is possible. Series of optical sections taken in the Z-axis with the TSM were reproduced as stereo images and three-dimensional reconstructions. These data show that the TSM is potentially a powerful tool in fluorescence microscopy for determining three-dimensional relationships of complex structures within cells labeled with multiple fluorochromes.

4

Welzel, J., Raphaela Kästle, and Elke C. Sattler. "Fluorescence (Multiwave) Confocal Microscopy." Dermatologic Clinics 34, no. 4 (October 2016): 527–33. http://dx.doi.org/10.1016/j.det.2016.06.002.

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5

Nie, Shuming, Daniel T. Chiu, and Richard N. Zare. "Real-time observation of single molecules by confocal fluorescence microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 53 (August 13, 1995): 60–61. http://dx.doi.org/10.1017/s0424820100136672.

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The ability to detect, identify, and manipulate individual molecules offer exciting possibilities in many fields, including chemical analysis, materials research, and the biological sciences. A particularly powerful approach is to combine the exquisite sensitivity of laser-induced fluorescence and the spatial localization and imaging capabilities of diffraction-limited or near-field optical microscopes. Unlike scanning tunneling microscopy (STM) and atomic force microscopy (AFM), which lack molecular specificity, optical spectroscopy and microscopy techniques can be used for real-time monitoring and molecular identification at nanometer dimensions or in ultrasmall volumes.We report the use of confocal fluorescence microscopy coupled with a diffraction-limit laser beam and a high-efficiency photodiode for real-time detection of single fluorescent molecules in solution at room temperature. Rigler and Eigen have also demonstrated single-molecule detection with a confocal microscope and fluorescence correlation spectroscopy. The probe (or sampling) volume is effectively an elongated cylinder, with its radius being determined by optical diffraction and length by spherical aberration.

6

Jason Kirk. "Beyond the Hype - Is 2-Photon Microscopy Right for You?" Microscopy Today 11, no. 2 (April 2003): 26–29. http://dx.doi.org/10.1017/s1551929500052469.

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Confocal microscopes have come a long way in the past decade. Not only are they more stable and easier to use than ever before, but their cost has dropped enough that multi-user facilities are finding competition from individual labs using the new breed of "personal" confocals. In fact it has, in some cases, become the de facto standard for fluorescence imaging regardless of whether the user actually has requirements for it or not.But, researchers always have an ear out for something better. Enter 2-photon microscopy (2PLSM). The “bigger & badder” cousin of the confocal microscope has become a new weapon in the arsenal of a microscopy industry that caters to researchers who can't wait to fill their labs with the latest and greatest imaging systems. Advertised by the industry and researchers alike as a technique that seems to solve most of the problems that plague confocal, “2-photon” has become the new buzzword in the vocabulary of researchers eager to enhance their fluorescence applications.

7

Cheng, P. C., S. J. Pan, A. Shih, W. S. Liou, M. S. Park, T. Watson, J. Bhawalkar, and P. Prasard. "Two-Photon Laser Scanning Confocal Microscopy." Microscopy and Microanalysis 3, S2 (August 1997): 847–48. http://dx.doi.org/10.1017/s1431927600011120.

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Two-photon fluorescence microscopy has become an important research tool in both biological and material sciences. The technique uses long wavelength, typically in the near IR, as the excitation light to obtain shorter wavelength fluorescence (e.g. visible light). Because of the low linear absorption coefficient of most biological and polymeric specimens, this technique allows deeper penetration of the excitation beam, achieving optical sectioning to a depth of 250μm or more into the specimen. As a result of the quadratic dependency of the two-photon induced fluorescence to the excitation intensity, the fluorescent emission and photobleaching are limited to the vicinity of focal spot. This capability of addressing a specimen’s 3D space allows exciting possibilities in biological researches, such as 3D photobleaching recovery experiment.Two-photon confocal fluorescence microscopy is ideal for the study of thick biological and material specimen in 3D. For example, Figure 1 shows a three-dimensional isosurface rendered image of a vascular bundle from a maize stem.

8

Oostveldt, P., and S. Bauwens. "Quantitative fluorescence in confocal microscopy." Journal of Microscopy 158, no. 2 (May 1990): 121–32. http://dx.doi.org/10.1111/j.1365-2818.1990.tb02985.x.

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9

VISSCHER, K., G. J. BRAKENHOFF, and T. D. VISSER. "Fluorescence saturation in confocal microscopy." Journal of Microscopy 175, no. 2 (August 1994): 162–65. http://dx.doi.org/10.1111/j.1365-2818.1994.tb03479.x.

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10

Ragazzi, Moira, Simonetta Piana, Caterina Longo, Fabio Castagnetti, Monica Foroni, Guglielmo Ferrari, Giorgio Gardini, and Giovanni Pellacani. "Fluorescence confocal microscopy for pathologists." Modern Pathology 27, no. 3 (September 13, 2013): 460–71. http://dx.doi.org/10.1038/modpathol.2013.158.

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11

Heintzmann, R., and C. Cremer. "Axial tomographic confocal fluorescence microscopy." Journal of Microscopy 206, no. 1 (April 2002): 7–23. http://dx.doi.org/10.1046/j.1365-2818.2002.01000.x.

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12

Raarup, Merete Krog, and Jens Randel Nyengaard. "QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY." Image Analysis & Stereology 25, no. 3 (May 3, 2011): 111. http://dx.doi.org/10.5566/ias.v25.p111-120.

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This paper discusses recent advances in confocal laser scanning microscopy (CLSM) for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm) tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging Microscopy), FCS (Fluorescence Correlation Spectroscopy) and FRAP (Fluorescence Recovery After Photobleaching) are introduced and their applicability for quantitative imaging of biomolecular (co-)localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

13

Sanai, Nader, Laura A. Snyder, Norissa J. Honea, Stephen W. Coons, Jennifer M. Eschbacher, Kris A. Smith, and Robert F. Spetzler. "Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas." Journal of Neurosurgery 115, no. 4 (October 2011): 740–48. http://dx.doi.org/10.3171/2011.6.jns11252.

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Object Greater extent of resection (EOR) for patients with low-grade glioma (LGG) corresponds with improved clinical outcome, yet remains a central challenge to the neurosurgical oncologist. Although 5-aminolevulinic acid (5-ALA)–induced tumor fluorescence is a strategy that can improve EOR in gliomas, only glioblastomas routinely fluoresce following 5-ALA administration. Intraoperative confocal microscopy adapts conventional confocal technology to a handheld probe that provides real-time fluorescent imaging at up to 1000× magnification. The authors report a combined approach in which intraoperative confocal microscopy is used to visualize 5-ALA tumor fluorescence in LGGs during the course of microsurgical resection. Methods Following 5-ALA administration, patients with newly diagnosed LGG underwent microsurgical resection. Intraoperative confocal microscopy was conducted at the following points: 1) initial encounter with the tumor; 2) the midpoint of tumor resection; and 3) the presumed brain-tumor interface. Histopathological analysis of these sites correlated tumor infiltration with intraoperative cellular tumor fluorescence. Results Ten consecutive patients with WHO Grades I and II gliomas underwent microsurgical resection with 5-ALA and intraoperative confocal microscopy. Macroscopic tumor fluorescence was not evident in any patient. However, in each case, intraoperative confocal microscopy identified tumor fluorescence at a cellular level, a finding that corresponded to tumor infiltration on matched histological analyses. Conclusions Intraoperative confocal microscopy can visualize cellular 5-ALA–induced tumor fluorescence within LGGs and at the brain-tumor interface. To assess the clinical value of 5-ALA for high-grade gliomas in conjunction with neuronavigation, and for LGGs in combination with intraoperative confocal microscopy and neuronavigation, a Phase IIIa randomized placebo-controlled trial (BALANCE) is underway at the authors' institution.

14

Suzuki, Takeshi, Keiko Fujikura, Tetsuya Higashiyama, and Kuniaki Takata. "DNA Staining for Fluorescence and Laser Confocal Microscopy." Journal of Histochemistry & Cytochemistry 45, no. 1 (January 1997): 49–53. http://dx.doi.org/10.1177/002215549704500107.

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We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treatment completely prevented the cytoplasmic RNA staining. In conventional fluorescence microscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and rhodamine (or Texas red).

15

Dolber, P. C., and M. S. Spach. "Conventional and confocal fluorescence microscopy of collagen fibers in the heart." Journal of Histochemistry & Cytochemistry 41, no. 3 (March 1993): 465–69. http://dx.doi.org/10.1177/41.3.7679127.

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The arrangement of collagen fibers has previously been studied with picrosirius red (PSR) staining and brightfield microscopy. We discovered that PSR staining can also be visualized by fluorescence microscopy. PSR-stained collagen was strongly fluorescent using excitation and barrier filters for rhodamine, and distracting background cytoplasmic fluorescence was drastically reduced with phosphomolybdic acid (PMA) treatment before PSR staining. The PMA-PSR fluorescence method was more sensitive than the brightfield PSR or PMA-PSR method, and permitted confocal microscopic study. We applied the method to the study of collagen fiber three-dimensional arrangement in perimysial and endomysial septa of the heart, showing the three-dimensional course of the fibers in stereo views generated by confocal microscopy. The PMA-PSR fluorescence method should be generally useful for accurately determining collagen fiber three-dimensional arrangement, a necessary prelude to mechanical modeling of collagen-reinforced tissues.

16

Wessendorf, Martin W., and T. Clark Brelje. "Multicolor Fluorescence Microscopy Using the Laser-Scanning Confocal Microscope." Neuroprotocols 2, no. 2 (April 1993): 121–40. http://dx.doi.org/10.1006/ncmn.1993.1017.

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17

Hell, Stefan W., Pekka E. Hanninen, Martin Schrader, Tony Wilson, and Erkki Soini. "Resolution beyond the diffraction limit: 4PI-confocal-, STED-, and GSD- fluorescence microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 53 (August 13, 1995): 56–57. http://dx.doi.org/10.1017/s0424820100136659.

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In far-field light microscopy resolution is determined by diffraction. In a far-field light microscope such as the confocal scanning light microscope, the resolution is governed by the extent of the squared intensity distribution in the focal region. Precise measurements of the confocal PSF have shown that the axial and lateral resolution of a confocal microscope (NA=1.4 oil, 1= 633 nm) is 520nm and 200nm (FWHM), respectively. At a wavelength of 375nm, this amounts to a resolution of 300 nm (axial) and 120 nm (lateral), obtainable with a standard confocal microscope of high aperture.A 3-7 fold increase in axial resolution is achieved with a 4Pi-confocal microscope. The 4Piconfocal microscope uses two high numerical aperture objective lenses that are used coherently for illuminating or detecting the same point in the object space. The present paper deals with the latest developments in the field of 4Pi-confocal microscopy. The Optical Transfer Functions (OTF) of 4Piconfocal microscopies with 4Pi-illumination (type A), 4Pi-detection (type B), and 4Pi illumination and detection (type C) are measured and compared with their standard confocal counterpart.

18

Yu Wang, Konstantin Maslov, Chulhong Kim, Song Hu, and Lihong V. Wang. "Integrated Photoacoustic and Fluorescence Confocal Microscopy." IEEE Transactions on Biomedical Engineering 57, no. 10 (October 2010): 2576–78. http://dx.doi.org/10.1109/tbme.2010.2059026.

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19

Sheppard, C. J. R. "Axial resolution of confocal fluorescence microscopy." Journal of Microscopy 154, no. 3 (June 1989): 237–41. http://dx.doi.org/10.1111/j.1365-2818.1989.tb00586.x.

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20

Pedley, Kevin C. "Applications of Confocal and Fluorescence Microscopy." Digestion 58, no. 2 (1997): 62–68. http://dx.doi.org/10.1159/000201546.

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21

Hepler, Peter K., and Brian E. S. Gunning. "Confocal fluorescence microscopy of plant cells." Protoplasma 201, no. 3-4 (September 1998): 121–57. http://dx.doi.org/10.1007/bf01287411.

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22

Deerinck, Thomas J., Maryann E. Martone, Varda Lev-Ram, David P. L. Green, Roger Y. Tsien, David L. Spector, Sui Huang, and Mark H. Ellisman. "3-Dimensional immunolabeling and in situ hybridization detection using fluorescence photooxidation and intermediate-voltage Electron Microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 52 (1994): 164–65. http://dx.doi.org/10.1017/s0424820100168554.

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The confocal laser scanning microscope has become a powerful tool in the study of the 3-dimensional distribution of proteins and specific nucleic acid sequences in cells and tissues. This is also proving to be true for a new generation of high contrast intermediate voltage electron microscopes (IVEM). Until recently, the number of labeling techniques that could be employed to allow examination of the same sample with both confocal and IVEM was rather limited. One method that can be used to take full advantage of these two technologies is fluorescence photooxidation. Specimens are labeled by a fluorescent dye and viewed with confocal microscopy followed by fluorescence photooxidation of diaminobenzidine (DAB). In this technique, a fluorescent dye is used to photooxidize DAB into an osmiophilic reaction product that can be subsequently visualized with the electron microscope. The precise reaction mechanism by which the photooxidation occurs is not known but evidence suggests that the radiationless transfer of energy from the excited-state dye molecule undergoing the phenomenon of intersystem crossing leads to the formation of reactive oxygen species such as singlet oxygen. It is this reactive oxygen that is likely crucial in the photooxidation of DAB.

23

Fritzky, Luke, and David Lagunoff. "Advanced Methods in Fluorescence Microscopy." Analytical Cellular Pathology 36, no. 1-2 (2013): 5–17. http://dx.doi.org/10.1155/2013/569326.

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It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

24

White, J. G., W. B. Amos, and M. Fordham. "An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy." Journal of Cell Biology 105, no. 1 (July 1, 1987): 41–48. http://dx.doi.org/10.1083/jcb.105.1.41.

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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.

25

W. Piston, David. "Two-Photon Excitation Imaging of Glucose Metabolism in Living Tissue." Microscopy and Microanalysis 3, S2 (August 1997): 305–6. http://dx.doi.org/10.1017/s1431927600008412.

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Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. It provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter, whose absence enhances fluorescence collection efficiency. This results in inherent submicron optical sectioning by excitation alone. In practice, TPEM is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy. Because of the intensity-squared dependence of the two-photon absorption, the excitation is limited to the focal volume.

26

Keith, Charles H., and Mark A. Farmer. "Visualization of the Microtubules of Glutaraldehyde-Fixed Cells by Reflection-Enhanced Backscatter Confocal Microscopy." Microscopy and Microanalysis 12, no. 2 (December 9, 2005): 113–23. http://dx.doi.org/10.1017/s1431927606060016.

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Performing reflection-mode (backscatter-mode) confocal microscopy on cells growing on reflective substrates gives images that have improved contrast and are more easily interpreted than standard reflection-mode confocal micrographs (Keith et al., 1998). However, a number of factors degrade the quality of images taken with the highest-resolution microscope objectives in this technique. We here describe modifications to reflection-enhanced backscatter confocal microscopy that (partially) overcome these factors. With these modifications of the technique, it is possible to visualize structures the size—and refractility—of individual microtubules in intact cells. Additionally, we demonstrate that this technique, in common with fluorescence techniques such as standing wave widefield fluorescence microscopy and 4-Pi confocal microscopy, offers improved resolution in the Z-direction.

27

Stelzer, Ernst H. K., and Steffen Lindek. "3D Microscopy using Confocal Microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 270–71. http://dx.doi.org/10.1017/s0424820100163812.

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The importance of confocal fluorescence microscopy in modem biological research results from its optical sectioning capability, which allows the three-dimensional analysis of thick specimens. This property is due to the combination of a point-like light source and a point-like detector, which restrict the illumination and detection volumes, respectively. Only the volume that is illuminated and detected is relevant to the confocal observation volume. The smaller it is, the better is the resolution. The performance of a confocal microscope is thus primarily specified by the spatial extent of the confocal point spread function (PSF). The extent can be estimated, e.g., by the volume enclosed by the isosurface at half maximum of the PSF (VHM – volume at half maximum).The relationship of the parameters that determine the lateral resolution of a microscope has been described by Ernst Abbé. The diameter of a light spot in the focal plane Δx is proportional to the wavelength λ of the incident light and inversely proportional to the numerical aperture of the optical system (N.A. = n, ∙ sin α).

28

Martin, Sonya, Antonio Virgilio Failla, Udo Spöri, Christoph Cremer, and Ana Pombo. "Measuring the Size of Biological Nanostructures with Spatially Modulated Illumination Microscopy." Molecular Biology of the Cell 15, no. 5 (May 2004): 2449–55. http://dx.doi.org/10.1091/mbc.e04-01-0045.

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Spatially modulated illumination fluorescence microscopy can in theory measure the sizes of objects with a diameter ranging between 10 and 200 nm and has allowed accurate size measurement of subresolution fluorescent beads (∼40–100 nm). Biological structures in this size range have so far been measured by electron microscopy. Here, we have labeled sites containing the active, hyperphosphorylated form of RNA polymerase II in the nucleus of HeLa cells by using the antibody H5. The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting. The hyperphosphorylated form of polymerase II was found in structures with a diameter of ∼70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.

29

Mikami, Hideharu, Jeffrey Harmon, Hirofumi Kobayashi, Syed Hamad, Yisen Wang, Osamu Iwata, Kengo Suzuki, et al. "Ultrafast confocal fluorescence microscopy beyond the fluorescence lifetime limit." Optica 5, no. 2 (January 29, 2018): 117. http://dx.doi.org/10.1364/optica.5.000117.

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Wang, Chun-Yang, Jui-che Tsai, Ching-Cheng Chuang, Yao-Sheng Hsieh, and Chia-Wei Sun. "Aorta Fluorescence Imaging by Using Confocal Microscopy." ISRN Cardiology 2011 (July 9, 2011): 1–7. http://dx.doi.org/10.5402/2011/215627.

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The activated leukocyte attacked the vascular endothelium and the associated increase in VEcadherin number was observed in experiments. The confocal microscopic system with a prism-based wavelength filter was used for multiwavelength fluorescence measurement. Multiwavelength fluorescence imaging based on the VEcadherin within the aorta segment of a rat was achieved. The confocal microscopic system capable of fluorescence detection of cardiovascular tissue is a useful tool for measuring the biological properties in clinical applications.

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Shavlokhova, Veronika, Christa Flechtenmacher, Sameena Sandhu, Michael Vollmer, Andreas Vollmer, Maximilian Pilz, Jürgen Hoffmann, Oliver Ristow, Michael Engel, and Christian Freudlsperger. "Feasibility and Implementation of Ex Vivo Fluorescence Confocal Microscopy for Diagnosis of Oral Leukoplakia: Preliminary Study." Diagnostics 11, no. 6 (May 26, 2021): 951. http://dx.doi.org/10.3390/diagnostics11060951.

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Background: Oral leukoplakia is a potentially malignant lesion with a clinical impression similar to different benign and malignant lesions. Ex vivo fluorescence confocal microscopy is a developing approach for a rapid “chairside” detection of oral lesions with a cellular-level resolution. A possible application of interest is a quick differentiation of benign oral pathology from normal or cancerous tissue. The aim of this study was to analyze the sensitivity and specificity of ex vivo fluorescence confocal microscopy (FCM) for detecting oral leukoplakia and to compare confocal images with gold-standard histopathology. Methods: Imaging of 106 submosaics of 27 oral lesions was performed using an ex vivo fluorescence confocal microscope immediately after excision. Every confocal image was qualitatively assessed for presence or absence of leukoplakia by an expert reader of confocal images. The results were compared to conventional histopathology with H&E staining. Results: Leukoplakia was detected with an overall sensitivity of 96.3%, specificity of 92.3%, positive predictive value of 93%, and negative predictive value of 96%. Conclusion: The results demonstrate the potential of ex vivo confocal microscopy in fresh tissue for rapid real-time assessment of oral pathologies.

32

Jovin, Thomas M., Michel Robert-Nicoud, Donna J. Arndt-Jovin, and Thorsten Schormann. "3-D imaging of cells using a confocal laser scanning microscope and digital image processing." Proceedings, annual meeting, Electron Microscopy Society of America 46 (1988): 96–97. http://dx.doi.org/10.1017/s0424820100102560.

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Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

33

Piston, David W. "Two-photon excitation fluorescence microscopy in living systems." Proceedings, annual meeting, Electron Microscopy Society of America 51 (August 1, 1993): 154–55. http://dx.doi.org/10.1017/s0424820100146618.

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Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

34

Masters, Barry R. "Three-dimensional confocal microscopy in living cells: historical development, practical application and limitations." Proceedings, annual meeting, Electron Microscopy Society of America 50, no. 2 (August 1992): 1120–21. http://dx.doi.org/10.1017/s0424820100130237.

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Confocal microscopy is a rapidly evolving technique which is solving problems in the biological and material sciences. This tutorial focuses on the confocal microscopy of living cells. Both in vivo and in vitro applications of confocal microscopy will be reviewed. Applications of confocal microscopy of the eye will illustrate the concepts. Fluorescence and back scattered confocal microscopy are critically reviewed. A bibliography on confocal microscopy is given to aid the users of this technique.Books (Optical Theory, Image Formation, Fluorescence Techniques) •Theory and Practice of Scanning Optical Microscopy, (eds. T. Wilson, C. Sheppard), Academic Press, London, 1984.•Confocal Microscopy, (ed. by T. Wilson), Academic Press, London, 1990.•Handbook of Biological Confocal Microscopy, (ed. J.B. Pawley), Plenum Press, New York, 1989.•New Techniques of Optical Microscopy and Microspectroscopy (ed. R.J. Cherry), CRC Press, Inc., Boca Raton, Florida, 1991.•Noninvasive Techniques in Cell Biology, (eds. J.K. Foskett, S. Grinstein), Wiley-Liss, New York, 1990.

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Cakir-Aktas, Canan, Sefik Evren Erdener, Büşra Teke, Sibel Bozdag Pehlivan, Naciye Dilara Zeybek, Aslihan Taskiran-Sag, Zeynep Kaya, Turgay Dalkara, and Melike Mut. "Confocal reflectance microscopy for metal and lipid nanoparticle visualization in the brain." Nanomedicine 17, no. 7 (March 2022): 447–60. http://dx.doi.org/10.2217/nnm-2021-0350.

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Aim: A requirement for nanoparticle (NP) research is visualization of particles within cells and tissues. Limitations of electron microscopy and low yields of NP fluorescent tagging warrant the identification of alternative imaging techniques. Method: Confocal reflectance microscopy (CRM) in combination with fluorescence imaging was assessed for visualizing rhodamine B-conjugated silver and fluorescein isothiocyanate-conjugated lipid core-stearylamine NP uptake in vitro and in vivo. Results: CRM successfully identified cellular uptake and blood–brain barrier penetration of NPs owing to their distinguishing refractive indices. NP-dependent reflectance signals in vitro were dose and incubation time dependent. Finally, CRM facilitated the distinction between nonspecific fluorescence signals and NPs. Conclusion: These findings demonstrate the value of CRM for NP visualization in tissues, which can be performed with a standard confocal microscope.

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Aguilar, Alberto, Adeline Boyreau, and Pierre Bon. "Label-free super-resolution imaging below 90-nm using photon-reassignment." Open Research Europe 1 (March 24, 2021): 3. http://dx.doi.org/10.12688/openreseurope.13066.1.

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Background: Achieving resolutions below 100 nm is key for many fields, including biology and nanomaterial characterization. Although nearfield and electron microscopy are the gold standards for studying the nanoscale, optical microscopy has seen its resolution drastically improve in the last decades. So-called super-resolution microscopy is generally based on fluorescence photophysics and requires modification of the sample at least by adding fluorescent tags, an inevitably invasive step. Therefore, it remains very challenging and rewarding to achieve optical resolutions beyond the diffraction limit in label-free samples. Methods: Here, we present a breakthrough to unlock label-free 3D super-resolution imaging of any object including living biological samples. It is based on optical photon-reassignment in confocal reflectance imaging mode. Results: We demonstrate that we surpass the resolution of all fluorescence-based confocal systems by a factor ~1.5. We have obtained images with a 3D (x,y,z) optical resolution of (86x86x248) nm3 using a visible wavelength (445 nm) and a regular microscope objective (NA=1.3). The results are presented on nanoparticles as well as on (living) biological samples. Conclusions: This cost-effective approach double the resolution of reflectance confocal microscope with minimal modifications. It is therefore compatible with any microscope and sample, works in real-time, and does not require any signal processing.

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Cannell, M. B., and C. Soeller. "Optical Sectioning in Fluorescence Microscopy by Confocal and 2-Photon Molecular Excitation Techniques." Microscopy Today 5, no. 8 (October 1997): 12–15. http://dx.doi.org/10.1017/s1551929500056741.

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Fluorescence microscopy has proved to be an invaluable tool for biomedical science since it is possible to visualise small quantities of labeled materials (such as intracellular ions and proteins) in both fixed and living cells, However, the conventional wide field fluorescence microscope suffers from the disadvantage that objects outside the focal plane also fluoresce (in response to the excitation light) and this leads to a marked loss of contrast for objects in the focal plane, This is especially a problem when the fluorescent probe is distributed throughout the thickness of the cell and the cell is thicker than about 1 µm. The confocal microscope overcomes this problem by illuminating the preparation with a point source of excitation light and limiting the collection of light with a pinhole that is confocal with the illumination source.

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Shaw, Peter, Alison Beven, David Rawlins, and Martin Highett. "Confocal microscopy, image restoration, and nuclear structure." Proceedings, annual meeting, Electron Microscopy Society of America 51 (August 1, 1993): 146–47. http://dx.doi.org/10.1017/s0424820100146576.

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Fluorescent in situ hybridization and 3-D confocal microscopy have been used to study the arrangement of genes and transcripts in interphase plant nuclei. Wide-field fluorescence microscopy coupled with computer deconvolution has also been used on the same specimens and gives comparable results to confocal imaging. When image deconvolution is applied to confocal images, these show a significant improvement. A comparison of confocal and wide-field (CCD) images of the same specimen (a plant nucleolus labelled with probe to the rRNA genes) is shown in Figure 1. The deconvoluted wide-field data (Fig 1b) and the raw confocal data (Fig 1c) are remarkably similar; the former is somewhat clearer. The deconvoluted confocal data (Fig 1d) is clearest of all.Double-stranded DNA and single-stranded RNA probes to the 45S and 5S RNA genes have been used to investigate the functional organization of the nucleolus in pea root tissue. In situ hybridization was carried out on vibratome slices approximately 50μm thick (about 3 cells).

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Kwon, Y. H., K. S. Wells, and H. C. Hoch. "Fluorescence Confocal Microscopy: Applications in Fungal Cytology." Mycologia 85, no. 5 (September 1993): 721. http://dx.doi.org/10.2307/3760603.

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NAKANISHI, Mamoru. "Confocal fluorescence microscopy for studying immune respones." Seibutsu Butsuri 34, no. 1 (1994): 1–5. http://dx.doi.org/10.2142/biophys.34.1.

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Puliatti*, Stefano, Laura Bertoni, Luca Reggiani Bonetti, Antonino Maiorana, Ahmed Eissa, Paola Azzoni, Ahmed Zoheir, et al. "PD54-12 EX-VIVO FLUORESCENCE CONFOCAL MICROSCOPY." Journal of Urology 203 (April 2020): e1108-e1109. http://dx.doi.org/10.1097/ju.0000000000000956.012.

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Kwon, Y. H., K. S. Wells, and H. C. Hoch. "Fluorescence Confocal Microscopy: Applications in Fungal Cytology." Mycologia 85, no. 5 (September 1993): 721–33. http://dx.doi.org/10.1080/00275514.1993.12026326.

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Lindek, Steffen, Christoph Cremer, and Ernst H. K. Stelzer. "Confocal theta fluorescence microscopy with annular apertures." Applied Optics 35, no. 1 (January 1, 1996): 126. http://dx.doi.org/10.1364/ao.35.000126.

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Thiele, Jan Christoph, Dominic A. Helmerich, Nazar Oleksiievets, Roman Tsukanov, Eugenia Butkevich, Markus Sauer, Oleksii Nevskyi, and Jörg Enderlein. "Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy." ACS Nano 14, no. 10 (October 9, 2020): 14190–200. http://dx.doi.org/10.1021/acsnano.0c07322.

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Nie, S., D. Chiu, and R. Zare. "Probing individual molecules with confocal fluorescence microscopy." Science 266, no. 5187 (November 11, 1994): 1018–21. http://dx.doi.org/10.1126/science.7973650.

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Földes-Papp, Zeno, Ulrike Demel, and Gernot P. Tilz. "Laser scanning confocal fluorescence microscopy: an overview." International Immunopharmacology 3, no. 13-14 (December 2003): 1715–29. http://dx.doi.org/10.1016/s1567-5769(03)00140-1.

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Jonkman, James. "Rigor and Reproducibility in Confocal Fluorescence Microscopy." Cytometry Part A 97, no. 2 (November 18, 2019): 113–15. http://dx.doi.org/10.1002/cyto.a.23924.

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Chattoraj, Shyamtanu, and Kankan Bhattacharyya. "Biological oscillations: Fluorescence monitoring by confocal microscopy." Chemical Physics Letters 660 (September 2016): 1–10. http://dx.doi.org/10.1016/j.cplett.2016.07.007.

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Wright, S. J., and G. Schatten. "Confocal fluorescence microscopy and three-dimensional reconstruction." Journal of Electron Microscopy Technique 18, no. 1 (May 1991): 2–10. http://dx.doi.org/10.1002/jemt.1060180103.

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Pedroso, M. Cristina, Michael B. Sinclair, Howland D. T. Jones, and David M. Haaland. "Hyperspectral Confocal Fluorescence Microscope: A New Look into the Cell." Microscopy Today 18, no. 5 (August 24, 2010): 14–18. http://dx.doi.org/10.1017/s1551929510000854.

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Confocal microscopy is widely used in cell biology. Like other filter-based systems, traditional confocal microscopes are limited by the spectral bands established by each optical filter. As a result, emission spectra from labels and/or autofluorescence can be overlapped leading to spectral crosstalk and inability to quantify the amount of signal originating from each individual fluorescent species. The need for accurate quantification of in vivo cellular processes and in-depth knowledge of organelle development and microstructure led Monsanto to search for non-commercial microscopes that could achieve those goals. Through a cooperative research and development agreement (CRADA) established between Monsanto and Sandia Corporation in August 2006, we built a new 3D-hyperspectral confocal fluorescence imaging system, specifically designed to meet the analytical requirements of plant specimens.