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Optical Imaging (Basics) (BIOE 498/598 DP) 03/31/2014. Outline. Understand light Light propagation in biological tissues Fluorescence, phosphorescence… Importance of NIR Tissue oximetry Exogenous contrast agents Imaging with light Therapy with light Dual modality systems.

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Optical Imaging (Basics)

(BIOE 498/598 DP)




  • Understand light
  • Light propagation in biological tissues
  • Fluorescence, phosphorescence…
  • Importance of NIR
  • Tissue oximetry
  • Exogenous contrast agents
  • Imaging with light
  • Therapy with light
  • Dual modality systems

Comparative energy spectra and location of non-ionizing light in the electromagnetic spectrum


2008 Nobel in Chemistry Awarded for“in vivo Optical Contrast Agent”

-- Optical contrast

-- In vivo imaging

-- Spectroscopy

-- Metabolism-based


Light propagation in biological tissues

In this equation, A is a function of refractive index, µa and µs are the absorption and scattering coefficients of biological tissues, respectively.


Energy diagram for photo-physical events related to absorption and fluorescence. The fluorescence lifetime is the average time that a population of fluorophores remains in the excited sate (S1-S3) after absorption of photons


How PhotonsInteract with Biological Tissue

Scattered and reflected



Scattered and absorbed

mal, msl, g

Scattered and transmitted


In NIR Region, Hb and HbO are Major Sensitive Absorber

The NIR window is ideally suited for in vivo imaging because of minimal light absorption by hemoglobin (<650 nm) and water (>900 nm). 


Advantage of NIR-NIR imaging system

Near infrared (NIR) emission by NIR excitation is observed using a NIR-NIR system. Due to weaker scattering and absorption, NIR light can penetrate deeper into/from tissues. In contrast, excitation light in the visible (VIS) region cannot reach the imaging target in tissues in the conventional VIS-VIS imaging. In upconversion (NIR-VIS) imaging, although NIR excitation light can reach its target in tissues, only a weak VIS emission can be obtained.


What Near Infrared Light Can Measure?

  • Absorption measurement
    • Tissue hemoglobin concentration
    • Tissue oxygen saturation
    • Cytochrome-c-oxidase concentration
    • Melanin concentration
    • Bilirubin, water, glucose, …
  • Scattering measurement
    • Lipid concentration
    • Cell nucleus size
    • Cell membrane refractive index change

Why Tissue Oximetry?

  • Tissue oxygenation and hemoglobin concentration are sensitive indicators of viability and tissue health.
  • Many diseases have specific effects on tissue oxygen and blood supply: stroke, vascular diseases, cancers, …
  • Non-invasive, real time, local measurement of tissue O2 and HbT is not commercially available

Why Near Infrared? Pros and Cons

  • Advantages:
    • Non-invasive
    • Non-radioactive
    • Real time functional imaging
    • Portable
    • Low cost
    • Tissue physiological parameters
    • Potential of molecular sensitivity
  • Disadvantages:
    • Low spatial and depth resolution
    • Hard to quantify

Schematic diagram of how scattered, absorbed, or re-emitted photons can be used to obtain diagnostic information in living tissue.


Optical Imaging Detects Single Stem Engraftment

Hematopoesis from a single stem cell

Cao et al. Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc Nat AcadSci USA. 2004;101(1):221-226.


Optical Imaging Detects Single Stem Cells

Optically labeled stem cells can be seen singly in vivo in bone marrow

Proc Natl AcadSci 2009 from University of Tsukuba, Japan and Univ. of Michigan Medical School.


Cells can be detected in whole blood with ordinary pathology labels

Real-time imaging of labeled probes in 1-10 cc whole blood

  • 4 billion cells per cc of blood
  • Large volume cell imaging
  • 1 min collection, 5 sec imaging time
  • Useful for:
  • − Circulating rare cell detection − Early sepsis detection

(Benaron et al, 2011 Project with Stanford Stem Cell Center, Sloan-Kettering Cancer Center)


First clinical translation of fluorescent probes

(Top) Clinical IBMI system installed for breast cancer surgery at the University Medical Center, Groningen, Netherlands. (Bottom) Real-time visualization of ovarian cancer surgery on a patient injected with a fluorescent folate-targeting probe (from van Dam G., et. al. Nature Medicine 2011).


ICG Intraoperative Coronary Imaging System

  • A post-CABG intraoperative image shows no flow in graft
  • (arrow)
  • Revised, and graft working prior to closure
  • (from Novadaq, 2008)

Three-dimensional rendering of bones, skin and lung based on XCT data and FMT reconstruction of a K-ras mouse with lung tumors.

Nature Methods 29(6); 615-620 (2012).


Optical imaging geometries for fluorescence detection demonstrating (a) Planar Reflectance, (b) Diffuse Reflectance and (c) Diffuse Transillumination with multiple source (S1-S4) and detector (D1-D4) locations.


Planar Reflectance Imaging

Example planar reflectance imaging system setup for detection of fluorescence in mouse cancer model. (b) Bright field and (c) fluorescence images of mouse after intravenous administration of tumor-targeted molecular probe in mouse with subcutaneous tumor (arrow).


Planar Reflectance Imaging

  • Camera-based, full-field detection
  • Good for fast and low-cost screening of PK and bio-d of probes
  • Simplest and most common geometry for preclinical instrumentation used for fluorescand biolumin imaging
  • Can provide the highest acquisition speed and resolution for superficial structures
  • Spatial resolution quickly diminishes with depth

Ntziachristos, Ripoll et al. 2005)


Planar Reflectance Imaging

(Clinical Use)

  • Fluorescence endoscopy for urologic surgery(van den Berg, van Leeuwen et al. 2012)
  • Robot-assisted laparoscopic surgery(Tobis, Knopf et al. 2012)
  • Fluorescence guided surgery for brain cancer(Roberts, Valdes et al. 2012)
  • Ovarian cancer(van Dam, Themelis et al. 2011).

Planar Reflectance Imaging

(Clinical Use)-Obstacles

  • In the visible wavelength region-background signal from endogenous fluorophores.
  • Multispectral imaging can be used to separate the signal of interest from these background signals for improved visualization and quantification

Carotid endarterectomy specimen in white light (left), near-infrared fluorescence signal before (autofluorescence, middle) and after incubation with MMP-sensitive activatableprobe (MMPSense, right) within the IVIS Spectrum.


Diffuse reflectance imaging

  • Utilizes reflectance geometry but with focused excitation and detection of light.
  • uses the diffuse nature of light propagation in tissues as a means to extend the depth sensitivity.
  • DRI gave better contrast than planar reflectance systems for imaging at depths greater than 6 mm.(de la Zerda, Bodapati et al. 2010)
  • The depth sensitivity of DRI is related to the separation of the excitation source from the detector.

Approaches of NIR fluorescent imaging probes

Isotope and fluorochrome reporters can be used interchangeably for nonspecific and targeted agents; however, fluorochromes can also be used to make activation-sensitive agents for read-out of protein function.


Exogenous and Endogenous Contrast Agents