Biophotonics Laboratory
California Institute of Technology
   

 

Optofluidic Microscopy (OFM)
Turbidity Suppression by Optical Phase Conjugation in Biological Media
Coherence Domain Probing Systems

Research

Molecular Contrast OCT

Optical coherence tomography (OCT) is an important non-invasive biomedical tool for high resolution imaging of biological samples to a depth of a few millimeters. In recent years, a new and exciting functional OCT method, known as molecular contrast optical coherence tomography (MCOCT), has been introduced that combines the major advantages of fluorescence microscopy (chemical contrast detection and imaging capability) and OCT (higher spatial resolution and depth penetration). Our current research is focused on the use of pump-probe schemes for MCOCT. In these types of schemes, a baseline OCT scan of the sample containing a contrast agent is acquired (by a probe beam), followed by photo-excitation of the sample (with a pump beam). The photo-illumination alters the absorption coefficient of the contrast agent. A second OCT scan is then acquired. The two OCT scans are slightly different as the absorption spectrum of the contrast agent has changed. The two OCT scans are thus processed to determine the distribution of the contrast agent within the sample.

Figure 1: (a) Pump-probe setup for MCOCT. (b) Timing diagram for the pump-probe scheme.

Recently, we have successfully demonstrated the use of indocyanine green (ICG) as a contrast agent using the MCOCT setup shown in Figure 1. ICG is a frequently used dye in medical diagnostics and photodynamic therapy. As opposed to previous pump-probe schemes which achieve contrast from dye molecules that accumulate in the triplet state, we achieve contrast as dye molecules are sent to the photobleached state. This method is advantageous because the photobleached state is permanent, and dye molecules can be sent to this state using relatively low levels of illumination intensity. Figure 2 shows the change in absorption cross-section, for ICG solutions in both DI water and 1% BSA, after photo-bleaching.

Figure 2: Absorption cross section spectra of ICG in DI water and BSA before and after the photobleaching.

From the change in absorption cross section (see Fig. 2), we are able to determine the distribution of the contrast agent as follows. The OCT interferogram is given by:

By taking OCT scans before and after photobleaching, we can write the change in the absorption coefficient of ICG as a function of the two scans:

Finally, the depth resolved distribution of bleached dye molecules within the sample can be determined as:

Figure 3(b) shows A-scans of a glass cuvette filled with an ICG solution before (blue) and after (red) photobleaching. The first two interfaces show no contrast as anticipated. The last two interfaces of the glass cuvette yield a measured contrast of ~ 7 dB. In Fig. 3(c), the cuvette is filled with a mixture of ICG and latex microspheres, and the contrast increases with ICG sample depth as it is cumulative in nature.

Figure 3: (a) Schematic of glass cuvette sample. (b) and (c) show A-scans of the cuvette filled with ICG alone and a mixture of ICG with latex microspheres, respectively, before (blue) and after (red) photbleaching. (d) shows the ratio of A-scans shown in Fig. 3 (c).

We have also demonstrated the suitability of this technique for biological imaging. Figure 4 shows OCT images of the gill arch cavities of a stage 54 Xenopus laevis. Part (a) shows the initial OCT scan, while (b) shows increased backscatter, attributable to change in the absorption coefficient of ICG from within the gill arch cavities after photobleaching.

Figure 4: (a) Standard OCT image of the gill arch cavities of a stage 54 Xenopus laevis. (b) MCOCT image showing increased contrast due to photobleached ICG molecules.

We are also investigating other dyes that absorb in a similar region of the spectrum (near IR), but appear to photobleach significantly faster than ICG.

Reference:
Zahid Yaqoob, Jigang Wu, Emily J. McDowell, Xin Heng, Changhuei Yang. 'Methods and application areas of endoscopic optical coherence,' Journal of Biomedical Optics, accepted (2006).