Cyanine dyes are an important tool in biomedical research due to their fluorescent properties, which are particularly useful for in vivo imaging. This article details the preparation and experimental steps for using various cyanine dyes in mouse in vivo imaging.
Preparation of Cyanine Dyes
Cyanine dyes with different active groups are utilized to react with biomolecules or small molecule drugs possessing corresponding active groups. This results in the formation of conjugated compounds suitable for in vivo imaging. The specific linking of cyanine dyes to molecules is carried out as follows:
- Cyanine3 NHS ester is linked to drug molecules containing NH2 groups.
- Cyanine7 maleimide is linked to drug molecules or biomolecules with SH groups.
- Cyanine5 amine is linked to drug molecules or small molecules with NHS groups.
- Cyanine5.5 azide and Cyanine3.5 alkyne are used for linking DNA and oligonucleotides.
Experimental Steps
Animals
Subjects: SPF BALB/C nude mice, 6-8 weeks old, weighing 18-20 grams.
Conditioning: Mice are allowed free access to food and water 24 hours before the experiment.
Anesthesia
Agent: 2% sodium pentobarbital.
Dosage: Inject 300 μL (215 mg/kg) intraperitoneally to anesthetize the animals.
Imaging Setup
Positioning: Place the nude mice in a prone position within the recording darkroom of a small animal multispectral in vivo imaging system.
Injection: Dilute Cy7 or Cy7-labeled biomolecules or drugs with DMSO. Inject 200 μL (0.5 mg/g) into the tail vein of nude mice. Optimal dosage and timing need to be optimized based on the specific instruments and reagents used.
Imaging and Analysis
Recording: Capture an imaging picture of the animal emitting fluorescence in vivo every 5 minutes to analyze the distribution of fluorescent drugs.
Control Group: Use control mice not injected with drugs and record them simultaneously for comparison.
Dissection and Imaging: After recording, quickly dissect the heart, liver, spleen, lungs, kidneys, and other organs of the nude mice and perform imaging.
Detection Parameters
- Excitation Wavelength: 700-770 nm bandpass.
- Emission Wavelength: 790 nm longpass.
- Scanning Range: 780-950 nm with a scanning step of 10 nm.
- Exposure Time: 500 ms.
Metabolism and Fluorescence Distribution
Different drugs have varying metabolism times. Post-injection, fluorescence is initially distributed throughout the body, then gradually accumulates in the bladder, indicating significant renal excretion. Typically, this process takes 4 to 6 hours, although some cases may show distribution as quickly as 30 minutes. For Cy7-labeled drugs targeting bones or other specific parts, fluorescence can be detected for up to a week.
Fig. 1 Cyanine3/5/7-drug molecules or small molecule peptides are used in in vivo imaging experiments in mice.
Organ Slice Observation
Fixation: Place dissected organs in 4% paraformaldehyde for over 4 hours.
Slicing: Precipitate in 0%, 20%, and 30% PBS sucrose solutions. Cut 20 μm slices.
Mounting and Staining: Mount slices on polylysine-washed slides, dry, and stain with DAPI.
Microscopy: Use a confocal microscope with a helium-neon 633 nm laser for observation.
This detailed methodology ensures accurate and reproducible imaging of cyanine dye-labeled compounds in vivo, providing valuable insights into drug distribution and targeting within live animals.
