CEST - Chemical Exchange Saturation Transfer

Chemical Exchange Saturation Transfer (CEST) is a technique where one resonance, in slow exchange with a second resonance, is saturated with a selective low power pulse followed by a hard non-selective 90° pulse.  The intensity of the second resonance is then diminished due to the transfer of saturation from the first resonance as the result of  chemical exchange.  The figure below demonstrates this for a 25 mM solution of salicylic acid in H₂O/D₂O buffered at pH 7.

The left-hand panel of the figure is a stacked plot of extracted spectra collected in a pseudo 2D acquisition as a function of saturation frequency.  The saturation frequency was varied from an initial value of 20 ppm to a final value of -20 ppm in steps of 0.2 ppm.  The spectra are plotted such that only the water resonance is on scale.  One can see that the intensity of the water resonance dips when a saturation frequency of ~14 ppm is applied, corresponding to the resonance frequency of the –COOH and –OH protons of the salicylic acid (which appear to be in fast or intermediate exchange with one another).  The water resonance of course also dips to zero when a saturation frequency of ~4.7 ppm is used, corresponding to a simple presaturation of the water.  The right-hand panel of the figure is a plot of the integral of the water resonance as a function of saturation frequency, showing again a dip at ~14 ppm.

CEST is used in MRI to provide image contrast where a chemical exchange agent is introduced and images are collected with and without saturation of the exchange agent.  The difference provides an image enhanced by the presence of the chemical exchange agent.

Thank you to Dr. Mojmir Suchy of Prof. Adam Shuhendler’s group at the University of Ottawa for arousing my interest in the use of CEST for MRI and preparing the sample used in this post. 

INEPT

The sensitivity of a low γ, spin I = ½ nucleus is determined by the difference in populations between the low energy and high energy states, governed by the Boltzmann distribution. If the low γ, spin I = ½ nucleus is coupled to a proton the energy level diagram is more complicated than simply two levels and is shown in the figure below where a ¹³C-¹H spin pair is used as an example.

The populations of the states involved in the ¹³C transitions and hence the sensitivity of the ¹³C signal can be altered by inverting the H1 or H2 ¹H transitions with 180° pulses. This is illustrated in the figure below.

In the left panel, the H1 transition of a ¹³C-¹H spin pair is inverted (i.e. the populations of the two energy levels of the H1 transition are swapped). This also affects the populations of the energy levels involved in the C1 and C2 ¹³C transitions. After inversion of the H1 ¹H transition, the intensities of the C1 and C2 ¹³C transitions have changed from their equilibrium value of 1:1 to an enhanced value of 5:-3. If the H2 transition is inverted (right-hand panel), the C1:C2 intensity ratio is -3:5. In both cases the sensitivity of the ¹³C doublet has been enhanced compared to its equilibrium value. This enhancement is called INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) and is one of the most common sensitivity enhancement techniques used in NMR pulse sequences. The simplest implementation of INEPT is shown in the figure below along with the vector diagrams.

Phase cycling can be employed to obtain a -4:4 anti-symmetric doublet, rather than doublets with components of unequal magnitude. This is represented in the figure below.

A refocusing element can be added to the end of the sequence to refocus the anti-symmetric doublets and data can be collected with proton decoupling.

The result is a singlet with 4 times  (i.e. γ_H/γ_C) the intensity of the singlet one would expect under equilibrium conditions without an NOE.  For ¹⁵N, one obtains a sensitivity gain of ~10. The results of these implementations of INEPT are compared to the equilibrium situation in the figure below.

INEPT has the additional advantage that its repetition rate is determined by the ¹H T₁ rather than the ¹³C T₁.  This is a tremendous additional sensitivity improvement when multiple scans are collected because the ¹H T₁ is often shorter than the ¹³C T₁ by an order of magnitude.  One can collect approximately ten times as many scans per unit time.  This advantage is even more significant for ¹⁵N.  Reverse INEPT is used in the collection of ¹H data for  carbon-proton pairs to suppress the protons bound to ¹²C.