Signs of hemoglobin in low power cw-laser induced human skin detected by diffuse reflectance spectroscopy and

multi-spectral imaging

Janis Lesins, Inesa Ferulova, Dainis Jakovels,

Alexey Lihachev, Janis Spigulis

Institute of Atomic Physics and Spectroscopy

University of Latvia, Riga, Latvia

This work has been supported by the European Social Fund

within the projects

«Support for Doctoral Studies at University of Latvia» and

No. 2009.0211/1DP/1.1.1.1.2.0/09/APIA/VIAA/077

Laser-skin interactions

• Laser induced skin autofluorescence photobleaching on focus

• This study:

o 405nm, 473nm, 532nm lasers

o Multi-spectral imaging (non-contact method)

o Single spot spectrometry (contact method)

o Power densities on skin 20 ÷ 120 mW/cm2

• Signatures of low power laser irradiation?

Skin surface

405 nm laser

473 nm laser

Laptops

Optical

fiber

𝜆1 𝜆2

𝜆3 𝜆4

𝜆, nm Pix

el (

x,y

) in

tensi

ty

Spectra

at pixel (x,y)

Pixel (x,y)

Hologen lamp

Multi-spectral imaging setup

Single Spot Spectrometry Setup • Spectral registration method

Laser

Probe

Y-shaped

optical fiber

bundle

SMA connectors

PC

Spectrometer

Skin

Filters

Light source

Skin autofluorescence photobleaching effects during cw laser excitation

5

𝐼𝜆=𝑐𝑜𝑛𝑠𝑡 = 𝑓(𝐼𝑜, 𝑡)

405 nm laser excitation (3 min)

𝐼 = 𝑓(𝜆, 𝑡)

Inte

nsi

ty

Wavelength

Laser excitation

Autofluorescence spectrum

t = 0

t = 3min

It=0 > It=3min

𝐼𝑡=𝑐𝑜𝑛𝑠𝑡 = 𝑓(𝐼𝑜, 𝜆)

Flu

ore

scen

ce I

nte

nsi

ty

Time, s

0

5

10

15

20

470 520 570 620 670 720

AF

In

ten

sity

(re

l.u

.)

Wavelength (nm)

3 min 405 nm

3 min 532 nm

AF intensity after 3 min

Initial AF intensity

Skin autofluorescence photo-bleaching effects

6

),( oconstt IfI

Impact of 3 min irradiation: 473nm-excited skin AF

0 120 240 360 480 600

0,6

0,8

1,0

Flu

ores

cenc

e in

tens

ity, r

el.u

.

Time, s

1- 680 nm

2- 600 nm

1

2

Skin photo-memory

7

Skin «photo-memory» - photo-induced irreversible processes in skin

),( tIfI oconst

Skin AF intensity recovery after low-power

532 nm laser irradiation (at 600nm ~ max)

0 25 50 75 100 1250

20

40

60

80

100

Rec

ov

ery

of

AF

in

ten

sity

(%

)

Time (Hours)

Relative optical density

9

500 550 600 650 700 750

0.00

0.05

0.10

0.15

0.20

Op

tica

l den

sity

Wavelength, nm

130 mW/cm2

115 mW/cm2

90 mW/cm2

532 nma)

500 550 600 650 700 750

0.00

0.05

0.10

0.15

0.20

Op

tica

l d

en

sity

Wavelength, nm

100 mW/cm2

45 mW/cm2

20 mW/cm2

473 nmb)

500 550 600 650 700 750

0.00

0.05

0.10

0.15

0.20

Op

tica

l den

sity

Wavelength, nm

70 mW/cm2

40 mW/cm2

20 mW/cm2

405 nmc)

532 nm

473 nm

405 nm

MSI results (optical density)

10

500 550 600 650 700 750

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Op

tica

l d

en

sity

Wavelength, nm

Irradiated skin

Not irradiated skin

473 nm473 nm

500 550 600 650 700 750

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Op

tica

l den

sity

Wavelength, nm

Irradiated skin

Not irradiated skin

405 nm405 nm

Hemoglobin absorption

• Hemoglobin absorption impact during and after

laser-irradiation

• Local skin heating – blood volume changes

• Arterial occlusion – blood flow after release

• Recovery after photobleaching:

o skin autofluorescence at the laser-irradiated spot

o diffuse reflectance spectra

Photobleaching Mechanisms

• Direct degradation of specific fluorophores (?)

• Changes in absorption properties, quenching (?)

• Photo-induced increase/generation of chromophores

(like UV-enhanced melanin) (?)

Conclusions

• Visible laser-excitated skin autofluorescence (AF) photo-

bleaching takes place even at irradiation power

densities well below the laser-skin safety limits

• Mechanism of skin AF photo-bleaching still under study;

seems like microcirculation is involved

• Future work will involve combined experiments of diffuse

reflectance, fluorescence emission spectrum and

fluorescence lifetime data.

• Interesting enough to continue ...

Thank you for your attention!

14

Institute of Atomic Physics and Spectroscopy

Riga, Latvia