Confocal microscopy reveals uniform male reproductive anatomy in eriophyoid mites (Acariformes, Eriophyoidea) including spermatophore pump and paired vasa deferentia

2015 ◽  
Vol 66 (4) ◽  
pp. 555-574 ◽  
Author(s):  
Philipp E. Chetverikov
Zootaxa ◽  
2015 ◽  
Vol 3919 (1) ◽  
pp. 179 ◽  
Author(s):  
PHILIPP E. CHETVERIKOV ◽  
ALEXEY G. DESNITSKIY ◽  
DENISE NAVIA

Due to the higher resolution, confocal microscopy (CLSM) can be applied to refine the origin of tiny structures of the autofluorescent exoskeletons of microarthropods (mites in particular) which are hard to visualize using traditional differential interference contract light microscopy (DIC LM) and phase contrast light microscopy (PC LM). Three-dimensional (3D) reconstructions of the prodorsal shield topography of eriophyoid mites using Neoprothrix hibiscus Reis and Navia as a model, suggest that the structures originally treated as paired setae vi are two internal rod-like apodemes. Based on this, the genus Neoprothrix is excluded from the subfamily Prothricinae Amrine and transferred to the subfamily Sierraphytoptinae Keifer. Observations on partially cleared specimens of N. hibiscus showed that remnants of the central nervous system, paired glands and developing oocytes can be visualized using DIC LM and CLSM methods. New high quality microscope images are provided of recently described “flower-shaped” structures and two main components of yolk inclusions of the mature eggs inside the oviduct. 


Author(s):  
David W. Piston ◽  
Brian D. Bennett ◽  
Robert G. Summers

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. 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 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.


Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.


Author(s):  
W.F. Marshall ◽  
K. Oegema ◽  
J. Nunnari ◽  
A.F. Straight ◽  
D.A. Agard ◽  
...  

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.


Author(s):  
J. Holy ◽  
G. Schatten

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.


Author(s):  
David W. Piston

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.


1998 ◽  
Vol 189 (1) ◽  
pp. 98-99
Author(s):  
Alan Entwistle
Keyword(s):  

Author(s):  
Richa Choudhary ◽  
Rishikant Sinha

Objectives: Hysterosalpingography and laparoscopy both are the diagnostic methods for assessment of female infertility.  The present study was to compare the evaluation of hysterosalpingography (HSG) versus laparoscopy in determination of tubal factors in female infertility. Methods: Detailed assessment, physical examination and clinical investigations were performed in all 100 infertile female with age 20 years to 40 years. All patients were advised to perform digital HSG. Patients with an abnormal HSG underwent laparoscopy without delay, whereas in patients with a normal HSG, laparoscopy was performed three months after HSG. HSG is best scheduled during the 2nd -5th day interval immediately following the end of menstruation, to minimize risk for infection, avoid interference from intrauterine blood and clot, and to prevent any possibility that the procedure might be performed after conception. Results: Data was analysed by using IBM SPSS version 23 software.  All data was tabulated and percentages were calculated. Mean ± standard deviation was observed. Conclusions: Diagnostic laparoscopy is the gold standard in diagnosing tubal pathology and other intra-abdominal causes of infertility. Other hand, Hysterosalpingography is a frequently utilized diagnostic tool in the assessment of tubal status and detection of uterine anatomical defects in infertility. Hysterosalpingography and laparoscopy are not alternatives but complimentary investigations. But, inadequacy of hysterosalpingography (HSG) in determining the state of tubal patency, emphasizes the need for laparoscopy. Laparoscopy provides both a panoramic view of the pelvic reproductive anatomy and a magnified view of pelvic organs and peritoneal surfaces. Keywords: Female infertility, Tubal patency, HSG, Laparoscopy


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