Use of coherent control methods through scattering biological tissue to achieve functional imaging
- Departments of *Chemistry and †Physics and Astronomy, Michigan State University, East Lansing, MI 48824
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Communicated by James L. Dye, Michigan State University, East Lansing, MI, October 18, 2004 (received for review June 30, 2004)
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Fig. 1.Coherence degradation and pulse transformation as a function of scattering path length. As a short pulse of light enters a scattering medium, coherent, or ballistic, photons (narrow black peaks) are lost exponentially. The scattered photons (broad gray peaks), which lag in time, lose their coherence and are randomly delayed.
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Fig. 2.Molecular formula and absorption spectra of HPTS in acidic and alkaline pH. Note that the loss of the hydroxylic proton leads to a large change in the absorption spectrum. a.u., arbitrary units.
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Fig. 3.Experimental setup used for selective two-photon (2P) excitation through scattering tissue. The sample consists of three 1-mm capillaries containing an acidic solution (soln.) of HPTS submerged in a quartz cell filled with an alkaline solution of HPTS. The sample was raster scanned (without and with scattering tissue) in the focal plane of the beam during data acquisition to obtain the images.
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Fig. 4.Spectroscopic characterization of the laser pulses and the spectral phases used to control their nonlinear excitation properties. (A) Experimental spectrum of the femtosecond laser pulses centered near 830 nm. Three different phase functions were used in the experiment: TL pulses with a flat spectral phase and shaped pulses optimized for excitation of HPTS in acidic and alkaline environments, BPS06 and BPS10, respectively. (B) Experimental SHG spectra obtained from TL pulses and SHG spectra obtained when the phase functions BPS06 and BPS10 were introduced by the pulse shaper. Note that BPS06 suppresses SHG at longer wavelengths, and BPS10 suppresses SHG generation at shorter wavelengths. a.u., arbitrary units.
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Fig. 5.Experimental sample with and without biological tissue imaged by a conventional camera and by two-photon imaging. (A and B) Photographs of the sample obtained under white-light illumination in the absence and presence of biological tissue. In both of these photographs, the three capillaries in the sample are difficult to distinguish. (C and D) Two-photon excitation images obtained by raster scanning the sample without and with scattering tissue, respectively. The walls of the capillary tubes, which appear as vertical lines, are ≈300 μm thick and are clearly visible in both images. Note that comparable image quality is obtained in the presence and absence of biological tissue. C and D were obtained with TL pulses, which are not capable of discriminating between the two different pH solutions.
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Fig. 6.Coherent control was used here to obtain pH-sensitive functional imaging. (A and B) Images obtained by two-photon excitation using the optimized phases BPS06 and BPS10, respectively. Note that the signal from the 1-mm capillary tubes is enhanced for BPS06 (A) and suppressed for BPS10 (B). (C) A functional image highlighting the contrast possible by using coherent control. This image was obtained by taking the ratio of the data obtained by BPS06/BPS10. (D) The functional image obtained when a slice of biological tissue was placed in front of the sample. Note that the presence of the tissue reduces the overall signal-to-noise ratio, but the discrimination between acidic and alkaline HPTS is conserved. The contrast in the functional images can be further enhanced by using false color (C Upper and D Upper). Higher values are shown in red, and lower values are shown in blue.
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Fig. 7.Loss of coherent photons as a function of scattering. The transmitted ballistic photons from TL (squares) and shaped (circles) pulses were measured as a function of scattering. The phase shape for these measurements was chosen to suppress SHG generation evenly through the entire spectrum (see text). The intensity of the ballistic photons measured at 800 nm (filled symbols) was found to decay exponentially for both TL and shaped pulses. The intensity of the coherent photons was measured after SHG in a thin SHG crystal (open symbols). Greater scattering by a factor of 2 was recorded in this case, given the square dependence of SHG, confirming that only ballistic photons generate the second harmonic light. The spectrum and phase for the TL and shaped pulses used in this measurement are shown in Upper Inset. The spectrum of the SHG output from TL and shaped pulses are shown in Lower Inset. Note that the spectra of TL and shaped pulses are virtually identical (Upper Inset), but the phase structure of the shaped pulses decreases their ability to generate second harmonic light (Lower Inset). We find the same slope for both TL and shaped pulses; hence, they experience the same rate of scattering. a.u., arbitrary units.
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Fig. 8.SHG spectra generated for TL pulses and shaped pulses optimized for pH 10 without a scattering medium and after propagating through a 15% skim milk-in-water solution (equivalent to 2.1 scattering lengths). The spectrum after scattering was normalized to the intensity of the unscattered spectrum. We found that scattering does not change the observed SHG spectrum except for the overall intensity. (Inset) Three FluoSpheres of 15-μm diameter behind a 1-mm-thick slice of biological tissue. This image demonstrates that high resolution (μm) can be achieved by the shaped pulses after they transmit through biological tissue. a.u., arbitrary units.
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Fig. 9.Characterization of spectral phase of the laser pulses by using the MIIPS method without (solid line) and with (dashed line) biological tissue. The raw data are shown in Insets. Note that the presence of the tissue reduces the overall signal-to-noise ratio in the bottom MIIPS trace. Phase distortion is minimal for the longer wavelengths (820–870 nm). For shorter wavelengths, phase distortion is more significant. However, even at 790 nm, the 0.2-rad distortion is 1 order of magnitude smaller than the value of π used for the BPS functions, which is why binary phase functions successfully achieved the selective two-photon excitation that is required for functional imaging. Int., intensity; a.u., arbitrary units.
Footnotes
- Copyright © 2004, The National Academy of Sciences
