8/6/2019 Molecular Imaging, Auto Fluorescence and Multi Spectral Unmixing- Dr.papineni

1/1 Carestream Health, Inc.

Non-invasive Multispectral Molecular Imaging in DistinguishingFluorescence Probes With Spectral Signatures in Close-Proximity

Rao V. L. Papineni*, Sean Orton, William McLaughlin, Douglas Vizard,Jens Waldeck, John Pizzonia, Seth Gammon, Gilbert Feke, and Xiaoning Si.

Carestream Molecular Imaging, 4 Research Dr., Woodbridge, CT, 06525 ,USA

*rao.papineni1@carestreamhealth.com

IntroductionA wealth of information on various macromolecules and signaling

pathways regulating diseases resulted from the scientific efforts during the

Omics era. Since then, the quest to elucidate the role of the diverse

players and pathways in vivohas been in the process. W ith optical

imaging, especially fluorescence imaging emerging as a keen front runner

in such discoveries of target molecules- investigation of multiple molecular

events simultaneously is becoming common. Researchers utilizing the

fluorescent molecular tools earlier used in the in vitro experiments,

microscopy and cell culture, face some challenges attempting in vivo

molecular imaging. Especially with molecular probes that fluoresce in the

visible wavelengths. The key challenges include the attenuation of light

by tissues, and light absorption by the endogenous macromolecules

(Fig.1). Also, it is likely that some of the molecular probes available might

have similar or overlapping spectral properties. Under these

circumstances, we propose that automated analysis of such multiple

probes can be undertaken using spectral unmixing process to overcome

issues from multiple signal identification and the tissue autofluorescence.

Here, we utilized a small animal in vivoimaging system to identify and

quantify the individual components from multiple fluorescent signals, and

demonstrate the feasibility to reliably quantitate fluorescent probes with

excitation optima as close as 20 nm.

Materials/Methods

Molecular Imaging - Wisdom To See For Maladies To Flee"Dr. Rao V. L. Papineni

estream is a trademark of Carestream Health, Inc. Carestream Molecular Imaging is a divisi on of Carestream Health, Inc. Alexa 680, and Alexa 700 are trademark of Invitrogen. Although the Carestream In-Vivo Multispectral Imaging System FXbe used for in vivoand in vitromolecular imaging of materials, researchers should be aware that the methods of preparing and viewing the materials for molecular imaging may be subject to various patent rights.

NIR Optical

autofluorescence

Multispectral unmixing to resolve multiple overlapping

signals and obtain reliable quantification.

pmole: 0

X-SIGHT 650

0

2.5

5.0

7.5

10pmole

1 53 42

6 108 97

11 1513 1412

16 2018 1917

21 2523 2422

2.5 5.0 7.5 10

Fig.1 Primary components in light attenuation of biological tissue.Adapted from Levi et al, Cancer 101 (1), 2843-2850, 2004.

Fig.2 Schematic showing wells allocated for the X-SIGHT650 and X-SIGHT 691 samples in the center of a 96-wellplate. X-SIGHTS are fluorescent dye encapsulated

nanoparticles used for targeting or as imaging agents.Previouslydescribed - Papineni and Wood, 2007.http://www.labmanager.com/?articles.view/articleNo/3153/article/Managing-and-Meeting-Challenges-With-Multiple-Fluorescence-Probe-Images

Excitation Spectra and Gaussian ModelsX-SIGHT650

Excitation Spectra and Gaussian ModelsX-SIGHT691

Fig. 4 Excitation spectra of individual probes. Thespectrum of each probe (yellow line) was measuredby averaging pixels from wells that containedsamples of only one probe or the other. A numericalmodel (green curve) was constructed by fitting a setof Gaussian functions to the measured spectra, twoGaussians (white lines) were used for each probe.

570 EX 590 EX 610 EX 630 EX

650 EX 670 EX 690 EX

Fig.3 Representative images from the rawdata cube showing the 5x5 grid of samplewells containing dilutions of X-SIGHT691and X-SIGHT650. Each image wasobtained using different excitation filterwith the emission set at 750nm. Theintensity scale used was identical to allimages in the series, and were imageswere not subjected to illuminationcorrection.

Fig. 5 Validation of the unmixing procedure. unmixed

images of the probes, and the images taken of a plateswith a 5x5 grid of plates containing only a single probe (totheir right).

Quantitative assessments of the fluorescence probe

intensities after multispectral unmixing were determinedand had an average error of only 3.22%.

UNMXED X-SIGHT 650 IMAGE X-SIGHT 650 Unmixed X-SIGHT 691 Image X-SIGHT 691

Automated Protocol User InterfaceThe automated system provides unique co-registration capabilities for precise localization of optical or nuclearmolecular signals on the anatomical X-ray images of an animal. To this 4 million pixel cooled CCD based capturesystem we have added motorized automation and programmable software control of the foll owing: 10, 15 or 29 position excitation filter wheel,

4 position emission filter wheel, Zoom, focus (including auto focus) and aperture of the 10x zoom lens 5 Position X-ray filter wheel Radioisotopic and Radiographic phosphor screen sliders

A software user interface links multiple types of image capture sessions in a step by step format. The UI enablesuser prompts to be included in each step and provides image math, multispectral cube unmixing and region ofinterest (ROI) image analysis steps after each image capture session. Simple multi-image protocols to highly

complex multispectral, multimodal, time lapse protocols have been created saved and recalled to be executed in a

highly repeatable, single push button manner.

in vitroApplication: X-SIGHT 650 (Absorption 650nm; Emission 673nm) and X-SIGHT 691 (Absorption691nm; Emission 715nm) were used individually or in combination for the purpose. 0, 2.5, 5.0, 7.5, and10 pmoles of the nanoparticles were dispensed i nto five wells (as in the schematic-Fig. 2).

Computational approach to quantitatively unmix overlapping Fluorescent spectra

In Vivo Spectral Unmixing

Fluorescent probes Alexa 680 and Alexa700 with excitation optima as close as 20nm ( Fig.6; right panels) was injected into

mice and used to demonstrate thefeasibility of multispectral unmixing

process. Multiple fluorescence signals fromprobes, endogenous entities are noted to

be unmixed reliably and quantitatively.

Alexa 680Alexa 700

Gaussian model Alexa 680

Gaussian model Alexa 700

Fluorescent Probe 700 nm Fluorescent Probe 680 nm

EX: 550 nm 600 nm 630 nm 650 nm 670 nm 690 nm

Fig. 7 Fluorescent images- Multispectralcube. EM: 790; 30 sec Exposure; f-Stop 2.8.

Fig. 8 Mouse image

Raw image showing skinautofluorescence, and the

two probes injected.

Fig. 9 Overlay of the spectralunmixed fluorescent signals (pseudo

colored) on the X-ray image.Quantification showed that 99% of

680 nm probe was separated fromthe skin and 700 nm probe signals.

While 95% of the fluorescence fromthe nearly overlapping Alexa 700nm

could be achieved using the sameprocedure.

Summary

A sensitive methodology to unmixmultiple spectrally overlapping fluorescentsignals (20 nm excitation maxima apart)along with the natural florescenceemanating from endogenous tissues isdetermined.

The quantification of the molecular

probes and visualization in its anatomicallocales demonstrate the feasibility of using

high-throughput optical imagingapproaches elsewhere- where one cansimultaneous analyse of multiple signaling

events in a non invasively manner.