530-Isobe-SpheroidCellPhotoconversion/Fig9a_BfConv_3D
Author reviewedZ-series images of x-y cross-sectional EpH4-spheroids expressing nucleus-localized PSmOrange2 before photostimulation of target cells
Dataset ID
Fig9a_BfConv_3D
Kind
Image data
Title
Z-series images of x-y cross-sectional EpH4-spheroids expressing nucleus-localized PSmOrange2 before photostimulation of target cells
Description
Z-series images of a living EpH4 cell spheroid expressing nucleus-localized PSmOrange2, acquired before photoconversion through two-photon laser-scanning microscopy system (2P-LSM).
The spheroids were from living mouse mammary epithelial EpH4 cells expressing PSmOrange2. EpH4 cells are a spontaneously immortalized, non-tumorigenic mouse mammary epithelial cell line.
PSmOrange2 here is a monomeric orange-to-far-red photoconvertible fluorescent protein used for optical labeling and tracking of selected EpH4 cells through photoconversion.
The images after photoconversion were included in the image dataset: Fig9a_AfConv_3D.
Folder Name
Fig9a_BfConv_3D
File Format
tif
File Size
42MB
File Separation Method
-
Figure Number
Figure9a
Dimensions
512x512x80x1x1
X Scale
0.586 micrometer
Y Scale
0.586 micrometer
Z Scale
1.0 micrometer
T Scale
-
Channel
1 channel
Quantitative Data
-
Paper(s)
Biosample
Description
EpH4 mouse mammary epithelial cells expressing nucleus-localized PSmOrange2 were cultured in Matrigel for 8–12 days to form three-dimensional spheroids. PSmOrange2 is a monomeric fluorescent protein for photoconversion from orange to red fluorescence.
Organism
Mus musculus
Strain
-
Cell
mammary gland epithelial cell (CL:0002327)
Cell line
EpH4
MeSH
Intrinsic Variable
-
Extrinsic Variable
Spheroids were generated from genetically modified EpH4 mouse mammary epithelial cells expressing nucleus-localized PSmOrange2. PSmOrange2 serves as a photoconvertible fluorescent protein tag fused to H2B.
Ontology
Anatomical Entity
-
UBERON
mammary gland (UBERON:0001911)
epithelium of mammary gland (UBERON:0003244)
Biological Process
cell growth (GO:0016049)
Cellular Component
nucleus (GO:0005634)
Molecular Function
-
MeSH Anatomy
MeSH BP
MeSH CC
MeSH MF
Genetics
Gene
-
Protein / Probe
human histone H2B
Method / Genotype
transfection
Treatments
Reagent / Compound
-
Concentration
-
Fold Dilution
-
Imaging Method
Imaging Method
Detection Method
photomultiplier tube (PMT)
Visualization Method
PSmOrange2
Illumination Method
single point scanning
Source of Contrast
Distribution of the orange form of PSmOrange2
Imaged Parameter
fluorescence emission
Sample Preparation Method
living cells
MeSH
Instrument
Body
Custom-built two-photon laser-scanning microscope, 2P-LSM
Module
Two-photon laser-scanning fluorescence imaging module
Light Source
As a laser source, a cutom-built Yb-doped fiber chirped pulse amplification (CPA) laser system, which produced a 108 fs pulse at a repetition rate of 7.0 MHz, was utilized.
Before patterned illumination, the 3D fluorescence image of the spheroids was acquired with the 2P-LSM system at an excitation wavelength of 1060 nm for the orange form of PSmOrange2.
Sufficiently low laser power (3.3 mW) was used for imaging to avoid the photoconversion of PSmOrange2 at a two-photon excitation wavelength of 1060 nm.
Detector
Hamamatsu H15460-40 PMT
Objective
Nikon MRD77225, 25× water immersion
Filter Set
Sigma Koki SHPF-25C-790
Dichroic
Sigma Koki DIMNQ-5172R03-P-R785-R565-T1325