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Nonlinear photoacoustic signal amplification from single targets in absorption background.

Sarimollaoglu M, Nedosekin DA, Menyaev YA, Juratli MA, Zharov VP - Photoacoustics (2014)

Bottom Line: This approach was demonstrated by using nonlinear PA flow cytometry platform for label-free detection of circulating melanoma cells in blood background in vitro and in vivo.Nonlinearly amplified PA signals from overheated melanin nanoclusters in melanoma cells became detectable above still linear blood background.Nonlinear nanobubble-based photoacoustics provide new opportunities to significantly (5-20-fold) increase PA contrast of single nanoparticles, cells, viruses and bacteria in complex biological environments.

View Article: PubMed Central - PubMed

Affiliation: Phillips Classic Laser and Nanomedicine Laboratories, Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR USA 72205.

ABSTRACT
Photoacoustic (PA) detection of single absorbing targets such as nanoparticles or cells can be limited by absorption background. We show here that this problem can be overcome by using the nonlinear photoacoustics based on the differences in PA signal dependences on the laser energy from targets and background. Among different nonlinear phenomena, we focused on laser generation of nanobubbles as more efficient PA signal amplifiers from strongly absorbing, highly localized targets in the presence of spatially homogenous absorption background generating linear signals only. This approach was demonstrated by using nonlinear PA flow cytometry platform for label-free detection of circulating melanoma cells in blood background in vitro and in vivo. Nonlinearly amplified PA signals from overheated melanin nanoclusters in melanoma cells became detectable above still linear blood background. Nonlinear nanobubble-based photoacoustics provide new opportunities to significantly (5-20-fold) increase PA contrast of single nanoparticles, cells, viruses and bacteria in complex biological environments.

No MeSH data available.


Related in: MedlinePlus

Principle of nonlinear PAFC. (a) Schematics. (b) Absorption profiles of an RBC and a melanoma cell. (c) Linear and nonlinear PA signal dependence on energy fluence from RBCs and melanoma cells. (d) PA signal traces in linear and nonlinear modes. (e) Dependence of signal-to-noise ratio (SNR) on averaging in the presence of photothermal bleaching.
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fig0005: Principle of nonlinear PAFC. (a) Schematics. (b) Absorption profiles of an RBC and a melanoma cell. (c) Linear and nonlinear PA signal dependence on energy fluence from RBCs and melanoma cells. (d) PA signal traces in linear and nonlinear modes. (e) Dependence of signal-to-noise ratio (SNR) on averaging in the presence of photothermal bleaching.

Mentions: When a CTC with intrinsic absorbing markers such as melanin or exogenous labels is illuminated by laser pulses, the absorbed energy is converted to heat which in turn induces thermal expansion of the heated zones leading to generation of PA waves referred as PA signals (Fig. 1a). Amplitude of a PA signal in linear mode is proportional to laser energy and absorption coefficient of the target. In positive-contrast PAFC, signal-to-background ratio (SBR) is determined by the ratio of PA signal amplitude from single CTC to background signal amplitude which is superposition of signals from individual red blood cells (RBCs) in the detected volume and noise of different origins (e.g., electronic, acoustic, fluctuating RBC number, or instability of laser energy [typically 3–5%]). According to multiple verifications using in vivo mouse model and ex vivo human blood spiked with melanoma cells, PAFC in near-infrared (NIR) spectral range (e.g., at 820 nm or 1064 nm) can detect single pigmented melanoma CTC in the presence of ∼100–300 RBCs that is also in line with coefficients of absorption for blood and melanin (see details in Ref. [14]). However, linear PAFC can miss low pigmented cells in blood background [36]. This problem can be partly overcome in nonlinear PAFC based on the differences in PA signal dependences on laser energy from targets and background. According to previous findings [8,9,16–21,26,29,37] and phenomenological model [8], absorbing targets (e.g., NPs, dyes, and chromophores) exhibit multistage behaviors when the energy fluence (E) is increased (Fig. 1c, black curve): (1) A gradual linear increase at a low fluence, En (n = 1); (2) absorption saturation (n ∼ 0.5–0.9); (3) slight nonlinear signal increase (n ∼ 1.2–1.6) related with temperature-dependent thermophysical parameters; (4) strong nonlinear signal amplification (n ∼ 2–5) related with nanobubble formation and its spatial and temporal overlapping; (5) secondary signal saturation (n ∼ 0–0.5) and even decrease related with nanobubble size saturation or target degradation, respectively; and (6) second strong signal amplification due to laser-induced explosion. Depending on the target's origin, only some of the aforementioned mechanisms may occur or are dominant, or the boundary between them may overlap. In view of the described phenomena, PA techniques can distinguish a target with strong nonlinearly amplified PA signals at certain laser energy which produce either linear or saturated PA signals from backgrounds (Fig. 1c). Specifically, the ability of nonlinear PAFC to detect low pigmented melanoma cells is based on high local absorption in melanin nanoclusters which at certain laser energy is sufficient to generate nanobubbles compared to lower local absorption of hemoglobin (Hb) in RBCs which generates linear PA signal only. Despite the average absorption of whole RBC with homogenously distributed Hb is relatively higher than of melanoma cell, local absorption of heterogeneously distributed melanin NPs and especially melanin nanoclusters in melanoma cell is higher (Fig. 1b), which leads to nanobubble formation, and hence PA signal amplification only in melanoma cells. As a result, at low laser energy the PA signal from a single low pigmented melanoma cell can be below blood background, while at higher energy nonlinearly amplified PA signals from overheated melanin nanoclusters become detectable above blood background (Fig. 1d). It should be noted that nanobubble-induced PA signal amplification is achieved when the laser energy fluence exceeds the nonlinear threshold by only 20–30%, while the cell photodamage threshold is at least 3–5 times higher. Nevertheless, at certain high energy, cells or NPs can be destroyed by strong PT and bubble related phenomena, that reduces the sample's absorption and hence PA signals. This photobleaching of PT origin (PT bleaching) is associated with overheating of absorbing zones and NPs that can lead to their melting, shape modification, destruction and explosion [9,14,16,18,31,38]. PT bleaching depends on both laser energy and number of laser pulses [16,17]. As a result, increasing the number of averaged PA signals (which is equal to number of laser pulses) from the same target may lead to increase SBRs when there is no PT bleaching effect, or can lead to decreased SBRs by PT bleaching due to PA signal degradation and increased average noise level (Fig. 1e).


Nonlinear photoacoustic signal amplification from single targets in absorption background.

Sarimollaoglu M, Nedosekin DA, Menyaev YA, Juratli MA, Zharov VP - Photoacoustics (2014)

Principle of nonlinear PAFC. (a) Schematics. (b) Absorption profiles of an RBC and a melanoma cell. (c) Linear and nonlinear PA signal dependence on energy fluence from RBCs and melanoma cells. (d) PA signal traces in linear and nonlinear modes. (e) Dependence of signal-to-noise ratio (SNR) on averaging in the presence of photothermal bleaching.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048727&req=5

fig0005: Principle of nonlinear PAFC. (a) Schematics. (b) Absorption profiles of an RBC and a melanoma cell. (c) Linear and nonlinear PA signal dependence on energy fluence from RBCs and melanoma cells. (d) PA signal traces in linear and nonlinear modes. (e) Dependence of signal-to-noise ratio (SNR) on averaging in the presence of photothermal bleaching.
Mentions: When a CTC with intrinsic absorbing markers such as melanin or exogenous labels is illuminated by laser pulses, the absorbed energy is converted to heat which in turn induces thermal expansion of the heated zones leading to generation of PA waves referred as PA signals (Fig. 1a). Amplitude of a PA signal in linear mode is proportional to laser energy and absorption coefficient of the target. In positive-contrast PAFC, signal-to-background ratio (SBR) is determined by the ratio of PA signal amplitude from single CTC to background signal amplitude which is superposition of signals from individual red blood cells (RBCs) in the detected volume and noise of different origins (e.g., electronic, acoustic, fluctuating RBC number, or instability of laser energy [typically 3–5%]). According to multiple verifications using in vivo mouse model and ex vivo human blood spiked with melanoma cells, PAFC in near-infrared (NIR) spectral range (e.g., at 820 nm or 1064 nm) can detect single pigmented melanoma CTC in the presence of ∼100–300 RBCs that is also in line with coefficients of absorption for blood and melanin (see details in Ref. [14]). However, linear PAFC can miss low pigmented cells in blood background [36]. This problem can be partly overcome in nonlinear PAFC based on the differences in PA signal dependences on laser energy from targets and background. According to previous findings [8,9,16–21,26,29,37] and phenomenological model [8], absorbing targets (e.g., NPs, dyes, and chromophores) exhibit multistage behaviors when the energy fluence (E) is increased (Fig. 1c, black curve): (1) A gradual linear increase at a low fluence, En (n = 1); (2) absorption saturation (n ∼ 0.5–0.9); (3) slight nonlinear signal increase (n ∼ 1.2–1.6) related with temperature-dependent thermophysical parameters; (4) strong nonlinear signal amplification (n ∼ 2–5) related with nanobubble formation and its spatial and temporal overlapping; (5) secondary signal saturation (n ∼ 0–0.5) and even decrease related with nanobubble size saturation or target degradation, respectively; and (6) second strong signal amplification due to laser-induced explosion. Depending on the target's origin, only some of the aforementioned mechanisms may occur or are dominant, or the boundary between them may overlap. In view of the described phenomena, PA techniques can distinguish a target with strong nonlinearly amplified PA signals at certain laser energy which produce either linear or saturated PA signals from backgrounds (Fig. 1c). Specifically, the ability of nonlinear PAFC to detect low pigmented melanoma cells is based on high local absorption in melanin nanoclusters which at certain laser energy is sufficient to generate nanobubbles compared to lower local absorption of hemoglobin (Hb) in RBCs which generates linear PA signal only. Despite the average absorption of whole RBC with homogenously distributed Hb is relatively higher than of melanoma cell, local absorption of heterogeneously distributed melanin NPs and especially melanin nanoclusters in melanoma cell is higher (Fig. 1b), which leads to nanobubble formation, and hence PA signal amplification only in melanoma cells. As a result, at low laser energy the PA signal from a single low pigmented melanoma cell can be below blood background, while at higher energy nonlinearly amplified PA signals from overheated melanin nanoclusters become detectable above blood background (Fig. 1d). It should be noted that nanobubble-induced PA signal amplification is achieved when the laser energy fluence exceeds the nonlinear threshold by only 20–30%, while the cell photodamage threshold is at least 3–5 times higher. Nevertheless, at certain high energy, cells or NPs can be destroyed by strong PT and bubble related phenomena, that reduces the sample's absorption and hence PA signals. This photobleaching of PT origin (PT bleaching) is associated with overheating of absorbing zones and NPs that can lead to their melting, shape modification, destruction and explosion [9,14,16,18,31,38]. PT bleaching depends on both laser energy and number of laser pulses [16,17]. As a result, increasing the number of averaged PA signals (which is equal to number of laser pulses) from the same target may lead to increase SBRs when there is no PT bleaching effect, or can lead to decreased SBRs by PT bleaching due to PA signal degradation and increased average noise level (Fig. 1e).

Bottom Line: This approach was demonstrated by using nonlinear PA flow cytometry platform for label-free detection of circulating melanoma cells in blood background in vitro and in vivo.Nonlinearly amplified PA signals from overheated melanin nanoclusters in melanoma cells became detectable above still linear blood background.Nonlinear nanobubble-based photoacoustics provide new opportunities to significantly (5-20-fold) increase PA contrast of single nanoparticles, cells, viruses and bacteria in complex biological environments.

View Article: PubMed Central - PubMed

Affiliation: Phillips Classic Laser and Nanomedicine Laboratories, Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR USA 72205.

ABSTRACT
Photoacoustic (PA) detection of single absorbing targets such as nanoparticles or cells can be limited by absorption background. We show here that this problem can be overcome by using the nonlinear photoacoustics based on the differences in PA signal dependences on the laser energy from targets and background. Among different nonlinear phenomena, we focused on laser generation of nanobubbles as more efficient PA signal amplifiers from strongly absorbing, highly localized targets in the presence of spatially homogenous absorption background generating linear signals only. This approach was demonstrated by using nonlinear PA flow cytometry platform for label-free detection of circulating melanoma cells in blood background in vitro and in vivo. Nonlinearly amplified PA signals from overheated melanin nanoclusters in melanoma cells became detectable above still linear blood background. Nonlinear nanobubble-based photoacoustics provide new opportunities to significantly (5-20-fold) increase PA contrast of single nanoparticles, cells, viruses and bacteria in complex biological environments.

No MeSH data available.


Related in: MedlinePlus