Femtosecond laser applied to biophotonics. Prof. Cleber R. Mendonca

Transcription

1 Femtosecond laser applied to biophotonics Prof. Cleber R. Mendonca

2 introduction short pulse duration ö high intensity (even at low energy)

3 introduction how short is a femtosecond pulse? 1fs= s

4 introduction how short is a femtosecond pulse?

5 introduction Ti:Sapphire lasers 100 fs 50 fs 20 fs Very intense light Laser intensities ~ 100 GW/cm 2 1x W/cm 2 Laser pointer: 1 mw/cm 2 (1 x10-3 W/ cm 2 )

6 introduction Ti:Sapphire lasers 100 fs 50 fs 20 fs Very intense light

7 Light matter interaction Semiclassical treatment electron on a spring harmonic oscillator k m e oscillation frequency ω 0 = k m e

8 Linear optical processes E << E radiation interatomic Induced polarization P = χe linear response ( ) absorption α = α λ refraction n = n( λ)

9 Nonlinear optical processes high light intensity E rad. ~E inter. How high should be the light intensity?

10 Nonlinear Optics Inter-atomic electric field cw laser 2P πw P = 20 W I = w o = 20 μm 2 0 I = W/m 2 e = C r ~ 4 Å E ~ V/m E o = V/m

11 Nonlinear Optics Inter-atomic electric field pulsed laser I = 10 GW/cm 2 = W/m 2 e = C r ~ 4 Å E ~ V/m E o = V/m

12 Nonlinear Optics high light intensity E rad. ~ E E inter. anharmonic oscillator nonlinear polarization response P = χ ( 1 ) E + χ ( 2 ) E 2 + χ ( 3 ) E

13 Second harmonic generation Second order processes χ ( 2 ) ω 2ω

14 Two-photon absorption Third order processes ( 3 ) χ α α + 2ω = 0 βi ω ω

15 Multi-photon absorption,...,, (7) (5) (3) χ χ χ = I I I I α α α β α α = I I I I α α α β α α

16 Spatial confinement of excitation Nonlinear interaction provides spatial confinement of the excitation fs-microfabrication α = α 0 α = α 0 + βi

17 fs-laser micromachining microstructuring t i microfabrication

18 fs-laser microstructuring photon energy < bandgap nonlinear interaction

19 fs-laser microstructuring nonlinear interaction E gap E f = hν

20 fs-laser microstructuring nonlinear interaction ti E gap E f =hν multiphoton absorption

21 fs-laser microstructuring amplified laser oscillator repetitive cumulative Micromachining the sample s s Volume or Surface

22 microstructuring surfaces CCD lens mirror fs-laser objective 0.65 NA sample

23 microstructuring surfaces

24 fs-laser micromachining Latex - natural rubber of the clones: GT 1 Production of latex-based scaffolds for cellular l growth

25 Microstructuring Latex influence of pulse energy in the micromachining of Latex 1.85 µj 1.21 µj 0.97 µj 0.90 µj 0.74µJ 0.61 µj

26 Microstructuring Latex High resolution and small collateral damage 1.85 µj 1.21 µj 0.97 µj 0.90 µj 0.74µJ 0.61 µj

27 Microstructuring Latex Some of the surface patterning produced d on latex 1.85 µj 1.21 µj 0.97 µj 0.90 µj 0.74µJ 0.61 µj no carbonization of the latex has been observed

28 Microstructuring Latex Relatively large areas can be produced with this method 1.85 µj 1.21 µj 0.97 µj 0.90 µj 0.74µJ 0.61 µj

29 Scaffolds for neuron growth fs-laser microfabrication to produce scaffolds for neuron growth

30 Scaffolds for neuron growth

31 Scaffolds for neuron growth Neuron growth platforms need very specific biopolymers approach 1 - Microstructure glass surface 2 Stamping with PDMS

32 Fabrication of the molds fs-micromachining y fs-micromachining x glass substrate master peel away pour on PDMS and cure

33 Fabrication of the molds examples of micromachined surfaces in glass 20 μm

34 Microfabrication Novel concept: build microstructures using fs-laser and nonlinear optical processes

35 Two-photon polymerization Monomer + Photoinitiator Polymer light Photoinitiator is excited by two-photon absorption R2 PA I The polymerization is confined to the focal volume. 2 High spatial resolution

36 Two-photon polymerization setup Ti:sapphire laser oscillator Ti:sapphire 100 fs 800 nm M illumination 130 fs 800 nm 76 MHz 20 mw z substrate objective sample x Objective scanning mirrors y 40 x 0.65 NA CCD

37 Two-photon polymerization

38 Resin preparation Monomers Monomer A Monomer B reduces the shrinkage upon polymerization gives hardness to the polymeric structure Photoinitiator iti t Lucirin TPO-L Appl. Phys. A, 90, (2008)

39 Two-photon polymerization 30 µm x 30 µm x 12 µm cube polymer glass

40 Two-photon polymerization After the fabrication, the sample is immersed in ethanol to wash away any unsolidified resin and then dried

41 Two-photon polymerization Microstructures fabricated by two-photon polymerization 50 μm 20 µm 20 μm 20 µm

42 Stem cell differentiation fabrication of specific 3D scaffolds for stem cell growth and differentiation

43 Stem cell scaffolds 20 μ m

44 Stem cell scaffolds

45 Stem cell differentiation Adhesion

46 Stem cell differentiation Proliferation

47 Stem cell differentiation Differentiation

48 Microstructures with active compounds monomer monomer Optical active dye Active Polymer

49 Doping microstructures F b i ti f i t t ith i l t l i l d Fabrication of microstructures with special topological and chemical design for bio-relates applications

50 Doping microstructures microstructures containing biopolymer - chitosan

51 Microstructures containing Rhodamine Rhodamine 6G H 3 C CH 3 NH O NH + C H 3 O CH3 O CH 3 High luminescence Used as dye laser gain medium

52 Microstructure containing Rhodamine

53 Microstructure containing Rhodamine

54 Microstructure containing Rhodamine

55 Microstructure containing Rhodamine

56 Microstructure containing Rhodamine

57 Microstructure containing Rhodamine 1.0 ce inten sity (arb b. units) fluo orescen wavelength (nm)

58 Microstructure containing Rhodamine fabrication of array of doped microstructures

59 Microstructure containing Rhodamine Fluorescent confocal microscopy planes separated by 6 μm

60 Guiding bacterial growth in a micro-environment microfabrication of multi- doped microstructures

61 Guiding bacterial growth in a micro-environment double doped microstructure Induce cell growth in distinct regions

62 Double doped microstructures fabrications microstructure containing Fluorescein and Rhodamine

63 Double doped microstructures fabrications

64 Viability of the Lactobacilus in the resin day 0 day 3 day 7

65 Viability of the Lactobacilus in the resin day 0

66 Viability of the Lactobacilus in the resin day 3

67 fs-laser spectroscopy py of bio-materials multi-photon absorption nonlinear refraction excited state absorption processes dynamics of ultrashort optical processes

68 fs-laser spectroscopy py of bio-materials all-trans retinal cytochrome c all-trans β-carotene Poster : Marcelo G. Vivas trans β-apo-8 carotenal

69 Summary

70 Acknowledgments FAPESP CAPES CNPq

71 Thank you!

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