In vitro and in vivo magnetic resonance imaging (MRI) detection of GFP through magnetization transfer contrast (MTC)

Abstract

Green fluorescent protein (GFP) is a widely utilized molecular marker of gene expression. However, its use in in vivo imaging has been restricted to transparent tissue mainly due to the tissue penetrance limitation of optical imaging. Here, we report a novel approach to detect GFP with Magnetization transfer contrast (MTC) magnetic resonance imaging (MRI). MTC is an MRI methodology currently utilized to detect macromolecule changes such as decrease in myelin and increase in collagen content. We employed MTC MRI imaging to detect GFP both in vitro and in in vivo mouse models. We demonstrated that our approach produces values that are protein specific, and concentration dependent. This approach provides a flexible, non-invasive in vivo molecular MRI imaging strategy that is dependent upon the presence and concentration of the GFP reporter.

Keywords: Green Fluorescent Protein (GFP); Magnetic Resonance Imaging (MRI); Magnetization Transfer; gene reporter.

Figure 1

Overview of Magnetization Transfer Contrast (MTC). Panel A shows the signal from protons associated with macromolecules. Panel B shows the signal from protons associated with the free water pool. When an RF pulse (blue wavyarrow) is applied off-frequency (red box), it leads to saturation of those macromolecular protons. Panel C is the saturated macromolecular pool. Notice the black dotted line drops in the area of the red square. Because this macromolecular pool is associated with the free water pool, the saturation can be transferred as is seen in Panel D.

Figure 2

GFP compared with BSA protein phantoms. For panels A-C top phantom is BSA and bottom phantom is GFP, both at a concentration of 0.1mg/mL. Panel A shows the unsaturated image while Panel B shows the saturated image at the 1 kHz offset frequency. Panel C is the pseudocolored pixel by pixel MTR calculation for the 1 kHz offset frequency. Panel D is also a pseudocolored pixel by pixel MTR calculation for the 1 kHz offset frequency of GFP at different concentrations. The central phantom is the initial 0.1 mg/mL concentration whereas the surrounding phantoms contain different GFP dilutions, indicated on the panel. Panel E shows the region based MTR calculations for the different frequency offsets and GFP concentrations. Significance was assessed using an ANOVA with a Dunnet post-test within each offset with ***p<0.001 and **p<0.01.

Figure 3

Ubiquitous expression GFP mouse model. Panels A and B are representative fluorescence images of the cortex and midbrain from GFP expressing (A) versus control (B) mice. Control sample (B) does not give appreciable signal at the settings used for GFP positive animals. Panel C shows the region based MTR calculations for the different frequency offsets for GFP vs. Control. Significance was assessed with a mixed model ANOVA followed by linear contrasts for each offset frequency with ***p<0.001, **p<0.01 and * p<0.05 (p=0.0113). Panels D and E contain the unsaturated images from GFP (D) and control (E) mice. The red outline depicts the region of interest for Panel C. Panel F and G show the pseudocolored pixel by pixel MTR calculation for 1 kHz offset from GFP (F) and Control (G) mice. Panel H is the subtraction of the MTR images in the form of GFP positive minus GFP negative MTR.

Figure 4

Cerebellar expression GFP mouse model. Panels A and B are representative fluorescence images of the cerebellum and brainstem from GFP expressing (A) versus control (B) mice. Control sample (B) does not give appreciable signal at the settings used for GFP positive animals. Panel C shows the region based MTR calculations for the different frequency offsets for GFP vs. Control. Significance was assessed a mixed model ANOVA followed by linear contrasts for each offset frequency with **p<0.01 and * p<0.05 (p= 0.0171 and 0.0321 for 1, and 10 kHz respectively). Panels D and E contain the unsaturated images from GFP (D) and control (E) mice. The red outline depicts the region of interest for Panel C. Panel F and G show the pseudocolored pixel by pixel MTR calculation for 1 kHz offset from GFP (F) and Control (G) mice. Yellow arrows point to the cerebellum while white arrows point to the brainstem.Panel H is the subtraction of the MTR images in the form of GFP positive minus GFP negative MTR.

Figure 5

Cortex expression GFP mouse model. Panels A and B are representative fluorescence images of the cortex and midbrain from GFP expressing (A) versus control (B) mice. The control sample (B) does not give appreciable signal at the settings used for GFP positive animals. Panel C shows the region based MTR calculations for the different frequency offsets for GFP vs. Control. Significance was assessed by a mixed model ANOVA followed by linear contrasts for each offset frequency with *** p<0.001. Panels D and E contain the unsaturated images from GFP (D) and control (E) mice. The red outline depicts the region of interest for Panel C. Panel F and G show the pseudocolored pixel by pixel MTR calculation for 1 kHz offset from GFP (F) and Control (G) mice. Panel H is the subtraction of the MTR images in the form of GFP positive minus GFP negative MTR. Yellow arrows point to the cortex while white arrows point to the midbrain. Panel H is the subtraction of the MTR images in the form of GFP positive minus GFP negative MTR.