In vivo fluorescence imaging: a personal perspective

Schematic of organic versus quantum dot–based contrast agents used for in vivo fluorescence imaging.

Absorption spectra of oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) in whole blood. Spectra are displayed in units of absorption extinction coefficient (M^-1 cm^-1) versus wavelength (nm) and are plotted on a log scale. The NIR window corresponds to a spectral region from 650 to 1000 nm where there is a nadir in the physiological absorption of light by endogenous chromophores. Utilizing exogenous NIR fluorescent contrast agents enables effective deep-tissue in vivo imaging.

Schematic depiction of the incorporation of various oligo(porphyrin)-based NIRFs within polymersomes. (a) The NIRFs vary with respect to the number of porphyrin subunits (N), the linkage topology between porphyrin monomers, and the nature and position of ancillary aryl-group substitutents (R). (b) Various diblock copolymer compositions have been utilized to form NIR-emissive polymersomes. (c) Membranous interactions between polymers and specific ancillary aryl group substituents vary the conformational populations assumed by the NIRF and can be used to tune its emission wavelengths. (d) Engineering the chemical composition and thickness of the polymersome membrane (d) helps to drive individual NIRFs into dielectric environments of matching polarity. (e) a family of nanoscale NIR-emissive polymersomes.

Schematic of the liquid tissue phantom arrangement. Imaging experiments on human breast phantoms were done with a GE eXplore Optix imaging instrument. Tris(porphyrinato)zinc(II) (PZn3)-based NIRFs (incorporated within aqueous suspensions of OB-18 polymersomes composed of a 1 : 100 molar ratio of NIRF-to-polymer) and equivalent numbers of ICG (dissolved in the same total volume of dilute bovine albumin solution) were utilized in order to quantify the tissue depths at which optical signals could be accurately detected for a given number of fluorophores. Samples were excited and emission was detected in the reflectance geometry; the liquid absorption/scattering media consisted of India ink in an intralipid solution (µa = 0.04 cm^-1, µs′ = 10.0 cm^-1 at λ = 785 nm).

Determination of fluorophore optical signal strength versus depth of tissue penetration. (a) Attenuation of optical signal sensitivity (signal-to-noise ratio; SNR) versus depth of immersion in the liquid phantom for various amounts of albumin-bound ICG emitters. (b) Attenuation of SNR versus depth of immersion in the liquid phantom for various amounts of PZn3-based NIRFs (incorporated within aqueous suspensions of OB-18 polymersomes composed of a 1 : 100 molar ratio of NIRF-to-polymer). (c) Comparison of the maximum tissue depth of fluorescence signal penetration versus number of albumin-bound ICG and polymersome-incorporated PZn3 emitters in solution. NIRF solutions were embedded within liquid tissue phantoms and compared on an emitter-to-emitter basis. Experimental conditions: emissive signals emanating from the liquid tissue phantoms were detected by NIR fluorescence imaging utilizing a GE eXplore Optix instrument (λex = 785 nm, λem = 830–900 nm).

In vivo fluorescence imaging experiments with nontargeted NIR-emissive polymersomes. (a) Image of a tumor-bearing mouse (in the supine position) taken 6 h after tail-vein injection of nontargeted, PZn3-based, NIR-emissive polymersomes. The 150-nm diameter polymersomes do not accumulate in the lungs. As such, they neither aggregate in vivo, as is often witnessed with quantum dots, nor do they cause any microvascular injury during their circulation, vide infra. (b) Images of a tumor-bearing mouse (in the prone position) taken 6 h after tail-vein injection of nontargeted, PZn3-based, NIR-emissive polymersomes. The 150-nm diameter polymersomes are able to preferentially accumulate and remain (for greater than 24 h) in tumor tissues as a result of the enhanced permeability and retention (EPR) effect. Experimental conditions: 1.5 nmol equivalent of the 3′,5′-alk-PZn3 NIRF loaded in 150-nm diameter nondegradable polymersomes (composed of a 1 : 1 molar mixture of PEO30-b-PBD46 and PEO80-b-PBD125; 1 : 40 NIRF-to-total-polymer molar ratio) were imaged after tail-vein injection of tumor-bearing mice utilizing a GE eXplore Optix instrument (λex = 785 nm, λem = 830–900 nm).