Aberration control in quantitative imaging of botanical specimens by multidimensional fluorescence microscopy

Background:Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole ovary can be observed by using 3D confocal microscopy.Methods:Whole ovaries were stained with LysoTracker Red (LT), fixed with 4% paraformaldehyde (PF) and 1% glutaraldehyde (Glut), dehydrated with methanol (MEOH), and cleared with benzyl alcohol and benzyl benzoate (BABB). Using this tissue preparation technique, the ovary becomes relatively transparent, allowing its morphology to be observed with confocal microscopes. A spectral imaging system (PARISS) located on a conventional microscope was used to interpret the LT dye spectra and fixation products in the tissues with different excitation wavelengths.Results:Apoptosis in the follicle was detected as clusters of intensely stained granulosa cells located in close proximity to the oocytes. The fixation with Glut and PF preserved morphological details, increased tissue fluorescence, thus increased the signal to noise of the background image.Conclusions:Thick tissues can be imaged after they are properly stained, aldehyde fixed, and BABB cleared. LT intensely stained single cells or clusters of apoptotic cells in the follicles and the nucleolus. Spectral differences between LT as an indicator of apoptosis and Glut‐PF fixation was used to visualize ovarian morphology and apoptosis. The PARISS spectrophotometer revealed spectral peaks for LT at 609.6 nm and for Glut‐PF at 471.3 nm. The proper use of the spectra from these fluorescence molecules is the foundation for high quality morphological images of apoptosis. By sequentially imaging the two probes with a 488 nm laser and a 543/568 nm laser, there was a reduction in fluorescent cross talk and an increase in image quality. © 2006 International Society for Analytical Cytology

Section: Discussionmentioning

confidence: 99%

Confocal laser scanning microscopy of whole mouse ovaries: Excellent morphology, apoptosis detection, and spectroscopy

Zucker

Jeffay

2006

“…By using a confocal laser microscope instead of a normal fluorescent microscope, the laser light can penetrate deeper into the tissue for better visualization. However, since the tissue is dense and not transparent, the light becomes scattered, reflected, refracted, and absorbed in the process of entering the tissue and passing back through the tissue into the objective (9,16). This results in decreased laser intensity and decreased optical quality as one penetrates deeper inside the tissue.…”

Section: Babb Clearingmentioning

confidence: 99%

“…After clearing, the resulting tissue is nearly transparent, and the refraction, reflection, scattering, and absorption of laser light are greatly reduced (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). This sample preparation procedure has effectively increased the laser penetration and minimized light scatter, refraction, and absorption by matching the refractive index of the tissue with the suspending medium (9,16). Other dyes (i.e.…”

mentioning

confidence: 99%

Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis

Zucker

2006

Background:After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three‐dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays.Methods:The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB.Results:Tissues as thick as 500 μm can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide).Conclusions:The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red‐emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were ∼500‐μm thick. © 2006 International Society for Analytical Cytology

“…To cope with reduced axial resolution and focus shifting with depth in confocal microscopy (16,17), the definition of the comet head was based on an ellipsoid geometry as a function of focal depth. Starting from the middle, the line projection of pixel intensities at Z max is evaluated in both directions for the location of the first zero pixels.…”

Section: Image Processingmentioning

confidence: 99%

“…These are related to the light distribution in the observed volume, which is affected by the distance of the plane of observation from the coverslip and the refractive index of the gel. Because the light distribution close to the coverslip medium transition is considered perfect, any deviation results in a loss of intensity, resolution, and a shift of focus (16,17). The ellipsoid head described as a function of focal depth allows the system to cope with the focus shift that would otherwise result in the overestimation of the comet tail due to the increased sampling rate deeper in the gel.…”

Section: Vertical Cometsmentioning

confidence: 99%

Miniaturizing the comet assay with 3D vertical comets

Baert

Oostveldt

2002

BackgroundThe comet or single‐cell gel electrophoresis assay is a sensitive method for the detection of DNA damage. The main drawback of comet sampling is the low cell density necessary to prevent nucleus overlap after electrophoresis, which limits large‐scale high throughput screening. Another problem may be inconsistent comet focusing. We investigated whether an approach based on three‐dimensional (3D) confocal microscopy might be beneficial for these concerns.MethodsA vertical comet assay enabling three‐dimensional confocal comet imaging of nuclei seeded at very high density was developed together with dedicated software algorithms to retrieve quantitative data at the single cell level.ResultsThree‐dimensional confocal comet imaging greatly relieved the user interactions of our nonautomated two‐dimensional comet sampling procedure. Batches of comets were blindly sampled, and confocal sectioning improved the clarity of the images and the accuracy of comet sampling. A 1‐Gy dose response was readily established. The sampling speed was competitive with that of commercial packages.ConclusionsVertical comet imaging is a new concept for fast and user‐friendly comet sampling that allows miniaturization of the assay. It may become an essential step toward high throughput screening and exploit the benefits of confocal imaging. Cytometry Part A 51A:26–34, 2003. © 2002 Wiley‐Liss, Inc.