Optical sensing of specific molecular targets and pathways deep inside living mice has become possible as a result of a number of advances. These include design of biocompatible near-infrared fluorochromes, development of targeted and activatable 'smart' imaging probes, engineered photoproteins and advances in photon migration theory and reconstruction. Together, these advances will provide new tools making it possible to understand more fully the functioning of protein networks, diagnose disease earlier and speed along drug discovery.

Shedding light onto live molecular targets

We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.

In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.

Optical imaging of live animals has grown into an important tool in biomedical research as advances in photonic technology and reporter strategies have led to widespread exploration of biological processes in vivo. Although much attention has been paid to microscopy, macroscopic imaging has allowed small-animal imaging with larger fields of view (from several millimeters to several centimeters depending on implementation). Photographic methods have been the mainstay for fluorescence and bioluminescence macroscopy in whole animals, but emphasis is shifting to photonic methods that use tomographic principles to noninvasively image optical contrast at depths of several millimeters to centimeters with high sensitivity and sub-millimeter to millimeter resolution. Recent theoretical and instrumentation advances allow the use of large data sets and multiple projections and offer practical systems for quantitative, three-dimensional whole-body images. For photonic imaging to fully realize its potential, however, further progress will be needed in refining optical inversion methods and data acquisition techniques.

Looking and listening to light: the evolution of whole-body photonic imaging.

The application of the chirped-pulse amplification technique to solid-state lasers combined with the availability of broad-bandwidth materials has made possible the development of small-scale terawatt and now even petawatt (1000-terawatt) laser systems. The laser technology used to produce these intense pulses and examples of new phenomena resulting from the application of these systems to atomic and plasma physics are described.

https://science.sciencemag.org/content/sci/264/5161/917.full.pdf

Terawatt to Petawatt Subpicosecond Lasers

We have imaged, in real time, fluorescent tumors growing and metastasizing in live mice. The whole-body optical imaging system is external and noninvasive. It affords unprecedented continuous visual monitoring of malignant growth and spread within intact animals. We have established new human and rodent tumors that stably express very high levels of the Aequorea victoria green fluorescent protein (GFP) and transplanted these to appropriate animals. B16F0-GFP mouse melanoma cells were injected into the tail vein or portal vein of 6-week-old C57BL/6 and nude mice. Whole-body optical images showed metastatic lesions in the brain, liver, and bone of B16F0-GFP that were used for real time, quantitative measurement of tumor growth in each of these organs. The AC3488-GFP human colon cancer was surgically implanted orthotopically into nude mice. Whole-body optical images showed, in real time, growth of the primary colon tumor and its metastatic lesions in the liver and skeleton. Imaging was with either a trans-illuminated epifluorescence microscope or a fluorescence light box and thermoelectrically cooled color charge-coupled device camera. The depth to which metastasis and micrometastasis could be imaged depended on their size. A 60-μm diameter tumor was detectable at a depth of 0.5 mm whereas a 1,800-μm tumor could be visualized at 2.2-mm depth. The simple, noninvasive, and highly selective imaging of growing tumors, made possible by strong GFP fluorescence, enables the detailed imaging of tumor growth and metastasis formation. This should facilitate studies of modulators of cancer growth including inhibition by potential chemotherapeutic agents.

/pdf/whole-body-optical-imaging-of-green-fluorescent-protein-3b45k7jjdj.pdf

Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases

Test bar charts hidden in a 5.5-cm-thick 2.5% Intralipid solution were imaged as a function of phantom depth and size with steady-state Fourier and picosecond Kerr–Fourier imaging systems. With time and space gating, a series of 250-μm bars placed in a thick highly scattering medium were resolved at a signal level of ~10−10 of the illumination intensity.

Kerr - Fourier imaging of hidden objects in thick turbid media.

The temporal profiles of ultra-short light pulses propagating through turbid media of different lengths were measured with a streak camera. Various Fourier spatial filters were used to select the spatial frequencies of the scattered pulses. The temporal profiles of the pulses scattered by a 0.4% Intralipid solution in a cell of 5-cm thickness were significantly narrowed because of the removal of the higher-frequency components by a Fourier spatial filter.

Fourier spatial filter acts as a temporal gate for light propagating through a turbid medium.

In this article, we have presented an overview of emerging novel techniques for early-light transillumination imaging as well as nonlinear optical tomography of body organs. The use of light for probing and imaging biomedical media offers the promise for development of safe, noninvasive, and inexpensive clinical imaging modalities with diagnostic ability. The strong scattering of light by biological tissues buries the shadowgram formed by forward-propatating image-bearing photons in the background noise of multiple-scattered light. Several methods for extraction of image-bearing light that capitalize on spatial, temporal and polarization characteristics of transmitted light are reviewed. More recently emerging nonlinear-optical histopathology methods for imaging subsurface structures of tissues in terms of its local spatial symmetry and molecular content are introduced. The progress made so far indicates that some of these techniques are apt to make a transition from laboratory to useful clinical modalities.

Time-Resolved and Nonlinear Optical Imaging for Medical Applicationsa

A system for imaging an object in or behind a highly scattering medium includes a light source for illuminating the highly scattering medium with a beam of light. The light emerging form the highly scattering medium consists of a ballistic component, snake-like component and a diffuse component. A 4F Fourier imaging system including a Fourier spatial filter located at 2F is used to form a time gated image of the emerging light, the time gated image consisting primarily of the ballistic component and the snake-like component.

Ultrafast optical imaging of objects in or behind scattering media

A method for detecting the presence of one or more calcifications within a portion of a turbid medium, such as a breast tissue. According to one aspect, the method involves illuminating at least a portion of the turbid medium with light, whereby light emerges from the turbid medium consisting of a ballistic component, a snake-like component and a diffuse component, temporally and/or spatially gating the emergent light to preferentially pass the ballistic and/or snake-like components, using the temporally and/or spatially gated light to form an image of the illuminated portion of the turbid medium, and examining the image for the presence of one or more calcifications. Wavelength difference images may also be used to highlight tumors and calcification regions. The foregoing method may be used to form optical images of breast tissues, with the presence in such images of calcifications suggestive of cancer being used to identify the corresponding breast tissues as good candidates for biopsy and screening for tumors.

X. Liang

Papers

Kerr - Fourier imaging of hidden objects in thick turbid media.

Fourier spatial filter acts as a temporal gate for light propagating through a turbid medium.

Time-Resolved and Nonlinear Optical Imaging for Medical Applicationsa

Ultrafast optical imaging of objects in or behind scattering media

Optical imaging of breast tissues to enable the detection therein of calcification regions suggestive of cancer