Biophotonics Laboratory
California Institute of Technology


Fourier Ptychographic Microscopy (FPM)
Wide Field of View Microscopy (WFOV)
Time-reversal Wavefront Engineering



Time-reversal Wavefront Engineering

Light scattering in biological tissues poses a significant challenge to our ability to study intact specimens beyond superficial depths (Fig. 1). Conventional optical focusing methods treat scattered light as noise and rely on unscattered light, which exponentially decreases with depth. However, scattering is a deterministic process and is reversible through optical phase conjugation (OPC). Using this technique, our group previously demonstrated focusing of light through biological tissues (Fig. 2)1, 2.

Fig. 1: Scattering in biological tissues limits focusing to superficial layers or thin samples.

Fig. 2: (Top) Wavefront distortions due to scattering can be reserved through optical phase conjugation. (Bottom) OPC foci obtained through 3 mm (a), 6 mm (b), and 10 mm (c) thick chicken breast tissues. (d) Photo of the 10 mm thick tissue sample.

Time Reversal of Ultrasound-Encoded Light (TRUE)

An important goal of biomedical optics, however, is to focus inside. To this end, Xu et. al. proposed combining optical phase conjugation with ultrasound encoding in a method named time-reversal of ultrasound encoded light (TRUE)3. To realize high resolution, high intensity TRUE focusing in thick tissues, our group utilized a digital optical phase conjugate mirror (DOPC) that enables high gain4 (Fig. 3). We illustrate the potential of our method for fluorescence bioimaging in the diffusive regime by imaging complex fluorescent objects and tumor microtissues ~ 2.5 mm deep in biological tissues, at a lateral resolution of ~ 40 µm (Fig. 4).

Fig. 3: A portion of the light passing through the ultrasound focus is frequency-shifted via the acousto-optic effect. The frequency-shifted light is selectively phase conjugated by the digital optical phase conjugation mirror (DOPC)4.

Fig. 4: Images of quantum dot “CIT” feature an fluorescently
dyed cancer microtissues (top) Epifluorescence before
embedding (middle) Epifluorescence after embedding (bottom)
Digital TRUE image.

Time Reversal of Variance-Encoded Light (TROVE)

The resolution of the TRUE method is fundamentally limited by the size of the ultrasound focus. In TROVE, we overcome this limitation and achieve single optical speckle resolution by using speckle statistics to encode the position of each optical speckle within the ultrasound focus. To do so, we illuminate the sample with a set of input wavefronts and measure the scattered ultrasound tagged wavefront that exits the sample. With the ability of the DOPC to digitally analyze and manipulate optical wavefronts, these wavefronts can be processed and decomposed into an eigenset of optimal wavefront solutions. When time-reversed, each of these wavefront solutions would focus to a single speckle-size limited spot. For example, the solution that exhibits the highest eigenvalue would time-reverse back to the exact center of the ultrasound focus. This essentially allows us to achieve optical speckle size limited, high resolution focusing.5

Fig. 5: TROVE makes use of speckle statistics to enable optical
speckle sized focusing.

Fig. 6: Results obtained with TROVE and comparison with TRUE.

Further information on the effects of Time-reversal Wavefront Engineering in Biological Media can be found here.


1. Yaqoob, Z., et al., Optical phase conjugation for turbidity suppression in biological samples. Nature Photonics 2(2): 110-115, (2008).

2. McDowell, E.J., et al., Turbidity suppression from the ballistic to the diffusive regime in biological tissues using optical phase conjugation. Journal of Biomedical Optics 15(2): 025004-025004, (2010).

3. Xu, X., Liu, H., and Wang, L.V., Time-reversed ultrasonically encoded optical focusing into scattering media. Nature Photonics 5, 154-157, (2011).

4. Wang, Y.M., Judkewitz, B., DiMarzio, C.A. and Yang, C., Deep-tissue focal fluorescence imaging with digitally time-reversed ultrasound-encoded light. Nature Communications 3: 928, (2012).

5. Judkewitz, B., Wang, Y.M., Horstmeyer, R., Mathy, A. and Yang, C., Speckle-scale focusing in the diffusive regime with time reversal of variance-encoded light (TROVE). Nature Photonics 7: 300-305, (2013).