Supplementary Video S6 from A Prospective Double-Blinded Comparison of Reflectance Confocal Microscopy with Conventional Histopathology for <i>In Vivo</i> Assessment in Oral Cancer
Daniella K. ZanoniPaula Demétrio De Souza FrançaCristina ValeroGary PetersonMarco ArdigòRonald GhosseinStephen W. DuszaDanielli MatsuuraDaniel W. ScholfieldDauren AdilbayPablo H. MonteroJocelyn MigliacciNagavarakishore PillarsettyKıvanç KöseIan GanlyMilind RajadhyakshaSnehal G. Patel
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<p>Supplementary Video S6 shows an example of an enlarged blood vessel on a coronal view. Blood cells can be observed flowing inside the vessel.</p>Keywords:
Histopathology
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Confocal theta microscopy is a novel method by which the axial resolution in confocal fluorescence microscopy can be substantially improved. The basic idea is to observe the sample with two or more objective lenses and to detect the emission light at an angle theta to the illumination axis. The observation volume is considerably decreased when the detection axis is rotated by 90 degree(s) relative to the illumination axis. This leads to an almost spherical observation volume, which is three times smaller than in a comparable confocal fluorescence microscope. Confocal theta microscopy can be combined with 4Pi-confocal microscopy and is the first viable method proposed for fully exploiting the resolution increase achievable with 4Pi-techniques. The axial side lobes of the 4Pi-point spread function are suppressed, and the observation volume is reduced by a factor of 4. The resolution properties of confocal theta fluorescence microscopies are investigated for single- and two-photon absorption. Evaluations of confocal theta point spread functions are presented and the resolution improvement achieved by theta observation is discussed.
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Precise examination of the corneal endothelium has become increasingly important due to the growing number of intraocular and corneal procedures. The purpose of this study was to compare prospectively the corneal endothelial cell count in normal eyes obtained by confocal and specular microscopy. Central corneal endothelial cell densities of 42 eyes from 42 patients were measured by confocal and contact specular microscopy. Endothelial cells were analyzed with the same software in a manual, an automated and a semi-automated mode. The mean endothelial cell density obtained by confocal microscopy was (in the manual, automated and semi-automated modes) 3,069 ± 285, 2,791 ± 344 and 3,077 ± 286 cells/mm<sup>2</sup>, and obtained by specular microscopy 3,076 ± 298, 2,796 ± 271 and 3,082 ± 282 cells/m<sup>2</sup>, respectively. No statistically significant difference of endothelial cell density between confocal and specular microscopy was found. Endothelial cell count was significantly lower in the automated than in the semi-automated and manual analysis both with confocal and with specular microscopy. In conclusion, endothelial cell count measurements with confocal and contact specular microscopy are comparable.
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Re-scan confocal microscopy (RCM) is a new super-resolution technique based on a standard confocal microscope extended with a re-scan unit in the detection path that projects the emitted light onto a sensitive camera. In this paper the fundamental properties of RCM, lateral resolution, axial resolution and signal-to-noise ratio, are characterized and compared with properties of standard confocal microscopy. The results show that the lateral resolution of RCM is ~170 nm compared to ~240 nm of confocal microscopy for 488 nm excitation and 1.49 NA. As the theory predicts, this improved lateral resolution is independent of the pinhole diameter. In standard confocal microscopy, the same lateral resolution can only be achieved with an almost closed pinhole and, consequently, with a major loss of signal. We show that the sectioning capabilities of the standard confocal microscope are preserved in RCM and that the axial resolution of RCM is slightly better (~15%) than the standard confocal microscope. Furthermore, the signal-to-noise ratio in RCM is a factor of 2 higher than in standard confocal microscopy, also due to the use of highly sensitive modern cameras. In case the pinhole of a confocal microscope is adjusted in such way that the lateral resolution is comparable to that of RCM, the signal-to-noise ratio in RCM is 4 times higher than standard confocal microscopy. Therefore, RCM offers a good alternative to standard confocal microscopy for higher lateral resolution with the main advantage of strongly improved sensitivity.
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Confocal laser scanning microscopy has been widely utilized for real-time, cytochemical, or immunofluorescence image analysis in many studies using various types of biological samples. Recently developed fluorophores and fluorescent proteins with higher fluorescence intensities in narrower ranges of excitation and emission wavelengths give researchers more choices for selections of fluorescent tags and labeling combinations. This chapter provides brief introduction of confocal microscopy, autofluorescence with focus on aging brains, signal and noise ratio, and common fluorophore selections/combinations. In addition, this paper describes general procedures for confocal image analysis using live cells or fixed samples, using examples for (1) real-time imaging analysis of intracellular ROS response of Neuro-2A cells to specific treatments and (2) triple-labeling immunofluorescence confocal microscopy using mouse brain sections.
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