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Addis Ababa UniversityAddis Ababa Institute of technologyCenter of Biomedical Engineering
Two-Photon Excited Fluorescence Lifetime Imaging and
Spectroscopy of Melanin in vitro and in vivo
By: Dawit Getahun and Samuel Getaneh
Febr
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Outline
Introduction
Method
Result
Conclusion ?
Eu &
Pheo-
melanin
msrt. By
HPLC
Cell &
raft
culture
2P
imaging
spectro-
scopy
Phasor
FlIM
analysis
2P
FLIM
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Introduction
Epidemiology studies have shown that red hair and light skin color are risk factors for melanoma
development.
high content of pheomelanin and low content of eumelanin in s
for melanoma
Knowledge of concentration of these two is useful for
melanoma
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Introduction
The first results for bulk melanin emission spectral propertie
skin were reported for simultaneous and stepwise two-pho
excitation using 810-nm light.
In this work they extend these previous studies by characte
naturally occurring eumelanin and pheomelanin using two-
excited fluorescence spectra and lifetimes. By comparing TPEF to conventional chemical separation me
say they were able to obtain new insight regarding the or
melanin fluorescence in vivo
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Introduction
Current methods in use
Currently, the total melanin content in biolo
specimens is typically determined by measuring
absorption at 450 to 475 nm using High-performa
liquid chromatography (HPLC) after alk
degradation (markers) is added to a sample
Because of the complicated processing stepsamplification constant used in this method, a s
error in the HPLC measurement can result in a l
error in the final reading.
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Introduction
Other techniques in use
Warren et al. have developed a pump-probe imaging ap
separating pheomelanin from eumelanin and have demons
performance.
Other optical approaches rely on reflectance spectroscopy
absorption spectroscopy
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Basics
melanin type and content => skin and hair color
The binding of MSH to Mc1R results in the formatio
of eumelanin binding of the agouti signaling protein
to Mc1R leads to the production ofpheomelanin
Melanocortin
receptor
(Mc1R)
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TWO PHOTON FLIM method
Sample
preparationHPLCmeasurement
Two-PhotonImaging and
Spectroscopy
Two-Photon
ExcitedFluorescenceLifetime
ImagingMicroscopy
Phasor FLIMAnalysis
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Sample preparation
MNT1 cells are used as sample MNT1 cells are
highly pigmented human melanoma cells
Scientific Application(s): Cell Culture / Growth Conditions
Dark in a culture.
MNT-46 and MNT-62 are two clones derived from
MNT-1 transfected Mc1R
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Eumelanin and Pheomelanin Measurements b
Chemical method of identifying melanin componnents
Make use of High-performance liquid chromatography (HPL
Use markers
Pyrrole-2, 3, 5-tricarboxylic acid (PTCA) > eumelanin
4-amino-3-hydro-xyphenylalanine (4-AHP) > pheomelanin
the ratio of PTCA to AHP serve as the ratio of eu- to
pheomelanin content.
Used to cross check other methods
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Two-Photon Imaging and Spectroscopy
Is used to calculate optical
melanin index (OMI) Emission peak of
pheomelanin = 615-625 nm
Emission peak of
eumelanin = 640-680 nm
OMI =eu FLUORESECE intensity @ 645 nmpheo FLUORESECE intensity @ 615 nm
Cell type OMI
MNT-1 1.6 +/- 0.22
MNT-46 0.5 +/- 0.05
MNT-62 0.17 +/- 0.03 LOWER OMI shows higher risk of CANCER
FIG SHOWS fluorescence emission spectra of
and eumelanin
eu
phe
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Two-Photon Excited Fluorescence Lifetime
Imaging Microscopy
Near infrared (NIR) two-photon excitation is advantageou
lower scattering
enhanced skin penetration
reduced photodamage.
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Phasor FLIM Anaalysis
clustering of pixels values in specific regions of the phasor
simplify the way data are analyzed in FLIM
Since every molecular species has a specific phasor, we ca
molecules by their position in the phasor plot.
The phasor method transforms the histogram of the time de
each pixel in a phasor, which is like a vector
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Phasor FLIM Anaalysis
In the case of a single exponential decay
the equation for points on the polar plane is given by
Simple rules to the Phasor plot:
Where
tau is the lifetime of the decayis the laser frequency
1. All single exponential lifetimes lie on the universal circle
2. Multi-exponential lifetimes are a linear combination of their components3. The ratio of the linear combination determines the fraction of the
components
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3. Results
3.1 TPEF Spectroscopy Approach Hair of different colors (red, black, and gray) were used a
source of naturally occurring eumelanin, pheomelanin, and
Melanins are produced by melanocytes located in the hair
in skin (dermis) and deposited in melanosomes,which then d
along the hair shaft during the hair growth.
The pigment in:
Blond and red hair contains predominantly pheomelanin,
Dark brown and black color is due to prevalence of eumelanin
Gray hair is devoid of any pigment.
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3. Results
Fluorescence was excited @ 1000 nmand the resulting emission spectra were
recorded and normalized to unity
@ 560 nm.
Fluorescence emission spectra of red (open
circle), black (filled circle) and gray (squares)human hair; excitation wavelength is 1000 nm
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3. Results
The spectrum of a colorless gray hair notassociated with melanin, and it was
subtracted from the spectra of pigmented
samples.
fluorescence emission spectra of pheomelanin
(circles, red hair) and eumelanin (squares, blackhair) after autofluorescence (gray hair) subtraction.
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3. Results
Pheomelanin fluorescence emission peaked
around 615 to 625 nm.
Eumelanin displayed red-shifted
(compared to pheomelanin) broad
fluorescence emission at 640 to 680 nm.
(possible limitations in detection range)
Samples with significant content ofeumelanin showed a very high susceptibility
to photodamage (burns) by a scanning
laser at a much lower incident power.
Excitation wavelengths > 1
practical because of a sha
laser output power and lig
properties of the microsco
@1000nm is approp
damage
To establish OMI (Optical
MNT-1, MNT-46, and
with different melanin
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3. Results
The ratios of eumelanin to pheomelaninwere measured by a novel high-performance liquid chromatographymethod (HPLC)
The OMI was defined as a ratio offluorescence signal intensities measured at645 nm (eumelanin) and 615 nm
(pheomelanin), and compared the experimental results
obtained by chemical (HPLC) and opticalmethods.
And it was in vitro
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3. Results
To test fluorescence
signatures of the two
melanins and measure their
ratios in vivo:
Microscopy imaging and
measurements of melanin
spectra on skin of fivehealthy human subjects
with different skin types(I, II-III, V, and VI).
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3. Results
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3. Results
Images and spectra were acquired at the epidermal-dermal junction aepidermis below the stratum corneum (data not shown).
Epidermal-dermal junction provided the most reliable data bebasal cellular layer containing pigmented cells (both melanocykeratinocytes) was easily discernable.
OMI were calculated for normal human skin in vivo by the same proced
used for cells in vitro: Average OMI for basal cells layers (melanocytes and keratino
measured in skin type I, II-III (not tanned and lightly tanned) we0.1, 1.05 0.2, and 1.16 0.1, respectively.
The epidermal-dermal junction in the highly pigmented skin type V-VI creached.
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3. Results
Thus, for type I/II skin, TPEF spectroscopy offers a noninvasive, relative
method of imaging and measurement of two forms of naturally occurritwo distinct components.
But, a complex mixture of fluorophores that coexist and co-emit in pigm
makes individual components in a raw spectrum impossible to resolve.
=> Requires additional assumptions and processing (involves subt
residual contribution to the spectra of other fluorophores presenas flavoproteins and keratin).
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3. Results
3.3 Phasor FLIM Approach
Fluorescence can also be characterized by lifetime.
Fluorescence lifetime measurements are independent of fluorophore conc
FLIM avoids some of the uncertainties and artifacts of fluorescence inten
measurements.
Moreover, the use of phasor FLIM reports not only the spatial localizatio
different components but also their relative concentration if they are mesimultaneously.
As in spectral fluorescence measurements, black, red, and gray human h
used as a major source of eumelanin, pheomelanin, and keratin, respecti
create a FLIM fingerprint database.
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3. Results
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3. Results
Non-pigmented keratin and the two different
types of melanin can be easily identified and
separated by their specific location in thephasor plot.
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3. Results
The broad lifetime distribution measured in Black (d) and Red (e) hair and h
characteristic linear-elongated pattern that reflects a mixture of keratin-eumof keratin-pheomelanin, respectively.
Or melanin Oligomerization
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3. Results
Phasor FLIM signatures of melanins in skin cells
(human melanocytes from skin type II, both
melanins should be present) show a good
correlation with the melanins measured in hair
samples, which was used as calibration
standard.
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4. Conclusions
Two-photon excited fluorescence emission spectra and lifetimes of two
melanin eumelanin, and pheomelanin, naturally occurring in human skin
measured.
The optically derived ratios of eumelanin to pheomelanin were compa
chemical extraction and separation methods using HPLC.
Spectral OMI values correlated well with HPLC-derived data over a ra
types. Spectral measurements of light human skin types (I to III) in vivo showed
increases in measured OMI values with Type I exhibiting the lowest OM
Phasor FLIM provided a clear fingerprint identification of keratin, eum
pheomelanin and straightforward quantitation of relative concentratio
4 C l
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4. Conclusions
These data suggest that a noninvasive TPEF spectral index and phasor
potentially be used for rapid melanin ratio characterization both in vit
R f
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References
[1] Principles of Fluorescence Spectroscopy, 3rd Ed,. By Joseph R Lakowicz
[2] The Phasor Approach: Application to FRET Analysis, by Enrico Gratton
[3] Tatiana et. al Two-photon excited fluorescence lifetime imaging and sp
melanins in vitro and in vivo, 2013
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Thank you!