Graduate Category: Physical and Life Sciences Degree Level: Master ...
Graduate Category: Physical and Life Sciences Degree Level: Master of Science Abstract ID#209
Power Dependence of Step-wise Multi-photon Activation Fluorescence (SMPAF) of Melanin ABSTRACT Melanin is regarded as the most enigmatic pigments/biopolymers found in most organisms. Our preliminary studies show that melanin goes through a step-wise three-photon absorption process when the fluorescence is activated with high laser intensity. The three-photon activation has been observed to require less laser power than what would be expected from a nonβlinear optical process. In this paper, we examined the power dependence of the activation process at 830nm and 920nm. We have conducted research using different laser powers to activate the melanin in a point-scanning mode and recorded as the functions of time. A sequence of experiments indicates the relationship between the activation power, energy and time dependence so that we can optimize the power level. The signal is not strongly dependent on the activation wavelength.
STATE OF THE ART Melanin is an ubiquitous biological pigment in most organisms produced by melanocytes. Malignant melanoma, the most aggressive skin cancer, is a malignant proliferation of melanocytes. Early visual detection of melanoma is critical for melanoma management. The current clinical diagnosis method is lengthy and painful and the histopathologic diagnoses vary depending on the expertise of the dermatopathologist. Confocal reflectance microscopy (CRM) is well-known for βoptical sectioningβ of tissue, which is the imaging of thin sections of the sample at both high resolution and contrast without physically dissecting the tissue. CRM has been used for detection of melanin, providing strong contrast. However, the specificity and sensitivity of melanin detection using CRM is low due to the background signal from other high-contrast materials.
πΉβ
πΌ3
Beam Splitter 1
πΉ β πΌ3
Excited State
S3
Zetong Gua,b, Zhenhua Laia,b,Xi Zhanga,b,Paula Arambel b,c,Charles DiMarzioa,b,c a Electrical and Computer Engineering Department, Northeastern University b Bernard M. Gordon Center for Subsurface Sensing and Imaging System c Mechanical Engineering Department, Northeastern University, Boston, MA, USA
Beam Splitter 2
Ti:Sapphire Laser
Quarter Wave Plate
Dichroic Mirror Objective
XY Scanners
Sample
S2
Virtual States
Real States
Cuvette
Reflected Light
S1
Simultaneous 3-photon excitation
from Sample Power
Ground State
S0
PMT / Camera
Figure 2 Layout of the KECK 3D fusion multi-modal microscope. Only modalities and functions related to this work are shown in the graph. (c) CRM
(b) SMPAF
50 um
APD
Meter
Stepwise 3-photon excitation
πΌ3 10 For Femtosecond laser , 3 = 10 πΌ Figure 1 Simultaneous vs. Stepwise
(a) MPFM
Filter Box
(d) Composite
50 um
50 um
(e) Brightfield
50 um
50 um
Figure 3 Images of mouse melanoma at the same position using different imaging modalities. (a) MPFM, (b) SMPAF, (c) CRM, (d) composite image of (b) (green channel) and (c) (blue channel), and e) Brightfield. By combining CRM with SMPAF, we can locate the melanin, with respect to other skin components. 1 11.3mw 15.9mw 20.7mw 39.8mw
0.9 0.8
1 0.9 y13 vs. x1 fitting curve
0.8 0.7
Other methods of melanin detection are either high-cost due to involving the use of pulsed lasers (e.g. MPFM lifetime imaging, pump-probe) or lowcost but with low-sensitivity and low-specificity (e.g. Skintel Melanin Reader).
0.7 y13
SMPAF number after normalized
0.6
0.6
0.5 0.4
0.5 0.3
0.4
0.2 0.1
0.3 0
THEORY
0.1
(a)
Kerimo et al. discovered enhanced melanin fluorescence by stepwise three-photon excitation. We have defined this process as stepwise multiphoton activated fluorescence (SMPAF). SMPAF can be activated using a low-cost CW laser. But the SMPAF signal is much stronger than simultaneous multi-photon excitation of fluorescence. The process of stepwise three-photon excitation fluorescence of melanin is different from the generally known process of simultaneous threephoton excitation of fluorescence. The former is a three-step process via a real intermediate excitation state, while the latter lacks such a real intermediate state (Figure 1). Stepwise three-photon excitation requires excitation intensity ten orders of magnitude lower than simultaneous excitation to obtain the same population density of the fluorescence. Therefore, while simultaneous three-photon excitation requires a highpowered short-pulsed laser, stepwise excitation can be accomplished with a low-cost CW diode laser.. The melanin SMPAF spectra showed a broad distribution from 450 nm to 1000 nm and beyond with a peak at 960nm (Figure 5). Kerimo's work showed an excitation process involving three photons, but at higher powers we have observed processes with higher numbers of photons.
0
0.2
(b)
Figure 4 SMPAF spectrum of (a) mouse hair melanin. πππ₯π = 920 ππ, πΆπ; (b) sepia melanin. πππ₯π = 1505.9 ππ, πΆπ
0
0
100
200
300
400
500
600
time(10-2s)
Figure 5 SMPAF power dependence of sepia melanin
100
200
300 x1
400
500
600
Curve fitting: f(x) = a*exp(-b*x)+c Coefficients (with 95% confidence bounds): a = 0.7562 (0.7475, 0.7649) b = 0.02647 (0.02601, 0.02692) c = 0.022 (0.02077, 0.02322)
METHODS
CONCLUSION
Keck 3D fusion multi-modal microscope The Keck 3d fusion multi-modal microscope (3DFM) in the Optical Science Laboratory (OSL) at Northeastern University allows us to image samples with multiple modalities on the same stage. In this work, only CRM and MPFM modalities are used. The layout of 3DFM is shown in Figure 2. Only the modalities and functions related to this work are shown in this figure.
β’ SMPAF images of mouse melanoma were compared with conventional MPFM and CRM. SMPAF images add specificity for melanin detection than MPFM images and CRM images.
Activation of melanin SMPAF The Tsunami Ti:Sapphire Laser was used to induce the emission from the melanin and to perform the SMPAF imaging. The SMPAF could be activated equally well in pulsed or CW mode using the same average laser power level. The laser focal volume was ~10-18 m3. The procedure used by Kerimo was followed to activate and image the specimen. The sample was exposure to a serial of power and we recorded the SMPAF number vs. time with different power.
β’ Considering the high performance and low-cost feature of the technology, melanin SMPAF is a promising technology for early detection of melanoma for dermatologists. β’ The number of SMAPF will reach the top after being laser exposure in ~2s. And then decreased rapidly. β’ SMPAF of Melanin will be photo-bleached, due to strong laser exposure. The photo-bleaching rate has a exponential decay with the power.
Power Dependence of Step-wise Multi-photon Activation Fluorescence (SMPAF) of Melanin ABSTRACT Melanin is regarded as the most enigmatic pigments/biopolymers found in most organisms. Our preliminary studies show that melanin goes through a step-wise three-photon absorption process when the fluorescence is activated with high laser intensity. The three-photon activation has been observed to require less laser power than what would be expected from a nonβlinear optical process. In this paper, we examined the power dependence of the activation process at 830nm and 920nm. We have conducted research using different laser powers to activate the melanin in a point-scanning mode and recorded as the functions of time. A sequence of experiments indicates the relationship between the activation power, energy and time dependence so that we can optimize the power level. The signal is not strongly dependent on the activation wavelength.
STATE OF THE ART Melanin is an ubiquitous biological pigment in most organisms produced by melanocytes. Malignant melanoma, the most aggressive skin cancer, is a malignant proliferation of melanocytes. Early visual detection of melanoma is critical for melanoma management. The current clinical diagnosis method is lengthy and painful and the histopathologic diagnoses vary depending on the expertise of the dermatopathologist. Confocal reflectance microscopy (CRM) is well-known for βoptical sectioningβ of tissue, which is the imaging of thin sections of the sample at both high resolution and contrast without physically dissecting the tissue. CRM has been used for detection of melanin, providing strong contrast. However, the specificity and sensitivity of melanin detection using CRM is low due to the background signal from other high-contrast materials.
πΉβ
πΌ3
Beam Splitter 1
πΉ β πΌ3
Excited State
S3
Zetong Gua,b, Zhenhua Laia,b,Xi Zhanga,b,Paula Arambel b,c,Charles DiMarzioa,b,c a Electrical and Computer Engineering Department, Northeastern University b Bernard M. Gordon Center for Subsurface Sensing and Imaging System c Mechanical Engineering Department, Northeastern University, Boston, MA, USA
Beam Splitter 2
Ti:Sapphire Laser
Quarter Wave Plate
Dichroic Mirror Objective
XY Scanners
Sample
S2
Virtual States
Real States
Cuvette
Reflected Light
S1
Simultaneous 3-photon excitation
from Sample Power
Ground State
S0
PMT / Camera
Figure 2 Layout of the KECK 3D fusion multi-modal microscope. Only modalities and functions related to this work are shown in the graph. (c) CRM
(b) SMPAF
50 um
APD
Meter
Stepwise 3-photon excitation
πΌ3 10 For Femtosecond laser , 3 = 10 πΌ Figure 1 Simultaneous vs. Stepwise
(a) MPFM
Filter Box
(d) Composite
50 um
50 um
(e) Brightfield
50 um
50 um
Figure 3 Images of mouse melanoma at the same position using different imaging modalities. (a) MPFM, (b) SMPAF, (c) CRM, (d) composite image of (b) (green channel) and (c) (blue channel), and e) Brightfield. By combining CRM with SMPAF, we can locate the melanin, with respect to other skin components. 1 11.3mw 15.9mw 20.7mw 39.8mw
0.9 0.8
1 0.9 y13 vs. x1 fitting curve
0.8 0.7
Other methods of melanin detection are either high-cost due to involving the use of pulsed lasers (e.g. MPFM lifetime imaging, pump-probe) or lowcost but with low-sensitivity and low-specificity (e.g. Skintel Melanin Reader).
0.7 y13
SMPAF number after normalized
0.6
0.6
0.5 0.4
0.5 0.3
0.4
0.2 0.1
0.3 0
THEORY
0.1
(a)
Kerimo et al. discovered enhanced melanin fluorescence by stepwise three-photon excitation. We have defined this process as stepwise multiphoton activated fluorescence (SMPAF). SMPAF can be activated using a low-cost CW laser. But the SMPAF signal is much stronger than simultaneous multi-photon excitation of fluorescence. The process of stepwise three-photon excitation fluorescence of melanin is different from the generally known process of simultaneous threephoton excitation of fluorescence. The former is a three-step process via a real intermediate excitation state, while the latter lacks such a real intermediate state (Figure 1). Stepwise three-photon excitation requires excitation intensity ten orders of magnitude lower than simultaneous excitation to obtain the same population density of the fluorescence. Therefore, while simultaneous three-photon excitation requires a highpowered short-pulsed laser, stepwise excitation can be accomplished with a low-cost CW diode laser.. The melanin SMPAF spectra showed a broad distribution from 450 nm to 1000 nm and beyond with a peak at 960nm (Figure 5). Kerimo's work showed an excitation process involving three photons, but at higher powers we have observed processes with higher numbers of photons.
0
0.2
(b)
Figure 4 SMPAF spectrum of (a) mouse hair melanin. πππ₯π = 920 ππ, πΆπ; (b) sepia melanin. πππ₯π = 1505.9 ππ, πΆπ
0
0
100
200
300
400
500
600
time(10-2s)
Figure 5 SMPAF power dependence of sepia melanin
100
200
300 x1
400
500
600
Curve fitting: f(x) = a*exp(-b*x)+c Coefficients (with 95% confidence bounds): a = 0.7562 (0.7475, 0.7649) b = 0.02647 (0.02601, 0.02692) c = 0.022 (0.02077, 0.02322)
METHODS
CONCLUSION
Keck 3D fusion multi-modal microscope The Keck 3d fusion multi-modal microscope (3DFM) in the Optical Science Laboratory (OSL) at Northeastern University allows us to image samples with multiple modalities on the same stage. In this work, only CRM and MPFM modalities are used. The layout of 3DFM is shown in Figure 2. Only the modalities and functions related to this work are shown in this figure.
β’ SMPAF images of mouse melanoma were compared with conventional MPFM and CRM. SMPAF images add specificity for melanin detection than MPFM images and CRM images.
Activation of melanin SMPAF The Tsunami Ti:Sapphire Laser was used to induce the emission from the melanin and to perform the SMPAF imaging. The SMPAF could be activated equally well in pulsed or CW mode using the same average laser power level. The laser focal volume was ~10-18 m3. The procedure used by Kerimo was followed to activate and image the specimen. The sample was exposure to a serial of power and we recorded the SMPAF number vs. time with different power.
β’ Considering the high performance and low-cost feature of the technology, melanin SMPAF is a promising technology for early detection of melanoma for dermatologists. β’ The number of SMAPF will reach the top after being laser exposure in ~2s. And then decreased rapidly. β’ SMPAF of Melanin will be photo-bleached, due to strong laser exposure. The photo-bleaching rate has a exponential decay with the power.
