Lecture 17 BIOE 498/598 DP 04/07/2014

1. Lecture 17BIOE 498/598 DP04/07/2014

2. 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.

3. 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).

4. 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 fluoresc and biolumin imaging • Can provide the highest acquisition speed and resolution for superficial structures • Spatial resolution quickly diminishes with depth Ntziachristos, Ripoll et al. 2005

5. 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).

6. 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

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

8. 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.

9. 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.

10. Exogenous and Endogenous Contrast Agents http://www.photobiology.info/Photomed.html

11. How Can These be Administered?

12. Influence of Administration Site on Optical Probes In Vivo Distribution

14. Labeling Mechanisms of Fluorescent Probes

15. Commonly Used Small Molecule Fluorophores Schematic representation of the region of optimal signal-to-background ratio in tissue. Hemoglobin can interfere below 700 nm, while water interferes above 900 nm. The excitation and emission regions for several dyes commonly used in optical imaging are also indicated.

16. Small Organic Fluorophores-FITC http://upload.wikimedia.org/wikipedia/commons/1/1c/Photobleaching.ogg

17. Small Organic Fluorophores-Alexa Fluor • Synthesized through sulfonation of coumarin, rhodamine, xanthene (such as fluorescein), and cyanine dyes. • Better photostability • Sulfonation makes them more hydrophilic and charged

18. Optical Properties for Commonly used Fluorophores Rhodamine Rhodamine core structure

19. Small Organic Fluorophores-Cyanine Dyes

20. Clearance Kinetics of Native IRDye 800CW

21. Time Course Accumulation of IRDye 800CW EGF in a subQ model IRDye 800CW DBCO

22. Clearance Kinetics of EGF Targeted IRDye 800CW in a non-tumor bearing mouse

23. Comparison of Different Implantation Methods in Preclinical Targeted Imaging

24. Subcutaneous and OrthotopicXenograft Models

25. Real-time in vivo imaging of invasive- and biomaterial-associated bacterial infections using fluorescently labeled vancomycin

26. (a) Imaging of a mouse with E. coli (Xen16)-induced myositis in the left hind limb and S. aureus (Xen36)-induced myositis in the right hind limb was performed with the IVIS SpectrumCT Imaging System at 8 and 24 h after intravenous administration of 1.8 mg kg−1 vanco-800CW. Left side: bioluminescence imaging (open filter, field of view (FOV) 12.8 cm, F-stop 1, 30 s acquisition time); right side: fluorescence imaging (excitation 745 nm, emission 840 nm, field of view (FOV) 12.8 cm, F-stop 2, 0.5 s acquisition time). (b) Excised muscle tissue of the E. coli-infected left leg (left) and the S. aureus-infected right leg (right) of the mouse shown in a. (c) Micro-computed tomography (CT) imaging of the mouse shown in a with bioluminescence (BLI; rainbow scale) and fluorescence (FLI; red–yellow scale) coregistration. A fluorescent signal from the bladder is detectable behind the spine. (d) Fluorescence microscopy of infected muscle tissue. A cluster of vanco-800CW-labelled Gram-positive cocci (that is, S. aureus) is indicated in the right panel (red) and a chain of Gram-negative rods (that is, E. coli) is indicated in the left panel (green). DAPI (4',6-diamidino-2-phenylindole)-stained cell nuclei are labelled green. Scale bar, 10 μm. Vanco-800CW imaging: excitation 710 nm, emission >785 nm; DAPI imaging: excitation 360 nm and emission >458 nm. Nature Communications 4, Article number: 2584

27. IR Dye 800 and SWNT Conjugates