New technologies for Molecular Contrast Optical Coherence Tomography and ultra-high resolution Photoacoustic Microscopy

High-resolution optical molecular imaging currently plays an essential role as a research tool for biology, biochemistry, and the biomedical sciences. Likewise, it is making in-roads into clinical practice, providing an optical biopsy for diagnosis and monitoring of various diseases. It provides an unparraleled glimpse into the biochemistry of living tissues on the cellular and subcellular levels, where the disease process begins. We are innovating in two areas, photoacoustic microscopy and optical coherence tomography, providing new technolgies for molecular imaging.

Molecular Imaging with Optical Coherence Tomography: Pump-Probe Optical Coherence Tomography (PPOCT)

Optical Coherence Tomography (OCT) is a relatively new medical imaging modality used clinically primarily in ophthalmology and cardiology. It can provide non-invasive high-fidelity morphological images of highly scattering tissues at depths of 1-2 mm. One drawback to OCT is that it cannot typically provide molecular information available in other optical imaging modalities such as fluorescence microscopy and photoacoustic microscopy. In order to overcome this drawback we have developed techniques based on pump-probe spectroscopy that has enabled imaging of endogenous melanin and hemoglobin as well as methylene blue, a common vital dye used in chromoendoscopy of the esophagus. We have focused on imaging natural occuring biomolecules (melanin and hemoglobin) or dyes already used in clincal practice (methylene blue) in order to facility clincal application of the developed technologies. 

Zebrafish kidneys tagged with methylene blue. Gray scale: OCT, Green/Blue scale: PPOCT (methylene blue).

Zebrafish kidneys tagged with methylene blue. Gray scale: OCT, Green/Blue scale: PPOCT (methylene blue).

High-Resolution Photoacoustic Microscopy: Transient Absorption Photoacoustic Microscopy (TAUM) 

Photoacoustic Microscopy (PAM) is a hybrid imaging modality that combines optical absorption contrast with ultrasonic detection to obtain the benefits of molecular imaging with the advantage of detecting sound waves. In tissue, the typical frequencies used for ultrasonic imaging are attenuated three orders of magnitude less than visible light. The molecular contrast afforded by photoacoustic microscopy is ideal for mapping the location of strong endogenous absorbers including hemoglobin, melanin, and cytochromes. The depth penetration benefit provided by converting optical energy to acoustic energy has enabled the development of several PAM techniques with varying resolution and penetration depth. A major limitation, of current high-resolution PAM techniques, is the generation of highly asymmetric point spread function. Using traditional approaches, subcellular transverse resolution imaging has previously been demonstrated, however due to the properties of the ultrasonic transducer, the axial resolution is typically limited to tens of microns. We recently demonstrated that integrating pump-probe spectroscopy with PAM, enables optical confinement of the point spread function in the axial dimension. This technique, termed transient absorption ultrasonic microscopy (TAUM), improves the axial resolution by over an order of magnitude enabling subcellular resolution photoacoustic microscopy in all three dimensions. Recent work has reduced the design of the system to the point where it is a very easy modification to a traditional PAM microscope that enabling greater than a 10 fold improvement in the axial resolution. We have also shown that variation in the molecular dynamics measured with TAUM can be used to distinguish between related molecular systems such as oxy and deoxy hemoglobin. 



  1. W. Kim and B. E. Applegate, “In Vivo Molecular Contrast Optical Coherence Tomography imaging of Methylene Blue,” Opt. Lett., 40, 1426-1429, (2015) PMID: 25831349    
  2. O. Carrasco-Zevallos, R. L. Shelton, W. Kim, J. Pearson, and B. E. Applegate, "In vivo pump-probe optical coherence tomography imaging in Xenopus laevis," J Biophotonics 8, 25-35 (2015) PMID: 24282110    
  3. S. P. Mattison*, W. Kim*, J. Park*, and B. E. Applegate, “Molecular Imaging in Optical Coherence Tomography” Current Molecular Imaging, 3, 1-18 (2014) PMID: 25821718    
  4. D. Jacob, R. L. Shelton, and B. E. Applegate, "Fourier domain pump-probe optical coherence tomography imaging of Melanin," Optics Express 18, 12399-12410 (2010) PMID: 20588366    
  5. Q. Wan, and B. E. Applegate, "Multiphoton coherence domain molecular imaging with pump-probe optical coherence microscopy," Opt Lett 35, 532-534 (2010) PMID: 20160808    
  6. B. E. Applegate, and J. A. Izatt, "Molecular imaging of endogenous and exogenous molecular chromophores with ground state recovery pump-probe optical coherence tomography," Optics Express 14, 9142-9155 (2006) PMID: 19529295    
  7. B. E. Applegate, C. Yang, and J. A. Izatt, "Theoretical comparison of the sensitivity of molecular contrast optical coherence tomography techniques," Optics Express 13, 8146-8163 (2005) PMID: 19498844
  8. S. P. Mattison and B. E. Applegate, "Simplified method for ultra high-resolution photoacoustic microscopy via transient absorption," Opt Lett 39, 4474-4477 (2014) PMID: 25078206
  9. R. L. Shelton, S. P. Mattison, and B. E. Applegate, "Molecular specificity in photoacoustic microscopy by time-resolved transient absorption," Opt Lett 39, 3102-3105 (2014) PMID: 24875987    
  10. R. L. Shelton, S. P. Mattison, and B. E. Applegate, "Volumetric imaging of erythrocytes using label-free multiphoton photoacoustic microscopy," J Biophotonics 10, 834-840 (2014) PMID: 23963621   
  11. S. P. Mattison, R. L. Shelton, R. T. Maxson, and B. E. Applegate, "Continuous real-time photoacoustic demodulation via field programmable gate array for dynamic imaging of zebrafish cardiac cycle," Biomed Opt Express 4, 1451-1463 (2013) PMID: 24010007    
  12. R. L. Shelton, and B. E. Applegate, "Ultrahigh resolution photoacoustic microscopy via transient absorption," Biomed Opt Express 1, 676-686 (2010) PMID: 21258499  
  13. R. L. Shelton, and B. E. Applegate, "Off-Axis Photoacoustic Microscopy," IEEE Trans Biomed Eng 57, 1835-1838 (2010)