Blinking - PowerPoint

Document Sample
Blinking - PowerPoint Powered By Docstoc
					  Photothermal Heterodyne Imaging of Individual Nonfluorescent
                  Nanoclusters and Nanocrystals

Stephane Berciaud, Laurent Cognet, Gerhard A. Blab, and Brahim Lounis

        Centre de Physique Moleculaire Optique et Hertzienne,
                    CNRS et Universite Bordeaux

                                             Ayca Yalcin
•   Motivation
•   Previous Technique
•   Method of Detection
•   Results and Applications
•   Conclusion
     Using scattering properties
• Fluorescent molecules:
  – Photobleaching  short observation times

• Semiconductor nanoparticles:
  – More stable, but blinking (On longer time scales (t>100
    ns), a semiconductor nanocrystal presents a succession
    of time intervals during which it emits light ("on-state") or
    not ("off-state").
  – Difficult to functionalize
      Using absorptive properties
• Large absorption cross section

• Short time interval between successive absorption events

Metal nanoparticles

 - excited near their plasmon resonance, large absorption cross section
  (~8x10-14cm2 for a 5nm diameter particle)
 - fast electron-phonon relaxation time (in the ps range).
 - luminescence is extremely weak, almost all the absorbed energy is
  converted into heat.
 - temperature rise induced by the heating leads to a variation of the local
  index of refraction.
                    PIC (Boyer et al.)

Heating beam modulated at high
frequency induces periodic phase
difference between two red
beams, which gives rise to
modulation   of   detected   red
                                   5nm particles
                Effect of scattering on
                Photothermal Image

• 300nm latex spheres, 80nm/10nm gold spheres
   – A: differential interference contrast
   – B: photothermal (30kW/cm2)
   – C: photothermal (1.5MW/cm2)
                Photothermal Effect
•   When a gold nanoparticle in a homogeneous medium is illuminated with an
    intensity modulated laser beam, it behaves like a heat point source with a
    heating power:
                            Pheat [1  Cost ]
Ω: modulation frequency
Pheat: the average absorbed laser power

•   It generates a time-modulated index of refraction in the vicinity of the particle
    with a spatiotemporal profile:

                       n Pheat                r  r Rth
          n(r , t )           [1  Cos(t      )e     ]
                       T 4 r               Rth
r: distance from particle                                         Rth  2 C
n: index of refraction
Rth: characteristic length for heat diffusion                      n
K: thermal conductivity                                               ~ 10 4 K 1
C: heat capacity per unit volume
                   Experimental Setup


• (red) probe beam (720 nm, single frequency Ti:sapphire laser)

• (green) heating beam (532 nm, frequency doubled Nd:YAG laser) intensity
modulated at (100 kHz to 15 MHz) by an acousto-optic modulator.

• high aperture objective (100X, Zeiss, NA=1.4)

• power of the heating beam: from less than 1 μW to 3.5 mW at the objective.
            Sample Preparation
• Samples prepared by spin coating a solution of gold
  nanoparticles [diameter of 1.4, 2, 5, 10, 20, 33, or
  75nm, diluted into a polyvinyl-alcohol (PVOH) matrix, 2%
  mass] onto clean microscope coverslips.
• The dilution and spinning speed were chosen such that the
  final density of spheres in the sample was less than 1 μm-2.
• Application of a silicon oil on the sample ensures
  homogeneity of the heat diffusion.
• The size distribution of the nanospheres was checked by
  transmission electron microscopy and was in agreement with
  the manufacturer’s specification.
                        Some Results
                                      •   3D representation of a photothermal
                                          heterodyne image of gold aggregates of
                                          67 atoms (1.4 nm diameter nanogold).

                                      •   Relatively small heating power (~3.5 mW)
                                          and a remarkably large signal-to-noise
                                          ratio (SNR > 10), shot noise limited.

• Confirmed that the peaks stem from single
particles by generating the histogram of the
signal height for 272 imaged peaks.
• Monomodal distribution with a width in
agreement with the spread in particle size.
          Comparison with Theory
•   In order to estimate the measured signal, theory of ‘‘scattering from a
    fluctuating dielectric medium’’ is used to calculate the field scattered by the
    modulated index profile.
•   The beating between the reference and scattered fields leads to a beating
    power S at the detector:

       n            Pheat 1
S  n    I inc Pref         [ f () cos(t )  g () sin(t )]
       T            C 

: geometry factor close to unity
Iinc: incident red intensity at the particle location
Pref: reference (backreflected) beam power
f, g: two dimensionless functions which depend on the modulation frequency
     and the thermal diffusivity of the medium
      Measured Signal vs. Frequency
•    The variations o fκ(Ω)/Ω and gκ(Ω)/Ω for

•    At low frequencies, Rth is larger than the
     probe spot size (~λ/2) and fκ(Ω)/Ω is

•    At high frequencies, Rth<< λ , gκ(Ω)/Ω
     dominates and decreases as 1/ Ω.

•    The magnitude of demodulated signal
     delivered by the lock-in amplifier is
     proportional to

    S dem    S (t ) 2              f  () 2  g  () 2
                         t       

•    The frequency dependence of this signal
     measured on single particles (5nm) is
Measured Signal vs. Heating Power

               • Single 2nm gold nanoparticle, σ~5x10-15cm2
               @ 532 nm, Pheat=10nW for 2MW/cm2

               • Pinc=70mW, Ω/2π =800kHz, Pref=100μW
               Calculated Sdem=5nW.
               Measured: ~2nW.

               • Linear dependence of the signal with heating
               power.     No    saturation   for    increased
               power, however, fluctuations in the signal
               amplitude and eventually irreversible damage
               on the particle.
        •   Size dependence of absorption
            cross    section    of   gold
            nanoparticles @ 532nm with
            diameter 1.4-75 nm.

        •   For each sample, histogram of
            signal amplitudes is generated.

        •   Histograms displayed bimodal
            distributions, the mean of each
            population was measured, and
            size    dependence     of   the
            absorption       cross  section
            normalized to that of 10 nm
            particles is obtained.
    Luminescence vs. Photothermal

•   Luminescent semiconductor nanocrystals or fluorescent molecules have radiative relaxation
    times in ns range, difficult to detect by absorption.

•   At high excitation intensities, efficient nonradiative relaxation pathways appear.

•   Fluorescent image of CdSe/ZnS quantum dots (peak emission at 640 nm) excited by the
    heating beam at 0.1 kW/cm2, blinking behavior visible. Photothermal heterodyne image at
    5kW/cm2 where quantum dots are no longer luminescent.

•   Two images correlate well, ensuring spots are individual quantum dots (>90% of the
    fluorescent spots correlate with a photothermal spot). No blinking behavior, initially non-
    fluorescent quantum dots are now detected.
          Biological Applications
• In the current configuration, a 5nm gold nanoparticle can be
  detected with a SNR>100 at a heating power of 1mW. At this
  power, a local temperature increase of 4K is estimated in
  aqueous solutions.

• As the temperature reduces as the inverse of distance, and
  most microscopy applications in biosciences do not require
  such a high SNR, imaging is possible by inducing a local
  heating of far less than 1K above the average temperature in
  the sample.

• Advantages of photothermal heterodyne detection
   – no photobleaching or blinking
   – Immune to background effects

• SNR>10 for 1.4nm gold particles, first detection

• As any far-field optical technique, it has wavelength limited
   – combine with near-field optical techniques  sensitivity+resolution

• Can apply to many diffusion and colocalization problems in
  physical chemistry and material science and to track labeled
  biomolecules in cells.

Shared By: