Guiding Principles of Specimen Preservation for Confocal Fluorescence Microscopy

Bacallao, Robert L.; Sohrab, Sadaf; Phillips, Carrie L.

doi:10.1007/978-0-387-45524-2_18

Cited by 57 publications

(37 citation statements)

References 57 publications

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“…Due to the low repetition rate of the laser (1 MHz), the pixel clock is roughly 15 microseconds, resulting in oversampling by our data acquisition card. For depth measurement, the slightly larger index of refraction in brain tissue (1.35 to 1.4 for the cortex 30,31 and as high as 1.467 for the white matter 31 ), relative to water (∼ 1.33), results in a slight underestimate (5–10%) of the actual imaging depth within the tissue because the imaging depths reported here are the raw axial movement of the objective. For image processing, a median filter with a 1 pixel radius was applied.…”

Section: Methodsmentioning

confidence: 89%

In vivo three-photon microscopy of subcortical structures within an intact mouse brain

et al. 2013

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Two-photon fluorescence microscopy (2PM)1 enables scientists in various fields including neuroscience2,3, embryology4, and oncology5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of 2PM within the mouse brain to the cortical layer, and imaging subcortical structures currently requires the removal of overlying brain tissue3 or the insertion of optical probes6,7. Here we demonstrate non-invasive, high resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue.

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Section: Methodsmentioning

confidence: 89%

In vivo three-photon microscopy of subcortical structures within an intact mouse brain

et al. 2013

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“…Cells were processed using Triton-X 100 (Roitbak et al ., 2004) or 1% SDS (Xu et al ., 2007), and primary and secondary antibody incubations were performed in a humidified chamber at 37°C. Primary NK1 cells immunostained for Rab11, PC1, and ASAP1 were fixed with 3% PFA or by the pH shift-fix method (Bacallao et al ., 2006), and labeled as previously described (Roitbak et al ., 2004). Cells costained with antibodies to α-ß-tubulin and to CD16 were first labeled for α-ß-tubulin and washed, then labeled with anti-CD16, and then washed with phosphate-buffered saline (PBS) and labeled with fluorescently tagged antibodies.…”

Section: Methodsmentioning

confidence: 99%

“…LSM 510 Image Acquisition software (Carl Zeiss) was used to acquire images. Specimens were mounted in Mowiol or 1,4-diazabicyclo[2.2.2]octane/glycerol (Bacallao et al ., 2006) mounting medium. Confocal Z-stacks were processed with the Zeiss Image Browser or Voxx2 (provided freely for noncommercial use by the Indiana Center for Biological Microscopy, Indianapolis, IN, www.nephrology.iupui.edu/imaging/voxx/index.htm), and assembled using Photoshop and Illustrator (Adobe, San Jose, CA).…”

Section: Methodsmentioning

confidence: 99%

A conserved signal and GTPase complex are required for the ciliary transport of polycystin-1

Ward

Brown-Glaberman

Wang

et al. 2011

MBoC

102

117

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“…Representative RI distributions of the live cells before processing, after PFA fixation and after the paraffin embedding removal process, are shown in Fig. 32,43,44 The average dry mass and volume of cells after the paraffin embedding removal process were reduced by 6.4% and 38.8%, respectively, compared to those of the live cells. The morphology of cells after PFA fixation showed no apparent alteration compared to that of the live cells.…”

Section: Preparation Of Human Oral Epithelial Tissuementioning

confidence: 99%

Investigation of influences of the paraformaldehyde fixation and paraffin embedding removal process on refractive indices and scattering properties of epithelial cells

Hsu

Tjiu

et al. 2014

J. Biomed. Opt

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The scattering properties and refractive indices (RI) of tissue are important parameters in tissue optics. These parameters can be determined from quantitative phase images of thin slices of tissue blocks. However, the changes in RI and structure of cells due to fixation and paraffin embedding might result in inaccuracies in the estimation of the scattering properties of tissue. In this study, three-dimensional RI distributions of cells were measured using digital holographic microtomography to obtain total scattering cross sections (TSCS) of the cells based on the first-order Born approximation. We investigated the slight loss of dry mass and drastic shrinkage of cells due to paraformaldehyde fixation and paraffin embedding removal processes. We propose a method to compensate for the correlated changes in volume and RI of cells. The results demonstrate that the TSCS of live cells can be estimated using restored cells. The percentage deviation of the TSCS between restored cells and live cells was only −8%. Spatially resolved RI and scattering coefficients of unprocessed oral epithelium ranged from 1.35 to 1.39 and from 100 to 450 cm−1, respectively, estimated from paraffinembedded oral epithelial tissue after restoration of RI and volume.

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Guiding Principles of Specimen Preservation for Confocal Fluorescence Microscopy

Cited by 57 publications

References 57 publications

In vivo three-photon microscopy of subcortical structures within an intact mouse brain

In vivo three-photon microscopy of subcortical structures within an intact mouse brain

A conserved signal and GTPase complex are required for the ciliary transport of polycystin-1

Investigation of influences of the paraformaldehyde fixation and paraffin embedding removal process on refractive indices and scattering properties of epithelial cells

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