Two photon excited fluorescence lifetime imaging

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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!

Two photon excited fluorescence lifetime imaging

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