Benjamin Judkewitz

Deep-tissue focal fluorescence imaging with digitally time-reversed ultrasound-encoded light

Fluorescence imaging is one of the most important research tools in biomedical sciences. However, scattering of light severely impedes imaging of thick biological samples beyond the ballistic regime. Here we directly show focusing and high-resolution fluorescence imaging deep inside biological tissues by digitally time-reversing ultrasound-tagged light with high optical gain (~5×105). We confirm the presence of a time-reversed optical focus along with a diffuse background—a corollary of partial phase conjugation—and develop an approach for dynamic background cancellation. To illustrate the potential of our method, we image complex fluorescent objects and tumour microtissues at an unprecedented depth of 2.5 mm in biological tissues at a lateral resolution of 36 μm×52 μm and an axial resolution of 657 μm. Our results set the stage for a range of deep-tissue imaging applications in biomedical research and medical diagnostics.

Mechanisms of scent-tracking in humans.

Whether mammalian scent-tracking is aided by inter-nostril comparisons is unknown. We assessed this in humans and found that (i) humans can scent-track, (ii) they improve with practice, (iii) the human nostrils sample spatially distinct regions separated by ∼3.5 cm and, critically, (iv) scent-tracking is aided by inter-nostril comparisons. These findings reveal fundamental mechanisms of scent-tracking and suggest that the poor reputation of human olfaction may reflect, in part, behavioral demands rather than ultimate abilities.

Speckle-scale focusing in the diffusive regime with time reversal of variance-encoded light (TROVE)

Focusing of light in the diffusive regime inside scattering media has long been considered impossible. Recently, this limitation has been overcome with time reversal of ultrasound-encoded light (TRUE), but the resolution of this approach is fundamentally limited by the large number of optical modes within the ultrasound focus. Here, we introduce a new approach, time reversal of variance-encoded light (TROVE), which demixes these spatial modes by variance-encoding to break the resolution barrier imposed by the ultrasound. By encoding individual spatial modes inside the scattering sample with unique variances, we effectively uncouple the system resolution from the size of the ultrasound focus. This enables us to demonstrate optical focusing and imaging with diffuse light at unprecedented, speckle-scale lateral resolution of ~ 5 μm.

Targeted single-cell electroporation of mammalian neurons in vivo

In order to link our knowledge of single neurons with theories of network function, it has been a long-standing goal to manipulate the activity and gene expression of identified subsets of mammalian neurons within the intact brain in vivo. This protocol describes a method for delivering plasmid DNA into single identified mammalian neurons in vivo, by combining two-photon imaging with single-cell electroporation. Surgery, mounting of a chronic recording chamber and targeted electroporation of identified neurons can be performed within 1–2 h. Stable transgene expression can reliably be induced with high success rates both in single neurons as well as in small, spatially defined networks of neurons in the cerebral cortex of rodents.

Mechanisms of pattern decorrelation by recurrent neuronal circuits

Decorrelation is a fundamental computation that optimizes the format of neuronal activity patterns. Channel decorrelation by adaptive mechanisms results in efficient coding, whereas pattern decorrelation facilitates the readout and storage of information. Mechanisms achieving pattern decorrelation, however, remain unclear. We developed a theoretical framework that relates high-dimensional pattern decorrelation to neuronal and circuit properties in a mathematically stringent fashion. For a generic class of random neuronal networks, we proved that pattern decorrelation emerges from neuronal nonlinearities and is amplified by recurrent connectivity. This mechanism does not require adaptation of the network, is enhanced by sparse connectivity, depends on the baseline membrane potential and is robust. Connectivity measurements and computational modeling suggest that this mechanism is involved in pattern decorrelation in the zebrafish olfactory bulb. These results reveal a generic relationship between the structure and function of neuronal circuits that is probably relevant for pattern processing in various brain areas.

Focusing on moving targets through scattering samples.

Focusing light through scattering media has been a longstanding goal of biomedical optics. While wavefront shaping and optical time-reversal techniques can in principle be used to focus light across scattering media, achieving this within a scattering medium with a noninvasive and efficient reference beacon, or guide star, remains an important challenge. Here, we show optical time-reversal focusing using a new technique termed Time Reversal by Analysis of Changing wavefronts from Kinetic targets (TRACK). By taking the difference between time-varying scattering fields caused by a moving object and applying optical time reversal, light can be focused back to the location previously occupied by the object. We demonstrate this approach with discretely moved objects as well as with particles in an aqueous flow, and obtain a focal peak-to-background strength of 204 in our demonstration experiments. We further demonstrate that the generated focus can be used to noninvasively count particles in a flow-cytometry configuration-even when the particles are hidden behind a strong diffuser. By achieving optical time reversal and focusing noninvasively without any external guide stars, using just the intrinsic characteristics of the sample, this work paves the way to a range of scattering media imaging applications, including underwater and atmospheric focusing as well as noninvasive in vivo flow cytometry.

Translation correlations in anisotropically scattering media

Controlling light propagation across scattering media by wavefront shaping holds great promise for a wide range of communications and imaging applications. But, finding the right shape for the wavefront is a challenge when the mapping between input and output scattered wavefronts (that is, the transmission matrix) is not known. Correlations in transmission matrices, especially the so-called memory effect, have been exploited to address this limitation. However, the traditional memory effect applies to thin scattering layers at a distance from the target, which precludes its use within thick scattering media, such as fog and biological tissue. Here, we theoretically predict and experimentally verify new transmission matrix correlations within thick anisotropically scattering media, with important implications for biomedical imaging and adaptive optics.

Dendritic Enlightenment: Using Patterned Two-Photon Uncaging to Reveal the Secrets of the Brain's Smallest Dendrites

It has been a longstanding challenge for experimentalists to manipulate precisely the spatial and temporal patterns of synaptic input to the dendritic tree in order to mimic activity occurring in the intact brain and determine their importance for synaptic integration. In this issue of Neuron, Losonczy and Magee have used rapid multisite two-photon uncaging of glutamate to define patterns of synaptic input on a submillisecond and micron scale to investigate the rules for summation of synaptic inputs in the fine oblique dendrites of pyramidal neurons.

Method for auto-alignment of digital optical phase conjugation systems based on digital propagation

Optical phase conjugation (OPC) has enabled many optical applications such as aberration correction and image transmission through fiber. In recent years, implementation of digital optical phase conjugation (DOPC) has opened up the possibility of its use in biomedical optics (e.g. deep-tissue optical focusing) due to its ability to provide greater-than-unity OPC reflectivity (the power ratio of the phase conjugated beam and input beam to the OPC system) and its flexibility to accommodate additional wavefront manipulations. However, the requirement for precise (pixel-to-pixel matching) alignment of the wavefront sensor and the spatial light modulator (SLM) limits the practical usability of DOPC systems. Here, we report a method for auto-alignment of a DOPC system by which the misalignment between the sensor and the SLM is auto-corrected through digital light propagation. With this method, we were able to accomplish OPC playback with a DOPC system with gross sensor-SLM misalignment by an axial displacement of up to~1.5 cm, rotation and tip/tilt of ~5° and in-plane displacement of ~5 mm (dependent on the physical size of the sensor and the SLM). Our auto-alignment method robustly achieved a DOPC playback peak-to-background ratio (PBR) corresponding to more than ~30 % of the theoretical maximum. As an additional advantage, the auto-alignment procedure can be easily performed at will and, as such, allows us to correct for small mechanical drifts within the DOPC systems, thus overcoming a previously major DOPC system vulnerability. We believe that this reported method for implementing robust DOPC systems will broaden the practical utility of DOPC systems.

Iterative Time-Reversed Ultrasonically Encoded Light Focusing in Backscattering Mode

The Time-Reversed Ultrasound-Encoded (TRUE) light technique enables noninvasive focusing deep inside scattering media. However, the time-reversal procedure usually has a low signal-to-noise ratio because the intensity of ultrasound-encoded light is intrinsically low. Consequently, the contrast and resolution of TRUE focus is far from ideal, especially in the backscattering geometry, which is more practical in many biomedical applications. To improve the light intensity and resolution of TRUE focus, we developed an iterative TRUE (iTRUE) light focusing technique that employs the TRUE focus itself as a signal source (rather than diffused light) for subsequent TRUE procedures. Importantly, this iTRUE technique enables light focusing in backscattering mode. Here, we demonstrate the concept by focusing light in between scattering layers in a backscattering configuration and show that the light intensity at the focus is progressively enhanced by a factor of ~20. By scanning across a fluorescent bead between these two scattering layers, the focusing resolution in the ultrasound axial and lateral directions was improved ~2-fold and ~3-fold, respectively. We further explored the application of iTRUE in biological samples by focusing light between 1 mm thick chicken tissue and cartilage, and light intensity enhancements of the same order were also observed.