Patent Application: US-201214232304-A

Abstract:
the invention relates to a method and a device for twin - focus photothermal correlation spectroscopy for the characterization of dynamical processes in liquids and biomaterials with the help of absorbing markers . thereby non - fluorescent absorbing nano objects are heated by an intensity - modulated heating laser which leads to a refractive index gradient lens around the object . this refractive index gradient is detected by a detection laser with a focal volume that , depending on the position of the heated object relative to the focal plane of the detection beam , splits into two - sub - volumes forming a twin - focus comprising two sharply separated parts of a focal volume showing no spatial overlap .

Description:
a detailed scheme of a photothermal microscopy setup for twin - focus photothermal correlation spectroscopy is depicted in fig1 . the setup has been used to image the twin focus photothermal volume as provided in fig2 . the twin - focus photothermal signal together with two examples of nanoparticle motion is depicted in fig3 . corresponding exemplary temporal correlation functions as obtained from an analysis with the twin - focus photothermal correlation spectroscopy under directed motion of particles along the optical axis are shown in fig4 . the influence of the position of a heated particle relative to the focal plane of a detection laser with regard to the beam shape and the relative photothermal signal is depicted in fig5 a - c . sp 1 , sp 2 : mirrors for the probe laser , highly reflecting at the probe laser wavelength ; se 1 , se 2 : mirrors for the excitation laser , highly reflecting at the excitation laser wavelength ; sm - fiber : single mode glass fiber for the probe or excitation wavelength ; aom : acousto optic modulator , the acousto optic modulator is modulating the excitation intensity in a periodic fashion . the intensity modulation could be either sinusoidal or rectangular or any other periodic shape ; s 1 , s 2 , s 3 , s 4 : mirrors , highly reflecting over a wide wavelength region including the probe and excitation laser wavelength ; d : dichroic mirror , highly reflecting at the excitation laser wavelength and highly transmitting at the probe laser wavelength ; p 1 , p 2 , p 3 : ports for reflecting out of the excitation , probe or transmitted beams at various positions of the setup ; lp : long pass optical filter transmitting the probe laser wavelength and rejecting the excitation laser wavelength . fig2 shows photothermal signal of a r = 30 nm gold nanoparticle in the x - z plane . the z - direction is the direction of the optical axis . the figure shows two lobes indicating the twin focus structure . signal and distance are in arbitrary units . fig3 is an illustration of possible processes in twin - focus photothermal correlation spectroscopy ( twin - phocs ) measurements . the two lobes are indicated with a “+” and a “−” corresponding to positive and negative photothermal signals , wherein it is shown in a ) left : simple brownian motion of a sample particle / molecule in one of the twin focus lobes . v z = 0 indicates no drift velocity of the particle / molecule along the z - direction ; t = 0 denotes the starting time of the process . at a time t 1 after the brownian motion has started , the particle / molecule has diffused a distance √ 4dt 1 right : simple brownian motion of a sample particle / molecule including a drift velocity v z & lt ; 0 in the negative z - direction . this causes the particle to change from the negative photothermal signal lobe to the positive photothermal signal lobe . the distance traveled due to the velocity v z is v z t 1 b ) experimental time trace for a r = 30 nm gold nanoparticle displaying the positive s + ( t ) and the negative photothermal signal time trace . the illustrations 1 - 3 indicate individual events observed ; 1 : motion from one of the lobes to the other , 2 : motion in the positive lobe , 3 : motion in the negative lobe . fig4 shows examples for the different correlation functions , which may be calculated from the data obtained by the twin - phocs method . the examples demonstrate the sensitivity of the different correlation function to a directional motion in z - direction ( positive or negative velocity v z ). graphs a )- c ) show the dependence of the correlation function on the time τ for different values of the velocity in z - direction . graph d ) shows the minimum value of the g ccf +− ( τ m ) and the temporal position τ m ( normalized to the average diffusion time τ d ) where this minimum occurs as a function of the velocity v z . in fig4 , the following parameters are shown : g acf abs ( τ ) is the autocorrelation of the magnitude of the photothermal signal . g acf ps ( τ ) is the autocorrelation of the phase sensitive photothermal signal , where phase sensitive means , that the signal can be either positive or negative depending on the position of the particle / molecule in the lobe . g ccf +− ( τ m ) is the cross - correlation of the positive part with the negative part of the phase sensitive photothermal signal . g ccf +− ( τ m ) is the value of cross - correlation of the positive part with the negative part of the phase sensitive photothermal signal at the time τ m where the cross - correlation is minimum . τ d is the average diffusion time of the sample particle / molecule through the focal volume of the twin - phocs setup . g acf abs ( 0 ) is the value of the autocorrelation function of the magnitude of the photothermal signal at τ = 0 . this value is used to normalize all correlation functions . fig5 shows an illustration of the working principle of the twin focal volume effect used for the twin - phocs method . the dot on the left side of the illustrations a , b , c depicts a sample particle / molecule which is surrounded by a temperature field and a refractive index gradient , respectively . the lines on the left side denote an incident probe laser beam focused on the particle / molecule . the dashed line represents the focal plane of the incident laser beam without the influence of the generated refractive index profile ( nanolens ). the two curved lines denote the shape of the probe laser beam modified by the nanolens . the illustration in the middle depicts the corresponding macroscopic analogue of the nanolens , which is a divergent lens . in illustration a of fig5 , the focal plane of the probe laser is behind the created refractive index gradient corresponding to a beam focused to the back side of a divergent lens . the focus position of the incident beam is moved towards the detector , the sample beam is more convergent and the relative photothermal signal increases and is thus positive ( right graph ). in illustration b of fig5 , the focal plane of the probe laser is at the position of the particle / molecule corresponding to a laser focused into the center of a divergent lens . this is not changing the position of the laser focus nor the divergence of the beam . the detected relative photothermal signal is therefore zero . in illustration c of fig5 , the focal plane of the probe laser is in front of the created refractive index gradient corresponding to a beam focused to the front side of a divergent lens . the focus position of the incident beam is moved away from the detector , the sample beam is more divergent , and the relative photothermal signal increases and is negative ( right graph ). seisenberger , g ., ried , m . u ., endreβ , t ., büning , h ., et al . real - time single - molecule imaging of the infection pathway of an adeno - associated virus . science 294 , 1929 ( 2001 ). 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