Dark-field Brillouin microscopy - OSA Publishing

1432

Letter

Vol. 42, No. 7 / April 1 2017 / Optics Letters

Dark-field Brillouin microscopy GIUSEPPE ANTONACCI1,2 1 2

Department of Physics, Imperial College London, London, UK Center for Life Nano Science, Istituto Italiano di Tecnologia, Rome, Italy ([email protected])

Received 21 December 2016; revised 14 January 2017; accepted 20 January 2017; posted 23 January 2017 (Doc. ID 283328); published 31 March 2017

Brillouin microscopy is a non-contact and label-free technique for mapping fundamental micro-mechanical properties in the volume of biological systems. Specular reflections and elastic scattering easily overwhelm the weak Brillouin spectra due to the limited extinction of virtually imaged phased array spectrometers, thereby affecting the image acquisition. In this Letter, a dark-field method was demonstrated to reject the elastic background light using an annular illumination and a confocal detection. To validate the method, images of polystyrene and liquid samples were obtained using both a confocal and the dark-field system. An extinction ratio of 30 dB was readily achieved. © 2017 Optical Society of America OCIS codes: (110.0110) Imaging systems; (290.5830) Scattering, Brillouin; (170.6510) Spectroscopy, tissue diagnostics. https://doi.org/10.1364/OL.42.001432

The mechanical properties of cells and tissues play a pivotal role in the pathophysiology of diseases, such as cancer [1], atherosclerosis [2], and glaucoma [3]. Standard methods to assess the mechanical properties, such as atomic force microscopy [4] and optical coherence elastography [5], typically involve physical contact to the sample. As a result, these techniques are invasive and are mostly limited to surface topologies. On the other hand, contactless techniques, such as ultrasound [6] and magnetic resonance imaging [7], are fundamentally limited by a low spatial resolution. In turn, Brillouin microscopy is a promising non-contact, label-free technique to non-invasively investigate the mechanical properties with a sub-micron resolution in the volume of biological samples [8,9]. In particular, light is used to probe the spontaneous acoustic waves that locally propagate through matter, and the Brillouin frequency shift associated with the longitudinal elastic modulus M 0 is measured by a high-throughput, high-resolution virtually imaged phased array (VIPA) spectrometer. This is a modified Fabry–Perot (FP) etalon, where the input beam is focused to an anti-reflectioncoated window and undergoes multiple internal reflections to interferometrically separate the individual spectral components. To date, Brillouin microscopy has been successfully applied to quantify atherosclerotic plaque stiffness [10], to assess corneal biomechanics [11], to screen bacterial meningitis [12], and to investigate subcellular biomechanics [13–15]. 0146-9592/17/071432-04 Journal © 2017 Optical Society of America

The collection of specular (Fresnel) reflections and elastically scattered light yet represents one of the main limitations in Brillouin microscopy. Although single-stage VIPA spectrometers are capable of measuring the Brillouin spectrum of transparent and liquid samples within a 100 ms data acquisition time [16], they are limited by a finite extinction ratio of approximately 30 dB. As a result, strong elastic background signals arising from turbid media (e.g., biological tissues) form undesired crosstalk signals that overwhelm the weak Brillouin peaks. Current methods to increase the spectral extinction involve the application of multiple disperser elements placed in tandem. For example, the gold-standard multi-pass FP interferometers can reach an extinction of 150 dB [17]; yet, they are limited by a few-second data acquisition time. On the other hand, twostage VIPA spectrometers use crossed etalons to rotate the dispersion axis from the elastic crosstalk lines [18], providing an extinction of 60 dB. Nevertheless, this comes at the cost of a reduced throughput efficiency of approximately 25%, i.e., half of that given by single-stage VIPA spectrometers. Given the lack of commercially available sub-GHz notch filters, other approaches, such as destructive interference [18], beam equalization [19], line absorption [20], and etalon filters [21], have been proposed to increase the extinction of the VIPA spectrometers. Stimulated Brillouin scattering is another promising technique; yet, current data acquisition time is still unpractical for attempting an imaging modality [22,23]. In this Letter, an optical dark-field method to minimize the collection of the elastic background signal is described. Unlike conventional bright-field and confocal microscopes, typical dark-field systems employ specific illumination schemes to reject the unscattered light [24], thereby increasing the image contrast of unstained or transparent specimens. In Brillouin imaging, this principle is useful to prevent the formation of the undesired crosstalk signals overwhelming the Brillouin spectra [25]. Since spontaneous Brillouin scattering is an inelastic process, rejection of specular reflections does not affect the signal strength. Figure 1(a) shows the principle and a schematic diagram of the confocal dark-field setup. An expanded linearly polarized beam (λ 561 nm) of a single longitudinal mode laser (Cobolt Jive) was reflected by a customized prism [Fig. 1(b)] that had a double cavity and a high-reflection-coated surface to yield an annular beam along the illumination arm. A microscope objective lens of numerical aperture NA 0.5 was used both to focus the annular beam to the sample and to collect the scattered


Letter b) R

R

U r; 0 −

NA0.5

ik r2 2J 1 krR∕f R2 expikf exp ik ; 2f 2f krR∕f (3)

while along the optical axis (r 0), we have 2 k 2f kz expikf exp −i 2 R 2 − 1 : U 0; z kz 2f 2f (4)

S Prism Laser

c) SM Fiber b a

VIPA Spectrometer

Fig. 1. (a) Principle and schematic of the dark-field setup. (b) The annular beam was generated using a customized prism. The prism had a double cavity of 5 mm diameter to reject on-axis specular reflected light and simultaneously enable collection of the scattered light. A SM fiber was used to enable confocality and flexible beam delivery to a single-stage VIPA spectrometer. (c) Illumination and detection pupil diagram. The annular illumination was defined by the outer radius b (matching the objective aperture radius) and the inner radius a. The collection of the scattered light occurred through the circular aperture of radius a.

light through the central aperture of radius a [Fig. 1(c)]. The prism filtered the scattered signal and simultaneously rejected the specularly reflected light propagating along the illumination optical path. The scattered light was delivered to a single mode fiber that enabled confocality and a flexible beam delivery to a high-throughput single-stage VIPA spectrometer. An sCMOS camera (Andor Neo 5.5) was used to detect the Brillouin spectrum with 200 ms of data acquisition time and 10 mW at the sample plane. To give an estimate of the spatial resolution of the dark-field microscope, the system point spread function (PSF) was evaluated as the product of the illumination and collection PSF: PSF PSFil l · PSFcoll :

(1)

ik expikf ik 2 U f r; z − exp r f 2f Z R ikz kρr ρdρ; exp − 2 ρ2 J 0 × 2f f 0

where the complex phase terms were neglected under the assumption that the two regions had equal phase. Since the collection of the scattered light occurs through the central aperture of radius a, the collection field distribution is given by 2J 1 kra∕f U coll r; 0 U a r; 0 ≃ a2 : (6) kra∕f Similarly, from Eq. (4), we have that the illumination distribution along the optical axis is given by kz U il l 0; z U b 0; z − U a 0; z ≃ exp −i 2 b2 2f kz (7) − exp −i 2 a2 ; 2f while the collection field distribution is kz U coll 0; z U a 0; z ≃ exp −i 2 a2 − 1 : 2f

I tot r; 0 jU il l r; z · U coll r; zj2z0 ;

where J 0 is the zero order Bessel function of the first kind and k 2π∕λ is the wavenumber. At the focal plane (z 0), Eq. (2) reduces to

(9)

and longitudinal [Fig. 2(b)],

a)

1

b)

I

tot

Iill

0.8

1

I

tot

Iill

0.8

I

Icoll

coll

0.6 0.4 0.2

(2)

(8)

Figure 2 illustrates the normalized PSF of our dark-field imaging system for both transverse [Fig. 2(a)],

I(r,0)

Scalar diffraction theory states that the amplitude distribution of a plane wave focused by a circular thin lens of radius R and focal length f is given by the Fresnel integral [26]

Assuming a perfectly coherent optical system of circular apertures, the illumination PSF of our dark-field system can be evaluated by subtracting the relative field amplitudes given by two circular apertures of radii b and a, respectively [Fig. 1(c)] [27]. Recalling Eq. (3), the illumination field distribution at the focal plane is thus given by 2J 1 krb∕f U il l r; 0 U b r; 0 − U a r; 0 ≃ b2 krb∕f 2J 1 kra∕f ; (5) − a2 kra∕f

I(0,z)

a)

1433

0 0

0.6 0.4 0.2

0.5

1 r (µm)

1.5

2

0 0

5

10 z (µm)

15

20

Fig. 2. (a) Longitudinal and (b) transverse PSF of the imaging system for NA 0.5. The total PSF is compared with the illumination and collection PSFs. The spatial resolution was estimated to be r ≃ 0.5 μm in the transverse and z ≃ 5 μm in the longitudinal direction.


a)

13

9

b)

20 15 10 5

Rayleigh Intensity (a.u.)

Letter

Brillouin shift (GHz)

1434

5

I tot 0; z jU il l r; z · U col l r; zj2r0 ;

(10)

intensity distributions using an objective lens of NA 0.5 and an aperture radius of b 1.5 · a 4 mm. In particular, the plots compare the total transverse and longitudinal intensity distributions with those given by the annular illumination (I il l jU il l j2 ) and collection (I coll jU coll j2 ). A transverse resolution of r ≃ 0.5 μm and a longitudinal resolution of z ≃ 6.2 μm were evaluated from the analysis. It is important to notice that, while the spatial resolution is comparable to that of standard confocal microscopes, the system efficiency is lower as a consequence of the reduced effective illumination and collection NAs. Figure 3 shows a comparison of two images of a polystyrene test object (Thorlabs, R3L3S1P) acquired with both a standard confocal and the dark-field system. Data acquisition was performed by scanning across the object mounted on a motorized stage and by mapping the intensity of the elastic light collected by the systems. Unlike the standard confocal image, the object imaged through the dark-field system results in being bright only along its edges [Fig. 3(b)], where a significant amount of the incident light is elastically scattered. This contrast enhancement results from the ability of the dark-field imaging system to reject specular reflections that are typically collected by standard bright-field and confocal microscopes. An extinction ratio of ∼30 dB for specular reflections was estimated by measuring the backscattering light from a mirror using both the modified prism and a standard beam splitter. Including the single-stage VIPA spectrometer, the overall system extinction was approximately 60 dB. The rejection capability of the dark-field system naturally matches the requirement in Brillouin imaging to minimize the amount of elastic background light delivered to the spectrometer. Figure 4(a) shows the Brillouin image of a liquid sample in contrast with the associated intensity dark-field image [Fig. 4(b)]. The liquid sample was made of layered regions of two immiscible liquids, i.e., distilled water and oil, placed in a glass bottom dish (ThermoFisher, 150682). The laser beam was focused in a backscattering geometry at the air-liquid interface, where specular reflections prevail due to the refractive index mismatch. Nonetheless, despite the limited extinction (∼30 dB) of the single-stage VIPA spectrometer, a high image contrast was obtained by mapping the Brillouin frequency sifts across the two liquids. Conversely, despite their different refractive indices, the two liquids were not distinguishable in the associated dark-field image given by the intensity of the elastically

d)

12

Brillouin linewidth (GHz)

c) Brillouin shift (GHz)

Fig. 3. (a) Confocal and (b) dark-field image of a test object. In the confocal image, the object appears bright as a result of the collection of specular Fresnel reflections arising across the surface. In turn, the object imaged through the dark-field system appears bright only along the edges, where the elastic scattering dominates. The scale bars are 20 μm.

11 10 Oil

9 8

Water

7 6

0

50

100

150

200

250

300

4 3.5 3 Water

2.5

Oil

2 1.5

0

50

100

150

200

250

300

Fig. 4. Dark-field Brillouin image of (a) a two-liquid interface and (b) the associated intensity image. The water drop (red spot) is distinguished from the oil in the Brillouin image. This contrast is lost in the associated intensity image due to the rejection of the unscattered light. The scale bars are 1 mm. 1D spatial distribution of (c) the frequency shift and (d) the linewidth of the Brillouin peaks along the two-liquid interface.

scattered light, thus further demonstrating the rejection capability of the technique. A one-dimensional (1D) line scan along the oil-water interface of both the frequency shift and the spectral linewidth of the Brillouin spectrum is shown in Figs. 4(c) and 4(d). The measured Brillouin frequency shift was νB 7.09 0.11 GHz for water and νB 10.85 0.13 GHz for oil, which gave an acoustic velocity of V water 1495.3 8.4 m/s and V oil 2008.9 9.6 m/s, respectively, in agreement with previous reports [28]. The spectral linewidth was measured to be ΔνB 1.88 0.23 GHz for water and ΔνB 3.29 0.32 GHz for oil, entailing a small instrumental broadening due to the use of a high NA lens [25]. In conclusion, a dark-field Brillouin microscope was demonstrated. An annular illumination and a custom prism were used to reject undesired specular reflections that easily overwhelm the Brillouin spectra. Given the increased extinction, dark-field Brillouin microscopy may enable the use of single-stage VIPA spectrometers, rather than lower efficient two-stage VIPA spectrometers, thus resulting in nearly a two-fold decrease in the data acquisition time. Besides specular reflections, elastic scattering from tissues may still affect the Brillouin spectrum, depending on the level of turbidity. Nevertheless, involving an alternative illumination scheme, the dark-field Brillouin microscope can be independently combined with the other extinction-enhancement techniques developed in the last decades. In turn, this technique paves the way for rapid three-dimensional Brillouin mechanical imaging heralding new potential biomedical applications. Funding. Engineering and Physical Sciences Research Council (EPSRC). Acknowledgment. I would like to thank Prof. Peter Török and Dr. Carl Paterson for their advice and guidance on and contributions to the design of the microscope and the experiment. Mr. Martin Kehoe and Mr. Simon Johnson

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