研究目的
To develop and validate a highly bright near-infrared BODIPY dye (BD) for enhanced in-vivo optical imaging of the inflammatory response to acute liver injury in C57Bl/6 mice, overcoming limitations of existing dyes in deep organ imaging.
研究成果
The BD dye significantly outperforms the CD dye in brightness, signal-to-noise ratio, and low cytotoxicity, enabling enhanced in-vivo imaging of inflammatory cell recruitment in deep-seated organs like the liver in immunocompetent mice. Its molecular structure and membrane localization contribute to superior optical properties, facilitating precise cell tracking and opening new possibilities for biomedical research and therapeutic assessment.
研究不足
The study is limited to acute liver injury models in C57BL/6 mice; applicability to chronic disease or other organ systems requires further validation. The dye's performance in multiplex imaging with other fluorophores is under investigation but not fully established. Tissue depth and pigmentation in wild-type mice remain challenges, though BD improves signal detection.
1：Experimental Design and Method Selection:
The study compared the performance of the novel BD dye with the gold standard CellVue Burgundy 710 (CD) dye for in-vivo cell tracking using IVIS and FACS. Theoretical models included TD-DFT calculations to understand the molecular orbitals and photophysical properties of BD.
2：Sample Selection and Data Sources:
C57BL/6 mice were used as the model organism. Neutrophils were isolated and labelled with either BD or CD dyes. Liver injury was induced by intraperitoneal administration of carbon tetrachloride (CCl4).
3：4). List of Experimental Equipment and Materials:
3. List of Experimental Equipment and Materials: Equipment included Shimadzu UV-1800 spectrophotometer, Shimadzu RF-6000 fluorimeter, PTI EasyLife apparatus for fluorescence lifetimes, Perkin Elmer IVIS Spectrum in-vivo imaging system, BD LSRFortessa? X-20 FACS system, Zeiss AxioImager upright microscope. Materials included BD dye, CD dye (CellVue Burgundy 710), solvents (cyclohexane, oleic acid, DMSO, Triton X-100), and reagents for cell labelling and fixation.
4：Experimental Procedures and Operational Workflow:
Mice were injured with CCl4, then injected intravenously with 10 million labelled neutrophils. After 2 hours, in-vivo IVIS imaging was performed under isoflurane anesthesia using specific excitation (675 nm) and emission (720 nm) filters. Mice were then sacrificed, livers harvested for ex-vivo imaging. FACS was used to assess cytotoxicity and fluorescence intensity. Confocal imaging was performed on fixed neutrophils.
5：Data Analysis Methods:
IVIS data were analyzed using Living Image 4.4 software by drawing regions of interest (ROI). FACS data were analyzed using FlowJo V10 software. Spectra deconvolution used PeakFit software. Statistical significance was determined using unpaired t-tests.
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UV-Vis Spectrophotometer
UV-1800
Shimadzu
Recording electronic absorption spectra at room temperature.
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Fluorimeter
RF-6000
Shimadzu
Acquiring fluorescence emission spectra at room temperature.
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In Vivo Imaging System
IVIS Spectrum
Perkin Elmer
Performing in-vivo and ex-vivo fluorescence imaging of mice and organs.
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Microscope
AxioImager upright
Zeiss
Confocal fluorescent imaging of fixed cells.
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Cell Labelling Dye
CellVue Burgundy 710
ThermoFisher Scientific
Gold standard lipophilic dye for cell membrane labelling, used as a comparator in IVIS experiments.
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Fluorescence Lifetime Apparatus
EasyLife
PTI
Collecting fluorescence lifetimes.
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Flow Cytometer
LSRFortessa X-20
BD Biosciences
Fluorescence-activated cell sorting (FACS) to separate and analyze fluorescently labelled cells.
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BODIPY Dye
BD
Novel super-bright membrane targeting dye for cell labelling and in-vivo imaging.
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