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Two photon optical fiber probe for detection and analysis of tumors

Although flow cytometry and confocal microscopy are two most important and widely used fluorescence-based technologies for biomedical applications, they have several limitations.  They require tissue processing and give out only semi-quantitative data.  Whole-body fluorescence imaging is also difficult because of the absorption and scattering of light in the tissue.  Our laboratory has developed a two-photon optical fiber fluorescence (TPOFF) system to quantify tissue fluorescence as an alternate approach to the conventional techniques.  The advantage of a “two-photon” over a “one photon” excitation system is that the former can be used for the simultaneous detection of fluorochromes with a wide range of excitation wavelengths.  Because of the small excitation volume determined by the nonlinear absorption, the two photon system will have increased spatial resolution in tissues.  Moreover, the near-IR laser light used in the two-photon system reduces photo-bleaching and photo damage comparing with the energetic UV photons used in one-photon system.

TPOFF instrumentation setup

Figure 1. TPOFF instrumentation setup.

The schematic diagram of the TPOFF system is shown Figure 1. A homemade Ti:sapphire laser providing 80-femtosecond pulses at 830 nm with an 80-MHz repetition rate is used for two-photon excitation. The laser pulses are delivered through an optical fiber, and the output end of the optical fiber is immersed in the tissue sample. The two photon fluorescence emitted from the tissue is collected through the same fiber and separated from the excitation light by a dichroic mirror. Then it is focused onto the entrance slit of a spectrometer, and filtered with a short-pass filter before being detected by a photon-counting photomultiplier tube. This unique detection configuration allows one to conduct in vivo biosensing in real-time.

The TPOFF counts (red) shown are the mean obtained for five different internal regions of the tumors.  The flow cytometric FL1 fluorescence (green) is the mean obtained for 10,000 cells.  The microscopic image pixel intensities (blue) shown are the mean of five different areas of 10 pixels wide and 640 pixels long.

We initially demonstrated the sensitivity of the TPOFF probe to quantify the fluorescence of tumors that express different levels of GFP.  TPOFF measurements were performed ex vivo in multiple internal regions of the tumors which express different levels of GFP, by inserting the fiber into the tumor through 27-gauge needle.  Figure 2 shows a comparison of the TPOFF counts taken for the GFP tumors, with flow cytometric values from isolated cells, as well as with microscopic image pixel intensity.  The results show that the TPOFF sensitivity is similar to conventional techniques such as flow cytometry and confocal microscopy.

Comparison of TPOFF measurements to flow cytometry and confocal microscopy

Figure 2. Comparison of TPOFF measurements to flow cytometry and confocal microscopy. 

.  Uptake of fluorescent nanoparticles in tumors of live mice, determined by the TPOFF probe

Figure 3.  Uptake of fluorescent nanoparticles in tumors of live mice, determined by the TPOFF probe.

15 nmols each of the targeted nanodevice G5-6T-FA or the non-targeted conjugate G5-6T were injected into the tail vein of KB tumor-bearing mice, and the TPOFF counts were taken at the different time points shown.

We have determined the uptake of the intravenously injected generation 5 (G5) dendrimer nanoparticles onto which the dye 6TMARA (6T) and the targeting molecule folic acid (FA) were conjugated covalently.  The real-time uptake of the synthesized “G5-6T-FA” performed in anesthetized live mice is shown in Figure 3.  The results show increased uptake of the targeted conjugate as compared to the control conjugate that reached a maximum at the 2 hour time point tested.

Our recent studies have shown a 4 to 6-fold increased sensitivity of a double-clad fiber vs. a single mode fiber that we have used in our previous studies.  We are currently testing the applicability of the double-clad fiber-based TPOFF probe as a minimally invasive technique to quantify the expression of cancer signatures such as HER2 in the breast tumor.
For more information, see:

Thomas P, Ye JY, Yang C, Myaing M, Majoros IJ, Kotlyar A, Cao Z, Norris TB, Baker JR, Jr.: Tissue distribution and real–time fluorescence measurement of a tumor–targeted nanodevice by a two photon optical fiber fluorescence probe. Proc. of SPIE, 2006:6095, 1–7.

Thomas TP, Myaing MT, Ye JY, Candido K, Kotylar A, Beals J, Cao P, Keszler B, Patri AK, Norris TB, Baker JR, Jr.: Detection and Analysis of Tumor Fluorescence Using a Two–Photon Optical Fiber Probe. Biophysical Journal, 2004:86(6), 3959–3965.

Myaing MT, Ye JY, Norris TB, Thomas T, Baker JR Jr., Wadsworth WJ, Bouwmans G, Knight JC, and Russell PS.: Enhanced two–photon biosensing with double–clad photonic crystal fibers. Optics Letters, 2003:28(14), 1224–1226.

Ye JY, Myaing MT, Norris TB, Thomas TP, Baker JR, Jr.:  Biosensing based on two–photon fluorescence measurements through optical fibers. Optic Letters, 2002:27(16), 1412–1414.

Ye, J.Y., Divin, C.J., Baker, JR, Jr., Norris, T.B.: Whole spectrum fluorescence detection with ultrafast white light excitation.  Optics Express, 15, No.16, 2007.

Eric R. Tkaczyk, Cheng Frank Zhong, Jing Yong Ye, Steve Katnik, Andrzej  Myc, Kathryn E. Luker, Gary D. Luker, James R. Baker, Jr., and Theodore B. Norris, "Two-photon, Two-color in Vivo Flow Cytometry to Noninvasively Monitor Multiple Circulating Cell Lines," Multiphoton Microscopy in the Biomedical Sciences VI, Proceedings of SPIE 6631, 2007.

Thomas TP, Ye JY, Chang Y, Kotlyar A, Cao Z, Majoros IJ, Norris TB, Baker, Jr., JR.  Investigation of Tumor cell targeting of a dendrimer nanoparticle using a double-clad optical fiber probeJ. Biomed. Optics., 2007, In press.

Eric R. Tkaczyk, Cheng Frank Zhong, Jing Yong Ye, Andrzej Myc, Thommey Thomas, Zhengyi Cao, Raimon Duran-Struuck, Kathryn E. Luker, Gary D. Luker, Theodore B. Norris and James R. Baker, Jr.  In Vivo Monitoring of Multiple Circulating Cell Populations Using Two-photon Flow Cytometry. Optics Communication, 2007, In press.



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