Small Animal In vivo Imaging FAQs

• Degree of Labeling (DOL) describes the number of fluorophores per antibody.   For in vivo labeling experiments, the DOL is restricted to a narrow range because it has significant consequences for the biodistribution and clearance of the probe.  We have determined that the DOL range for the NIR Alexa Fluor dyes is 1.5 to 3 molecules per antibody for optimal optical in vivo imaging.
  
  

• Antibodies should be in PBS buffer at a concentration of 0.5-3.0 mg/ml. The antibody must be free of preservatives (azide etc.), amine containing buffers and carrier proteins such as BSA.

• Due to light scattering caused by fur, hairless mice such as athymic nude (nu/nu) mice are recommended for in vivo imaging. If  this is not an option, the hair covering the area to be imaged should be removed using a clippers or chemical depilatory such as Nair™.
  
  

• The volume of reagent that can be injected varies according to the route of administration. (The following numbers are for a 25 gram animal)

What size needle should I use?

• We recommend the use of a 28-32 gauge tuberculin or insulin syringe (0.3 or 1.0 ml volume) with a fixed (non-removable) needle.
  
  

What dosage of QTracker non-targeted should I inject to image vasculature?

• A recommended starting dosage is 25-50 µl of QTracker diluted to the desired injection volume with PBS. QTracker should be diluted immediately prior to injection. DO NOT STORE DILUTED. The end user will need to determine the optimal dosage for their experimental models.
  
  

What dosage of conjugated antibody should I inject to image tumors?

• A recommended starting dosage is 50µg. The end user will need to determine the optimal dosage for their experimental models.
  
  

How do I excite quantum dots for in vivo imaging?

• Quantum dots can be excited by wavelengths of  light ranging from 410nm to 40nm below the emission peak wavelength. For example, QTracker 655 non-targeted quantum dots can be excited from 410-615 nm. Remember that the wavelength in the product name refers to the emission peak, not the excitation peak.
  
  

What happens to unconjugated dye in the mouse?

• Hydrolyzed dye will be eliminated via the bladder. The bladder signal is detectable within 3 minutes of IV injection.
  
  

How long after injection should I start imaging my mouse?

• The imaging time course varies with the nature of the injected agent. Vascular tracers are visible in the blood vessels immediately after injection and may be imaged for several hours. Conjugated whole IgG antibodies reach their targets within a few hours of injection and may be imaged for several days.
  
  

What are the fluorescent lifetimes of the Alexa Fluor dyes?

• The fluorescence lifetime (τ) of the Alexa Fluor 647 dye in H2O at 20°C is 1.0 nanoseconds and 1.5 nanoseconds in EtOH.
• The fluorescence lifetime (τ) of the Alexa Fluor 680 dye in pH 7.5 buffer at 20°C is 1.2 nanoseconds.
• The fluorescence lifetime (τ) of the Alexa Fluor 750 dye in H2O at 22°C is 0.7 nanoseconds.