Super-resolution photoacoustic imaging is a set of techniques used to enhance spatial resolution in photoacoustic imaging. Specifically, these techniques primarily break the optical diffraction limit of the photoacoustic imaging system. It can be achieved in a variety of mechanisms, such as blind structured illumination, multi-speckle illumination, or photo-imprint photoacoustic microscopy in Figure 1.
Photoacoustic (PA) imaging This particular biomedical imaging modality is a combination of optical imaging, and ultrasound imaging. In other words, a photoacoustic (PA) image can be viewed as an ultrasound image in which its contrast depends on the optical properties, such as optical resolution of biomolecules like hemoglobin, water, melanin, lipids, and collagen. The advantages of photoacoustic imaging are that it gives higher specificity than conventional ultrasound imaging and greater penetration depth than conventional ballistic optical imaging modalities. Photoacoustic imaging works by irradiating the target with a short-pulsed laser, or alternatively an intensity-modulated laser. The target absorbs this optical energy, which is converted into heat; in most cases the amount of heat is a fraction of approximately (1 - fluorescence quantum yield). Heat is further converted into a pressure rise via thermoelastic expansion, and the pressure rise behaves as an ultrasonic wave, the wave of which is called a "photoacoustic wave" that propagates throughout the surroundings of the target. The photoacoustic wave is then detected by ultrasonic transducers, and these signals are used by an image processor and computer to reconstruct an image of the target.
Super-resolution imaging and photoacoustics Recently, several techniques have broken the diffraction limit of light, enabling the observation of individual cellular structures, sub-cellular structures, and processes at the nanometer level, structures that were previously unresolvable by conventional microscopes due to resolutions finer than the optical diffraction limit (~250 nm in lateral direction at high optical NA). However, as super-resolution optical imaging generally relies on fluorophores, only fluorescence imaging by usage of multiple lasers or chemical manipulation of fluorophores is possible. This results in a set of complex configurations for the imaging system and limited use for fluorescent targets. Photoacoustic tomography, is able to complement these super resolution techniques and achieve much greater field of depth and remove the need for fluorescent molecules.
Super resolution techniques Photoacoustics relies on optical excitation of targets and detection of acoustic emission. Since the frequencies of electromagnetic waves (light) are significantly higher than that of acoustic waves, the optical excitation generally sets the absolute resolution. This absolute resolution is well known in optics and is called the diffraction limit of light. This diffraction limit shown below represents the minimum distance that can be resolved between two objects (or similarly the minimum separation distance between two objects excitated by a laser).
Super resolution techniques break this limit by generally using nonlinear techniques such as saturations, switchable quenching, or multiphoton absorption. Photoacoustics directly benefits from these techniques as the excitation can be held to a tighter spot and hence detect acoustic waves to a tighter spot, ultimately improving resolution.
Optical saturation and nonlinear thermal expansion Photoacoustics primarily relies on the pressure wave generated by the absorbing material. At higher excitation energy levels, the excitation becomes greatly nonlinear, leading to different effects on the pressure wave generated. The two equations below are the resulting pressure waves from either nonlinear thermal expansion or optical saturation.
(1)
(2)
Equation (1) above is the pressure wave generated by the non-linear thermal expansion. Equation (2) is the resulting wave generated from optical saturation.
The key idea is that the sample is pumped with high levels of energy such that all of its electrons are saturated to the excitated state. In this saturated state, the additional energy will be utilized in different ways such as nonlinear thermal expansion. From these pressure waves, images corresponding to the absorption of different molecules can be reconstructed. In Figure 2a and 2b, two 100 nm gold nanoparticles are resolved with super resolution enhanced photoacoustics and verified by atomic force microscopy, a different modality that can break the diffraction limit resolution since it does not rely on light.
Figure 2: Comparison of different imaging modalities on two 100 nm gold nanoparticle.
This can be extended to whole images with scanning techniques and Figure 3 shows the dramatic difference with the added super resolution technique. With super resolution, the smaller details of the melanoma cell pictured is visible.
Figure 3: Before and after super resolution techniques applied to photoacoustic phase microscopy.
Photoswitchable probes Reversibly photoswitchable proteins can switch back and forth between two optically separated states, making them useful for high-contrast and high resolution PA imaging. Yao et al. demonstrated differential imaging of the reversibly switchable phytochrome BphP1 in in vivo experiments (terming the technology RS-PAM). The two states of the BphP1 molecule are the Pfr and Pf states, referred to as the ON and OFF states, respectively. When stable, BphP1is at the On state. Upon 780 nm laser pulse train illumination, BphP1 molecules in the on state gradually switch to the off state. As a result, the amplitude of the generated PA signals decrease. The decay rate is proportional to the local excitation intensity. Thus, PA signal in the center of the excitation beam will decay faster compared to the surrounding. Using the difference image between the two states gives a high contrast image of the molecules (Figure 4), and sub-diffraction limited lateral and axial resolutions will be achieved. In the lateral direction, the decay dependence on the local excitation intensity results in a smaller FWHM of the decay PSF. The higher the order of the nonlinear decay, the higher the resolution enhancement will be. The effective lateral PSF for this imaging technique was shown to be where b is the power dependence of the switching-off rate on the excitation intensity (for BphP1, b=1), and m is the order of the polynomial fitting. This technique achieves finer resolutions with a factor of compared to conventional PAM.
In the axial direction, the nonlinear optical effect will determine the resolution (since only in focus molecules contribute in the signal decay), thus, making it independent from the acoustic detection. This allows this technology to have optical sectioning capability. For point targets, the achievable axial resolution is whereas for large targets, it is
Figure 4: PA images of BphP1 expressing U87 cells and HbO2 in scattering media. The differential image effectively removes the background signal, increasing the contrast of the cell area.
As a demonstration (figure 5) this technique showed much finer lateral and axial resolutions compared to conventional PAM. The lateral and axial resolutions were quantified to be ~141 nm and ~400 nm, respectively, which were about 2 and 75 times better than that of conventional PAM.
Figure 5: Comparison of lateral and axial resolution of conventional PAM and RS-PAM in imaging.
Two-photon photoacoustic mechanism Non-radiative two-photon absorption can be utilized to achieve high 3D resolution in in vivo experiments. In conventional PAM temporal or spatial filtering is needed to eliminate background signal and achieve high axial resolution. However, in 2PAM, only the area within the focal spot generates nonlinear photoacoustic signals (figure 6). Based on the two-photon absorption PSF, the lateral () and axial resolution () of the 2PAM system are determined to be...
(3)
(4)
where λex is the excitation wavelength, and n is the medium refractive index. The important property of the axial resolution is its independence from the frequency of the photoacoustic waves. Therefore, lower frequency ultrasound waves can be used for deeper detection.
Figure 6: Comparison between PAM and 2PAM.
Although two-photon excitation can potentially give a smaller PSF, but because one photon absorption is achieved easily by molecules, there will be a prominent 1PA signal from the area around the focal spot, making the detection of 2PA signals a hard problem. The 2PA signal can be separated from the 1PA background signal using a lock-in detection system, as demonstrated by Lee et al... After amplitude modulation of the input laser train (modulation frequency = f), nonlinear absorption of molecules within the focal excitation spot will generate high harmonics of the modulation frequency (2f, 3f….). Photoacoustic signals from the two-photon absorption can be extracted by locking at the second harmonic of the modulation frequency (2f). In this paper, lateral and axial resolutions of 0.51 μm and 2.41 μm in in vivo experiments were achieved, demonstrating the sub-femtoliter resolution (0.49 μm3 with NA=0.8) of the 2PAM imaging system. 1PA images had only 0.71 μm lateral resolution. The two-photon nature of the signals contribute to the 1.4 factor improvement of the 2PAM to 1PAM lateral resolution
Figure 7: Comparison between PAM and 2PAM images of melanin distribution.
Structured illumination Structured illumination is an imaging technique that when applied to microscopy, can double the spatial resolution of that of conventional fluorescence microscopy using the moiré interference pattern, the coarse pattern that is produced when two finer patterns are overlapped and provides easier viewing than either original pattern. Structured illumination occurs in the interaction of a three-dimensional modulated illumination pattern and high-frequency variations in the sample fluorescence caused by small structures, the interaction of which produces a lower-frequency Moiré pattern that contains non-resolvable structures present in the observed image. When these Moiré patterns are imaged in different positions and subsequently computationally post-processed, then the lower-diffraction sample information can be algorithmically decoded and reconstructed. When information above and below the focal plane are added, then spatial resolution will be enhanced and normally non-resolvable sample structures are more easily resolvable. One advantage of structured illumination is its ability to be used with any conventional fluorophores, and one disadvantage is its image acquisition speed that requires complex imaging and therefore compromises temporal resolution needed for live cell imaging
Blind illumination Blind structured illumination photoacoustic microscopy (BSIPAM) was employed as a feedback-free imaging method that uses random optical speckle patterns as a structured illumination for enhancing the spatial resolution of PA imaging within scattering media. Unlike structured illumination microscopy, where the spatial resolution enhancement was limited only to 2, BSIPAM has a higher spatial resolution. BSIPAM operates by the key principle: to recover absorber distribution ρ at spatial resolution close to speckle size.
Principle If there are M different speckle patterns, 𝚽1,...,𝚽M, and the assumption holds that speckle patterns and absorber distribution are represented by discrete vectors 𝚽m, , an expression for measured PA data can be written.
where m = 1,...,M. is the PSF of the photoacoustic imaging system in discrete form, is the pointwise multiplication, is the discrete deconvolution step, and εm is the noise in the data.