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19F MRI Probes with Tunable Chemical Switches

  • Kazuya KikuchiEmail author
  • Tatsuya Nakamura
Open Access
Conference paper

Abstract

Activatable 19F MRI small molecule probes have been developed to detect calcium ion, pH change, enzyme activity etc. However, small molecule based probes could not be applicable to in vivo applications owing to low sensitivity. Though PFC encapsulated nanoparticle are highly sensitive, activatable PFC encapsulated nanoparticles (switching OFF/ON-type probes) have not been reported. Thus, activatable PFC nanoparticles are highly desirable in order to realize various applications.

This thesis describes herein the development of switching OFF/ON type nanoparticles probes for detecting biological environment and biological functions. To develop activatable 19F MRI probes, the author utilized FLAME as highly sensitive contrast agent and PRE effect as modulation of 19FNMR/MRI signals. The PRE effects of Gd3+ complexes was efficient for decreasing the 19F NMR/MRI signals of fluorine compounds in FLAME. Based on this finding, the author attempted to develop an activatable 19F MRI probes (switching OFF/ON type probes) for the detection of reducing environment.

7.1 Magnetic Resonance Imaging

MRI is the imaging technique based on nuclear magnetic resonance (NMR) phenomena. MRI offers high resolution, deep tissue imaging, and no radiation exposure (Louie et al. 2000). To acquire high contrast images, contrast agents such as Gd3+ complexes and superparamagnetic iron oxide nanoparticle (SPIO) are widely used in the field of clinical and research (Fig. 7.1) (Lee et al. 2008). Gd3+ complexes shorten the longitudinal relaxation time (T1), results in enhancement of MRI signals. SPIO shorten the tranverse relaxation time (T2), results in attenuation of MRI signal intensities. Figure 7.2 shows the switching OFF/ON type probes based on Gd3+ complexes and SPIO (Perez et al. 2002). However, 1H MRI often suffers from high background signals derived from water and lipid etc. Therefore, there is a limitation of monitoring of biological signals.
Fig. 7.1

(a) Clinically utilized T1 contrast agent, Dotarem®, and T1 relaxation. (b) Clinically utilized T2 contrast agent, Resovist, and T2 relaxation

Fig. 7.2

The switching OFF/ON type probes based on (a) Gd3+ complexes and (b) SPIO

Recently, heteronuclear MRI has been attracted considerable attentions as the alternative 1H MRI. Several non proton MRI such as 13C, 15N, 19F, 29Si, 31P, and 129Xe has been utilized in biological analysis (Table 7.1) (Cassidy et al. 2013). Among these non proton MRI, 19F MRI has considerable attentions, because fluorine has a 100% natural abundance and a high gyromagnetic ratio (Ahrens et al. 2005). In our bodies, there are a large amount of fluorine atoms in bones and teeth and almost no fluorine atoms in tissues. However, these fluorine atoms are immobilized in a solid state, exhibits very short T2 which results in invisible MRI. Therefore, the 19F MRI can acquire the image without the background signals.
Table 7.1

NMR observable nucleus and the sensitivity

Nuclei

Resonant frequency (MHz·T−1)

Relative sensitivity

Natural abundance (%)

NMR sensitivity

1H

42.58

1

99.985

1

13C

10.71

1.59 × 10−2

1.108

1.76 × 10−4

15N

4.31

1.04 × 10−3

0.37

3.85 × 10−6

19F

40.05

8.33 × 10−1

100

8.33 × 10−1

29Si

8.46

7.84 × 10−3

4.70

3.69 × 10−4

31P

17.24

6.63 × 10−2

100

6.63 × 10−2

129Xe

11.78

2.12 × 10−2

26.4

5.60 × 10−3

Toward this ends, 19F MRI contrast agents (always ON type probes) have been utilized in visualization of foci, and cell tracker (Ahrens et al. 2005; Thurecht et al. 2010; Srinivas et al. 2007). In particular, perfluorocarbon (PFC) encapsulated nanoemulsions have attracted significant attention as highly sensitive 19F MRI contrast agents (Srinivas et al. 2010), and have been utilized as a cell tracker, and oxygen delivery. Recently, several activatable 19F MRI probes (switching OFF/ON type probes) have also been developed. However, there are only a few examples of in vivo applications owing to the low sensitivity of such probes.

7.2 Perfluorocarbon Encapsulated in Silica Nanoparticle (FLAME)

In the author’s research group, novel unique shape nanomaterials, which are perfluoro-15-crown-5 ether (PFCE)-encapsulated silica nanoparticles, FLAMEs (FLuorine Accumulated silica nanoparticle for MRI contrast Enhancement), were developed (Fig. 7.3) (Matsushita et al. 2014). FLAMEs are composed of a liquid PFCE, which shows the high molecular mobility to achieve the long T2, and a silica shell, which can be easily surface-modified for various functionalization. Although Ahrens et al. reported lipid-based PFCE nanoemulsions as 19F MRI contrast agents for immune cell tracking (Ahrens et al. 2005; Srinivas et al. 2007), the chemical modification of the lipid emulsion surface is limited due to the unstablity in organic solvents. In contrast, the silica shell fulfills the many demands such as high hydrophilicity, high stability in both aqueous and organic solutions, and chemically surface-modifiable property. In fact, various surface functionalization of FLAMEs was achieved and the functionalized FLAMEs were useful for monitoring a reporter protein expression in living cells and in vivo detection of a tumor. These biological applications represent only a fraction of the forthcoming applications.
Fig. 7.3

Illustration and transmission electron microscope image of FLAME. The molecular motion of PFC is highly retained and thus the sensitivity of the nanoparticles is high sensitive

7.3 Paramagnetic Relaxation Enhancement (PRE) Effect

There are three types of paramagnetic effects: paramagnetic relaxation enhancement (PRE) effect, pseudocontact shifts (PCSs), and residual dipolar couplings (RDCs) (Clore and Iwahara 2009). Since PCSs and RDCs are observed only in anisotropic electron systems, only PRE is effective in the case of SPIO and Gd3+ complexes (Keizer et al. 2007). The PRE decreases the spin-spin relaxation time (T2) and results in the broadening of the NMR signals and the decrease of the MRI signals. There are two types of the relaxation mechanism of PRE effect. One is PRE through dipole-dipole interaction and the other is PRE through Curie-spin relaxation. The PRE effect of Gd3+ complexes is occurred through dipole-dipole interaction. The transverse (Γ2) PRE rates of Gd3+ are described by the Solomon–Bloembergen (SB) equations (Solomon 1955; Bloembergen and Morgan 1961; Lipari and Szabo 1982):
$$ {\varGamma}_2=\frac{1}{15}{\left(\frac{\mu_0}{4\pi}\right)}^2{\gamma}_{\mathrm{I}}^2{g}^2{\mu}_{\mathrm{B}}^2S\left(S+1\right)\left\{4{J}_{\mathrm{SB}}(0)+3{J}_{\mathrm{SB}}\left({\omega}_{\mathrm{I}}\right)\right\} $$
where μ0 is the permeability of free space, μB is the magnetic moment of the free electron, γI the fluorine gyromagnetic ratio, g is the electron g-factor, S is the electron spin quantum number, and ωI/2π is the Larmor frequency of the fluorine compound. JSB(ω) is the spectral density function;
$$ {J}_{\mathrm{SB}}\left(\omega \right)={r}^{-6}\frac{\tau_C}{1+{\left(\omega {\tau}_C\right)}^2} $$
τC is the correlation time, defined as (τr−1 + τs−1)−1. τr is the rotational correlation time of the molecule, and τs is the effective electron relaxation time.
In contrast, Curie-spin relaxation arises from dipole-dipole interaction between a observable nuclide and the magnetization of the electron. The PRE effect of SPIOs is governed by Curie-spin relaxations owing to their high magnetic susceptibility. The Γ2 PRE rates of Curie-spin relaxation are given by (Bertinin et al. 2002):
$$ {\varGamma}_2=\frac{1}{5}{\left(\frac{\mu_0}{4\pi}\right)}^2\frac{\omega_I{g}^4{\mu}_{\mathrm{B}}^4{S}^2{\left(S+1\right)}^2}{{\left(3{k}_BT\right)}^2{r}^6}\left(4{\tau}_r+\frac{3{\tau}_r}{1+{\omega_I}^2{\tau_r}^2}\right) $$
where kB is the Boltzmann constant, T is temperature.
In both cases, PRE effect is effective over short distance due to its r−6 dependency, where r is the distance between NMR-observable nuclei and a paramagnetic center. When the T2 relaxivity of SPIO is compared with that of Gd3+ complexes, SPIOs have higher T2 relaxivity than Gd3+ complexes (Table 7.2). Thus, SPIO is efficient for decreasing the 19F NMR/MRI signals of PFCE near the FLAME core compared with Gd3+ complexes.
Table 7.2

Relaxivities (mM−1 s−1) of paramagnetic contrast agents in H2O at 37 °C (Rohrer et al. 2005)

 

0.47 T

1.5 T

3.0 T

4.7 T

 

r1

r2

r2/r1

r1

r2

r2/r1

r1

r2

r2/r1

r1

r2

r2/r1

Gd3+ complex

Magnevist

3.4

4.0

1.18

3.3

3.9

1.18

3.1

3.7

1.19

3.2

4.0

1.25

Gadovist

3.7

5.1

1.38

3.3

3.9

1.18

3.2

3.9

1.22

3.2

3.9

1.22

ProHance

3.1

3.7

1.19

2.9

3.2

1.10

2.8

3.4

1.21

2.8

3.7

1.32

MultiHance

4.2

4.8

1.14

4.0

4.3

1.08

4.0

4.7

1.18

4.0

5.0

1.25

Dotarem

3.4

4.1

1.21

2.9

3.2

1.10

2.8

3.3

1.18

2.8

3.7

1.32

OmniScan

3.5

3.8

1.09

3.3

3.6

1.09

3.2

3.8

1.19

3.3

4.1

1.24

Teslascan

1.9

2.1

1.11

1.6

2.1

1.31

1.5

2.3

1.53

1.6

2.7

1.69

Optimark

4.2

5.2

1.24

3.8

4.2

1.11

3.6

4.5

1.25

3.8

4.7

1.24

SPIO

Resovist

20.6

86

4.17

8.7

61

7.01

4.6

143

31.1

2.8

176

62.9

Feridex

27

152

5.63

4.7

41

8.72

4.1

93

22.7

2.3

105

45.7

7.4 Gadolinium Based-19F MRI Nanoprobe for Monitoring Reducing Environment

PRE effect is effective over short distance due to its r−6 dependency, where r is the distance between NMR-observable nuclei and a paramagnetic center (Clore and Iwahara 2009; Iwahara and Clore 2006). The author’s research group has employed PRE effect to develop activatable 19F MRI small molecule probes for detection of enzyme activity (Mizukami et al. 2008). The probes consist of fluorine compound, enzyme substrate, and Gd3+ complex. Gd3+ complex was conjugated with fluorine compounds through enzyme substrate. The distance between fluorine compound and Gd3+ complex was approximately 2.2 nm, determined by molecular mechanic method. Since PRE effect is effective at such close distance, 19F NMR/MRI signal of the probes were decreased. Upon addition of enzyme, Gd3+ complexes were away from fluorine compounds, which results in high 19F NMR/MRI signal enhancements.

In the case of FLAME, most of PFCE compounds are more than 50 Å away from the surface-modified Gd3+ complexes due to the thickness of the silica shell. Thus, it was assumed that the PRE effect might not sufficiently attenuate the 19F NMR/MRI signals of FLAME.

The authors first confirmed whether the PRE of the Gd3+ complexes on the FLAME surface was effective. Different concentration of Gd3+ diethylenetriaminepentaacetate (DTPA) complexes were attached to FLAME to yield FLAME-DTPA-Gd1–2 (Scheme 7.1). The 19F NMR spectrum of FLAME-DTPA without Gd3+ exhibited a sharp, single peak (T2 = 420 ms). Meanwhile, that of FLAME-DTPA-Gd became a broader peak as Gd3+ concentration increased (Fig. 7.4a). The T2 of FLAME-DTPA-Gds decreased in Gd3+ concentration dependent manner (T2 = 68, 40 ms for FLAME-DTPA-Gd1, 2 respectively). Although the 19F MRI signal of FLAME-DTPA were observed due to the long T2, that of FLAME-DTPA-Gd was decreased with Gd3+ concentration increasing (Fig. 7.4b). These results indicated that the 19F NMR/MRI signals of PFCE in FLAME were affected by the PRE from the surface-modified Gd3+ complexes. Therefore, the author expected that activatable 19F MRI probes with high 19F MRI signal enhancement would be achieved by introducing a cleavable linker between FLAME and the surface-modified Gd3+ complexes.
Scheme 7.1

Preparation of FLAME-DTPA-Gd. (a) diethylenetriaminepentaacetic acid dianhydride, TEA, DMF; (b) GdCl3·6H2O, methanol

Fig. 7.4

19F NMR spectra and 19F MRI phantom images of FLAME-DTPA and FLAME-DTPA-Gd. For 19F NMR, CPFCE = 0.6 mM, and the accumulation time was 1 min 22 s. For 19F MRI (Rapid Acquisition with the Refocused Echoes (RARE) method): TR was 3000 ms. TE,eff was 12 ms. The NEX was 64. The acquisition time was 12 min 48 s

This result was explained by the molecular mobility on the NMR/MRI measurement time scale. Iwahara et al. reported that the PRE effect was efficient in spite of the long average distance, when NMR-observable nuclei can occasionally enter the effective range of the PRE effect (Lee et al. 2008). The long T2 indicates that the PFCE in FLAME maintains high molecular mobility even in the nanoparticle structure (Matsushita et al. 2014). Although the PFCE at the center of the FLAME core is about 250 Å away from the surface Gd3+ complexes (where PRE is not efficient), the fluorine compounds can access the inner shell of FLAME on the measurement time scale. Near the inner shell, although the contribution of one Gd3+ complex to the PRE effect is small, the PRE effect from multiple surface Gd3+ complexes is combined, and thus the T2 of PFCE is efficiently decreased (Fig. 7.5). Although Grüll et al. observed the PRE of PFCE in Gd3+-modified nanoemulsions, where the distance between the Gd3+ complexes and the fluorine core was less than 22 Å (De Vries et al. 2014), we confirmed that the PRE was effective as such distance for the first time.
Fig. 7.5

Proposed relaxation mechanism of fluorine compounds in FLAME

Next, the authors designed activatable FLAMEs, FLAME-SS-Gd3+ (FSG), to image reducing environments. Gd3+ complexes were attached to the FLAME surface via disulfide linkers to reduce the T2 of the fluorine compounds by the PRE effect, which attenuates the 19F NMR/MRI signals (Fig. 7.6). When the disulfide of FSG was reduced, the Gd3+ complexes were cleaved from the FLAME surface. Then, the T2 of the encapsulated PFCE would be elongated and the 19F NMR/MRI signal intensity would increase.
Fig. 7.6

Design of activatable FLAME, FLAME-SS-Gd3+ (FSG)

To optimize the amount of Gd3+ complexes on the surface of FLAMEs, three types of FSGs with different concentrations of Gd3+ were prepared (Scheme 7.2). The synthetic intermediate FLAME-Py was prepared by the reaction of FLAME with different amounts of 2-((3-(trimethoxysilyl)propyl)dithio)pyridine (1 eq. for FSG1, 10 eq. for FSG2, and 100 eq. for FSG3). Then, 1 eq., 10 eq., or 100 eq. of Gd3+ complexes were conjugated to the FLAMEs via a thiol-disulfide exchange reaction to afford FSG1–3, respectively.
Scheme 7.2

Preparation of FLAME-SS-Gd3+ (FSG). (a) 2-((3-(trimethoxysilyl)propyl) dithio)pyridine, isopropanol; (b) Gd-DOTA-SH, MeOH

Next, the number of fluorine atoms and Gd3+ ions per nanoparticle were calculated as n19F and nGd, respectively (Table 7.3). The quantity of attached Gd3+ ions was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES), and the amount of the fluorine atoms was quantified by 19F NMR in comparison with that of an internal standard, sodium trifluoroacetate. The average diameter of FLAME was 53.4 nm with a 5 nm-thick silica shell, as measured by transmission electron microscopy. If FLAME has a single size of 53.4 nm, the mole of PFCE per one nanoparticle (mPFCE) could be calculated as follows:
$$ {m}_{\mathrm{PFCE}}=\frac{w_{\mathrm{PFCE}}}{M{W}_{\mathrm{PFCE}}}=\frac{d_{\mathrm{PFCE}}\times {V}_{\mathrm{core}}}{M{W}_{\mathrm{PFCE}}}=\frac{d_{\mathrm{PFCE}}\times \frac{4}{3}\pi {r}_{\mathrm{core}}^3}{M{W}_{\mathrm{PFCE}}}\approx 1.4\times {10}^{-19}\left(\mathrm{mol}/\mathrm{particle}\right) $$
where wPFCE is the weight of PFCE in FLAME, MWPFCE is the molecular weight of PFCE, dPFCE is the density of PFCE (1.86 g/cm3), Vcore is the volume of PFCE in FLAME, and rcore is the radius of the FLAME core (21.7 nm). Thus, the number of fluorine atoms per one nanoparticle (n19F) was calculated as:
$$ {n}_{{}{}^{19}\mathrm{F}}={m}_{\mathrm{PFCE}}\times 20\times {N}_{\mathrm{A}}\approx 1.7\times {10}^6\left({}{}^{19}\mathrm{F}\;\mathrm{atom}/\mathrm{particle}\right) $$
where NA is Avogadro’s constant. Since the amount of the Gd3+ ions was measured by ICP-AES, the molar ratio of the Gd3+ ions to PFCE for FSG1, FSG2, and FSG3 was calculated to be 0.011, 0.026, and 0.038, respectively. Therefore, the number of Gd3+ ions per nanoparticle (nGd) was calculated as:
$$ {\displaystyle \begin{array}{l}\mathrm{FSG}1:{m}_{{\mathrm{Gd}}^{3+}}/{m}_{\mathrm{PFCE}}=0.011\\ {}{n}_{Gd}={m}_{{\mathrm{Gd}}^{3+}}\times {N}_{\mathrm{A}}=0.011\times {m}_{\mathrm{PFCE}}\times {N}_{\mathrm{A}}\approx 9.1\times {10}^2\left({\mathrm{particle}}^{-1}\right)\\ {}\mathrm{FSG}2:{m}_{{\mathrm{Gd}}^{3+}}/{m}_{\mathrm{PFCE}}=0.026\\ {}{n}_{Gd}={m}_{{\mathrm{Gd}}^{3+}}\times {N}_{\mathrm{A}}=0.026\times {m}_{\mathrm{PFCE}}\times {N}_{\mathrm{A}}\approx 2.1\times {10}^3\left({\mathrm{particle}}^{-1}\right)\\ {}\mathrm{FSG}3:{m}_{{\mathrm{Gd}}^{3+}}/{m}_{\mathrm{PFCE}}=0.038\\ {}{n}_{\mathrm{Gd}}={m}_{{\mathrm{Gd}}^{3+}}\times {N}_{\mathrm{A}}=0.038\times {m}_{\mathrm{PFCE}}\times {N}_{\mathrm{A}}\approx 3.1\times {10}^3\left({\mathrm{particle}}^{-1}\right)\end{array}} $$
Table 7.3

Physical properties of FLAME and FSGs

 

ς-potential/mV

n19Fa

nGda

n19F/nGda

T2, TCEP−/ms

T2, TCEP+/ms

FLAME

−24.8 ± 1.7

1.7 × 106

0

420

b

FSG1

−12.6 ± 2.4

1.7 × 106

9.1 × 102

1.8 × 103

120

383

FSG2

3.9 ± 1.4

1.7 × 106

2.1 × 103

7.7 × 102

66

365

FSG3

5.7 ± 1.5

1.7 × 106

3.1 × 103

5.3 × 102

27

371

n19F: the number of 19F atoms in one nanoparticle, nGd the number of Gd3+ atoms in one nanoparticle

aThese values were predicted assuming that FSG has a single size of 53.4 nm (diameter)

bNot measured

The ς-potentials of FSGs gradually shifted towards the positive direction with increasing amounts of surface Gd3+ ions (Table 7.3). This was because the slightly electronegative silanol groups on the FLAME surface were decreased owing to the coupling with 2-((3-(trimethoxysilyl)propyl)dithio)pyridine. The nGd and ς-potential data indicated that different concentrations of Gd3+ complexes were successfully introduced on the FLAME surface.

The 19F NMR spectrum of FLAME without paramagnetic ions exhibited a sharp peak. In contrast, the 19F NMR peaks of FSGs were decreased and more broad according to the concentration of surface Gd3+ on account of the PRE effect (Fig. 7.7a). Although the 19F NMR of FSG1 exhibited a sharp peak, the T2 of FSG1 (120 ms) was shorter than that of FLAME (420 ms) (Table 7.3). The T2 of FSG2 and FSG3 was 66 ms, 27 ms, respectively. As such, the PRE effect was observed in all FSGs.
Fig. 7.7

(a) 19F NMR spectra of FSGs incubated with or without TCEP. CPFCE: 0.6 mM, CTCEP: 1.0 mM, incubation time: 4 h, accumulation time: 10 min 55 s. (b) 19F NMR signal to noise ratio of FSGs in the presence of TCEP (Blue: FSG1, Red: FSG2, Green: FSG3). CPFCE: 0.15 mM

19F NMR spectra and T2 of FSGs were measured after treatment with a reducing agent, tris(2-carboxyethyl)phosphine (TCEP) (Fig. 7.7). Addition of TCEP made the 19F NMR peaks of all FSGs sharper and taller as compared to those before the addition. The T2 values of FSG1–3 were significantly increased upon addition of TCEP within 2 h, and were comparable to that of FLAME. All Gd3+ complexes were cleaved upon addition of more than 2 mM TCEP (Fig. 7.7b). The highest 19F NMR SNR of FSG1–3 was obtained at 2 mM TCEP, and the values were 16.2 for FSG1, 19.5 for FSG2, and 17.9 for FSG3. The signal enhancement factors in response to the reductant were 3.1, 9.7, and 12.7 for FSG1–3, respectively. Thus, FSG3 was the most sensitive 19F NMR probe in the detection of the reducing environment.

The 19F NMR signals of the FSGs increased upon addition of other reducing agents such as glutathione, cysteine, and dithiothreitol (Fig. 7.8). In particular, addition of glutathione induced the greatest 19F NMR signal enhancement. Although there are some concerns about the stability of reduction-triggered nanoparticles in normal tissues, rational optimization of the disulfide linkage will lead to practical in vivo applications.
Fig. 7.8

19F NMR spectra of FSG2 (CPFCE = 0.15 mM) incubated with several thiol-based reducing agents (3 mM). Left to right, control (without reductant), glutathione (GSH), cysteine (Cys), 1,4-dithiothreitol (DTT). The accumulation time was 1 min 22 s. Incubation time was 4 h

Finally, 19F MR phantom images of FSGs solutions with or without TCEP were obtained by varying TE,eff. In general, the MRI signal of the long T2 component is well observed at both short and long TE,eff. In contrast, the MRI signal of samples with moderately short T2 is only visible at short TE,eff, and that of the extremely short T2 component is not observed even at short TE,eff. As expected from the 19F NMR results, almost no 19F MRI signals of FSG2 and FSG3 were detected without TCEP at any TE,eff due to the strong PRE effect (Fig. 7.9a, b). In contrast, the 19F MRI signals of FSG1 were observed at TE,eff ≤ 84 ms because of the moderately short T2. However, the measurement of FSG1 without TCEP at TE,eff ≥ 108 ms extinguished the undesired 19F MRI signals. Reductive reactions induced a noticeable 19F MRI signal enhancement in FSG1–3 at any TE,eff (filled circles). At TE,eff = 12 ms, approximately 60- and 40-fold increases were observed in FSG2 and FSG3, respectively. Although the signal the enhancement of FSG1 was only two-fold at TE,eff = 12 ms, a 50-fold increase was observed at TE,eff = 108 ms. These results indicated that FSG2 was the most effective probe for detecting reducing environments. One of the advantages of FSGs is the high sensitivity, because the 19F NMR/MRI signals of 1.7 × 106 fluorine atoms in the core were decreased by ca. 1.0 × 103 Gd3+ complexes on the FLAME surface. The ratios of fluorine atoms to Gd3+ complexes (Table 7.1) are the highest among known PRE-based probes, of which the ratios were single digits. This high ratio led to the high signal amplification.
Fig. 7.9

19F MRI signal enhancement of FSGs by TCEP. (a) 19F MRI phantom images of FSG1–3 with or without TCEP. (b) Plot of 19F MRI signal intensity of FSG1–3 at different TE,eff with (filled circles) or without (open circles) TCEP. 19F MRI RARE method: the matrix size was 128 × 64 and the slice thickness was 30 mm. TR was 3000 ms. The NEX was 64. The acquisition time was 25 min 36 s

Supplementary material

Video S1-6

(MP4 508249 kb)

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Authors and Affiliations

  1. 1.Graduate School of EngineeringOsaka UniversitySuita CityJapan

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