Fluorescence emission difference with surface plasmon-coupled emission applied in confocal microscopy

Kang Jiang; Xinrui Lei; Kuanguo Li; Yonghua Lu; Pei Wang

doi:10.1364/OE.26.002380

1. Introduction

Surface plasmon polaritons (SPPs) are tightly confined electromagnetic waves that propagate along a metal surface [1]. Their strong interaction with matter close to metal interfaces has led to a variety of applications from modern biological imaging to new developments in photovoltaics and quantum optics [2]. Surface plasmon-coupled emission (SPCE) is an effective technique that takes the advantage of surface plasmon resonance to intensify the fluorescence adjacent to the surface of metal film coated on glass slides. The coupling between fluorophore and SPPs leads to directional fluorescence emission at the surface-plasmon resonant (SPR) angle, which is certainly favorable for improving signal-to-noise ratio (SNR) of fluorescence [3]. The existence of SPPs modes can also increase the fluorescence decay rate and minimize photobleaching [4, 5]. SPCE microscopy has been successfully developed to image and study muscle fibers [6]. Despite all these advantages, SPCE microscopy suffers from a distorted donut-shaped point spread function (PSF) [7]. Several methods have been proposed to enhance the imaging resolution in SPCE microscopy by repairing the distorted PSF [8, 9]. A lateral resolution of around 200 nm at 532nm laser excitation can be obtained by introducing a vortex phase plate or a polarization converter to convert the hollow PSF into a solid pattern [9].

Fluorescence emission difference (FED) microscopy provides a simple approach to break the diffraction barrier through subtracting two fluorescence images excited by two differently-modulated beams of solid and donut shapes [10]. Subtraction of these two images with a proper subtractive factor will produce a high-quality FED image. FED is also an efficient method to improve resolution and contrast of laser scanning confocal microscope (LSCM) [10]. Segawa reported FED technique can improve lateral resolution of LSCM without deterioration due to excess subtraction by using the specific focusing characteristics of radially-polarized and azimuthally-polarized laser beam [11, 12].The donut-shaped PSF is undesired for traditional SPCE microscopy, but it is beneficial for FED microscopy. Ge et al. applied FED technique into SPCE microscopy to enhance its lateral resolution by introducing an additional vortex phase plate to produce solid PSF [13]. Moreover, they also reported FED of SPCE microscopy could be realized by simply deviating the sample away from the focal plane of objective lens because the transverse PSF of SPCE microscope was changeable along the axial position [14]. Different from fluorescence radiation mediated by surface plasmon in wide-field microscope, the focal spot of an oil-immersed objective lens through a plasmonic substrate (metallic film) is solid under illumination of radially-polarized light and donut-shaped with circularly-polarized light illumination in confocal microscopy [15]. Therefore, we ask whether it is feasible to improve resolution through combination of FED and SPCE into confocal microscopy.

In this paper, we propose a novel method to carry out super-resolution microscopy with FED technique. Replacing the glass substrate with a plasmonic substrate, we introduce SPCE into traditional scanning confocal microscopy, which will be termed as confocal surface plasmon coupled microscopy (C-SPCEM) hereafter. The PSF of C-SPCEM will be solid when radially-polarized light illuminates through a high NA oil-immersed lens and donut-shaped PSF is realized for circularly-polarized illumination. If FED technique is properly applied into C-SPCEM, the resolution and contrast of the images can be evidently enhanced. The paper is organized as following: firstly, Richards-Wolf vectorial diffraction theory and Surface-plasmon-mediated fluorescence dipole radiation research are used to characterize the PSFs of C-SPCEM illuminated by radially polarized (RP) and circularly polarized (CP) beam; then solid and donut-shaped PSFs of C-SPCEM are experimentally demonstrated by imaging tiny fluorescent bead; finally, FED technique is applied into C-SPCEM to enhance the resolution of confocal microscopic images of aggregated fluorescent beads.

2. Theory

2.1 Focal field of oil-immersed lens through silver layer

We used Richards and Wolf’s vectorial diffraction theory to calculate the focal fields of radially polarized (RP) and circularly polarized (CP) beam through a 40 nm-thick silver film. Starting from the angular spectrum representation of the electric field near the focus in cylindrical coordinates [16], we obtain all the electric field components of the focal field. The focal electric fields of radially-polarized incident beam in the medium above the silver layer are calculated using the following integrals:

E_{r} (r, φ, z) = 2 A \int_{0}^{θ_{\max}} P (θ) t_{p} (θ) \sin θ \cos θ J_{1} (k_{1} r \sin θ) \exp [i z {(k_{2}^{2} - k_{1}^{2} \sin^{2} θ)}^{1 / 2}] d θ

E_{z} (r, φ, z) = i 2 A \int_{0}^{θ_{\max}} P (θ) t_{p} (θ) \sin^{2} θ J_{0} (k_{1} r \sin θ) \exp [i z {(k_{2}^{2} - k_{1}^{2} \sin^{2} θ)}^{1 / 2}] d θ

Where t_p(θ) is transmission coefficient of p-polarization at incident angle θ; P(θ) is pupil apodization function, which is set as

\sqrt{\cos (θ)}

; A is a constant;

J_{m} (x)

is Bessel function of the first kind with order m;

θ_{\max} = \sin^{- 1} (N A / n)

is given by numerical aperture (NA = 1.45) of objective lens and refractive index (n = 1.51) of matching oil; and k₁ and k₂ are wave vectors in the medium above and below the silver layer, respectively. As shown in Fig. 1(a), transmission coefficient curve versus incident angle calculated for 40 nm-thick silver film indicates that surface plasmon resonance occurs at θ_sp = 45.8°. The excitation of surface plasmon polaritons is confirmed by measuring the image of samples at the back focal plane of objective lens. Because of the cylindrical symmetry of the polarization, a complete black ring is observed in the back focal plane image for both radially-polarized light [Fig. 1(b)] and circularly-polarized light [Fig. 1(c)]. The radius of the dark ring is well consistent with the surface plasmon resonance angle.

Fig. 1 (a) Calculated magnitude of the transmission coefficient versus incident angles for p-polarized light incident on the dielectric–metal interface. The surface plasmon resonance condition is satisfied at θ_sp = 45.8°. Measured intensity distribution at the back focal plane of the objective lens after reflection with RP beam (b) and CP beam (c). The dark rings correspond to the surface plasmon resonance excitation.

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Surface plasmon resonance plays the role of an angular spectrum filter for the focal field, just similar to an axicon for a fundamental Gaussian beam [17]. Given the very narrow linewidth of surface plasmon resonance (Fig. 1(a)), the transmission coefficient of silver film is approximated to delta function $t_{p} (θ) = t_{p} (θ_{s p}) δ (θ - θ_{s p})$ . Then, integrals of Eq. (1) and (2) are reduced to

E_{r} (r, φ, z) = 2 A P (θ_{s p}) t_{p} (θ_{s p}) \sin (θ_{s p}) \cos (θ_{s p}) J_{1} (k_{1} r \sin θ_{s p}) \exp [i z {(k_{2}^{2} - k_{1}^{2} \sin^{2} θ_{s p})}^{1 / 2}]

E_{z} (r, φ, z) = i 2 A P (θ_{s p}) t_{p} (θ_{s p}) (\sin^{2} (θ_{s p}) J_{0} (k_{1} r \sin θ_{s p}) \exp [i z {(k_{2}^{2} - k_{1}^{2} \sin^{2} θ_{s p})}^{1 / 2}]

The radial component defined by

J_{1}

Bessel function in Eq. (3) has a dark center [Fig. 2(a)]. On the other hand, the longitudinal component defined by

J_{0}

Bessel function in Eq. (4) has maximum in the center [Fig. 2(b)] and dominates the total focal field [Fig. 2(c)]. Figure 2(d) shows the intensity profiles of radial and longitudinal components and total focal field. It is advantageous to use Bessel beams because their spot size is non-diffractive in a relative longer region than the depth of field of Gaussian beams. Moreover Bessel beams are naturally resistant to scattering, thanks to their self-reconstruction properties [18], and can therefore be used for deeper imaging in confocal microscopy [19–21], and has potential applications in nano-lithography and nano-trapping [22].

Fig. 2 The electric field intensity on the focal plane for RP illumination. (a) The transverse component; (b) The longitudinal component; (c) The total field. (d) The intensity profiles of different field components.

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Similarly, the focal field of a circularly polarized incident beam through the thin silver film is found to be:

\begin{array}{l} E (r, φ, z) = - i A {[I_{0} + \cos (2 φ) I_{2} + i \sin (2 φ) I_{2}] e_{x} + [\sin (2 φ) I_{2} + i (I_{0} - \cos (2 φ) I_{2}] e_{y} \\ + (2 i \cos φ I_{1} - 2 \sin φ I_{1}) e_{z}} \end{array}

I_{0} = \int_{0}^{θ_{\max}} P (θ) t_{p} (θ) \sin θ \cos θ J_{0} (k_{1} r \sin θ) e^{i z \sqrt{k_{2}^{2} - k_{1}^{2} \sin θ^{2}}} d θ

I_{1} = \int_{0}^{θ_{\max}} P (θ) t_{p} (θ) \sin^{2} θ J_{1} (k_{1} r \sin θ) e^{i z \sqrt{k_{2}^{2} - k_{1}^{2} \sin θ^{2}}} d θ

I_{2} = \int_{0}^{θ_{\max}} P (θ) t_{p} (θ) \sin θ (- \cos θ) J_{2} (k_{1} r \sin θ) e^{i z \sqrt{k_{2}^{2} - k_{1}^{2} \sin θ^{2}}} d θ

The focal field is comprised of transverse field and longitudinal field as shown in Figs. 3(a) and 3(b), respectively. Due to the modulation of t_p(θ), the transverse and longitudinal field component is mainly defined by

J_{0}

and

J_{1}

Bessel function, respectively. Additionally, the transverse field has comparable intensity value compared with the longitudinal field. Thus, the focal field is the superposition of a solid spot and a hollow spot, and the final result is a slightly dented round spot shown in Fig. 3(c). Figure 3(d) shows the intensity profiles of different components.

Fig. 3 The electric field intensity on the focal plane for CP illumination. (a) The transverse component; (b) The longitudinal component; (c) The total field. (d) The intensity profiles of different field components.

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2.2 Radiation pattern of dipole emitter located near the silver film surface

We consider an oscillating electric dipole that is situated at a small distance d away from a silver film surface [Fig. 4(a)]. Since the diameter of fluorescence bead is approximately 150 nm, the dipole-metal distance d is chosen as 75 nm. The surrounding medium is air with refraction index of 1. The silver film of 40 nm in thickness is deposited on a glass substrate. Complex refraction index of silver film is chosen as 0.12 + 3.58i at the fluorescent wavelength of 580 nm. Refraction index of the glass substrate is 1.51. Radiation of the dipole will be simulated for in-plane and out-of-plane orientation with respect to the metal surface. The far-field radiation of out-of-plane dipole is mainly mediated and enhanced by surface plasmon, which result in a ring-shaped pattern [Fig. 4(b)]. The coupling between in-plane dipole yields a couple of crescent far-field radiation pattern along the surface plasmon resonant angle [Fig. 4(c)]. Although the radiation of in-plane dipole is obviously boosted by surface plasmon resonance, the enhancement is quite smaller than that of out-of-plane dipole. By integrating the intensity of in-plane and out-plane far-field radiation pattern respectively, we calculate the enhancement ratio between in-plane and out-of-plane dipoles as $\partial = I_{i n - p l a n e} / I_{o u t - o f - p l a n e} = 0.14$ . The different enhancement between in-plane and out-of-plane dipoles [23] has to be considered during the evaluating PSF for C-SPCEM.

Fig. 4 (a) An oscillating electric dipole is situated on the z axis at a distance d from a metal/dielectric interface in the x, y plane. The far-field patterns emitted by an out-of-plane and an in-plane dipole are plotted in Fig. 4(b) and 4(c), respectively.

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2.3 PSF of confocal surface plasmon-coupled emission microscopy (C-SPCEM)

Different from wide-field microscope, laser scanning confocal microscope scans the specimen point by point. This particular imaging way of point scanning determines that PSF of confocal fluorescence microscope (PSF_c) is composed of the contributions of both the illuminating beam path (PSF_ill) and the fluorescence detection beam path (PSF_det). For conventional laser scanning confocal microscope, without SPCE, the PSF_c can be mathematically described as follows [24]:

P S F_{c} (x, y) = P S F_{i l l} (x, y) \times [P S F_{\det} (x, y) \otimes p (x, y)]

where PSF_ill denotes the PSF for the objective lens used to excite the fluorescence. In other words, it is the normalized intensity distribution of the excitation pattern on the sample. PSF_det denotes the PSF of detection beam path projecting a point detail of the object into the image space, p(x,y) is the transmission function of the pinhole, and ⊗ represents the convolution operator. It is evident that the imaging properties of a confocal microscopy are determined by the interaction between PSF_ill and PSF_det when the diameter of confocal aperture is small enough. However, the diameter of confocal aperture in our experiment is set as 100

μ m

, which is about two Airy units, to ensure the collection efficiency of fluorescence. For such a large confocal aperture, the detection PSF has little effects on the total PSF, therefore, the resolution is mainly determined by the illumination PSF [25].

However, the story is a little different for C-SPCEM because surface plasmon mediates the fluorescence radiation in C-SPCEM. The illumination PSF is modified by the enhancement ratio between in-plane and out-of-plane dipoles due to the interaction between the fluorescent emitter and the vector focal field. Considering the random orientation for dipole momentum (μ) of fluorescent emitters, the averaged excitation rate ( $< {| μ \cdot E (r) |}^{2} >$ ) will depend mainly on the vectorial focal electric field of the illumination light. Because enhancement of fluorescence mediated by surface plasmon is quite different for emitters with in-plane or out-of-plane dipole momentum [26, 27], PSF for confocal microscope should be modified by including the effect of the enhancement ratio between in-plane and out-of-plane dipoles. Then, PSF of C-SPCEM can be written as:

P S F_{c - S P} (x, y) = {| E_{i l l}^{o u t -} |}^{2} + \partial \cdot {| E_{i l l}^{i n -} |}^{2}

The mediation of surface plasmon on fluorescent emission leads that longitudinal component of focal electric field contributes more to PSF_c-SP than that of transverse focal field component. The PSF_c-SP under RP and CP illumination are presented in Figs. 5(a) and 5(b) respectively. Because of the domination of longitudinal focal field component, the PSF_c-SP is solid under RP illumination and hollow under CP illumination which are quite different from that of conventional confocal microscope [10]. The capability to achieve solid or hollow PSF for C-SPCEM by simply changing the polarization of illumination brings convenience to improve resolution of C-SPCEM with FED technique.

Fig. 5 Calculated PSF for C-SPCEM according to Eq. (10). The illumination light is (a) radially polarized and (b) circularly polarized respectively.

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2.4 Principle of Fluorescence emission difference with C-SPCEM

The FED image is obtained by intensity subtraction of the image taken with the donut-shaped beam from the image taken with the solid beam [10],

I_{F E D} = I_{s o l i d} - γ \times I_{d o n u t}

where

γ

is an artificial subtractive factor, which is generally chosen between 0 to 1. A bigger

γ

will enhance resolution of the image, but artifacts will be introduced at the same time. Hence, the value of

γ

should be chosen to maintain the trade-off between high resolution and acceptable imaging quality. We assume that a negative side-lobe no larger than 10 percent of the peak can improve image quality while achieving a small FWHM. When the subtraction produces negative values, the value of FED is set to zero [28]. In this paper, I_solid and I_donut are represented by the PSF_c-SP of RP and CP beams, respectively. The effective PSF of C-SPCEM combined with the FED technique can be obtained by

P S F_{c - S P}^{F E D} = P S F_{c - S P}^{R P} - γ \times P S F_{c - S P}^{C P}

The subtraction of the two PSFs with different intensity distributions has the potential to create an imaging result with a sharper PSF. The excitation wavelength is chosen as 488 nm in all the theoretical calculation to keep consistent with the laser used in experiments. Figure 6(a) shows the subtracted PSF_c-SP excited by RP and CP beams. The intensity profiles of PSF_c-SP under RP and CP illumination are shown by black and red line in Fig. 6(b), respectively. The blue line represents the intensity profile of subtracted PSF_c-SP with

γ = 0

.25. Obviously, the FED technique yields a narrower PSF with full width at half-maximum (FWHM) of about λ/4.

Fig. 6 (a) The subtracted PSF for C-SPCEM excited by RP and CP beams. (b) Intensity profiles of different PSFs. The black line corresponds to PSF_c-SP for RP beam. The red line corresponds to PSF_c-SP for CP beam. The blue line corresponds to PSF_c-SP of FED microscopy with $γ = 0$ .25.

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3. Experimental results and discussions

3.1 Experiment preparation and C-SPCEM configuration

Figure 7(a) shows the schematic diagram of the experimental setup (built on Olympus FV300 LSCM). 40 nm-thick silver film is deposited onto the surface of glass slide to make a plasmonic substrate. A silver film with a thickness of 40 nm is adopted because of the tradeoff of local electric field enhancement and the fluorescence emission coupling [29]. Then, a 20 nm-thick polymethyl methacrylate (PMMA) spacer is spin-coated on the silver film. The dielectric spacer not only prevents oxidation of the Ag film in aqueous solution, but also blocks the fluorescence quenching by Ag film, hence increase the quantum yield of the fluorescent dyes [30]. A laser beam at 488 nm is expanded to fulfill the rear aperture of the objective (100 × , N.A. 1.45) and then focused to excite the fluorescence samples (fluorescence polystyrene spheres: FPSs, 150 nm in diameter, Duke Science).We use an S-waveplate (RPC, WOP, Lithuania) polarization converter [Fig. 7(b)] to produce RP illumination light. If the S-waveplate is replaced by a quarter wave plate (QWP), the LP incident light will be converted into a CP beam [Fig. 7(c)]. All polarization converter are placed along separate optical paths, hence we can easily switch from linear to radial/circular polarization without realignment. An x-y scanning galvanometer is used to ensure the system stability and to avoid the image mismatch. Confocal configuration has a point detector at the optically conjugated position of the focused spot for the excitation, differing from the wide-field microscopy equipped with a charge-coupled device (CCD). Photomultiplier tube (PMT) is set after a bandpass filter centered at 580 nm to detect the fluorescent radiation.

Fig. 7 (a) Schematic diagram of the experimental setup, (b) LP beam converted into RP by RPC and (c) LP beam converted into CP by QWP.

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3.2 Characterization of PSFs for C-SPCEM

Fluorescence polystyrene spheres (FPSs) on the plasmonic substrate are imaged by scanning confocal microscope under illumination in different polarizations conditions. Image of an isolated fluorescent bead can be approximately regarded as experimental PSF for C-SPCEM and will be compared with the theoretical prediction above. The images are repeatedly measured ten times in the same experimental condition and then averaged to remove random noise. Because of the tradeoff of sufficient fluorescence and small side-lobe of PSF_c-SP for RP beam, we chose confocal aperture with a diameter of 100 μm. As shown in Fig. 8(a), a solid homogeneous focal spot is generated with the RP illumination and the FWHM of PSF_c-SP for RP beam is determined to be 164 nm, which is consistent with the theoretical prediction of PSF_c-SP for RP illumination in term of the morphology [Fig. 5(a)]. This is very different from wide-field SPCE microscope, in which PSF for RP illumination is donut-shaped [7]. It should also be pointed out that the FWHM of PSF in conventional confocal microscopy illuminated by LP beam is measured to be 230 nm in our experimental system. About twenty-nine percent improvements in the resolution of C-SPCEM are obtained by RP beam compared to conventional LP beam based on confocal microscopy. Donut-shaped PSF for C-SPCEM is demonstrated experimentally by the image of 150 nm FPSs under CP illumination [Fig. 8(b)]. Subtracting the donut-shaped image from the solid image of FPSs with γ = 0.2, we get the PSF for C-SPCEM with FED technique [Fig. 8(c)]. The FWHM of PSF for FED microscopy (about 146 nm) with γ = 0.2 corresponds to λ/4, which is clearly beyond Rayleigh’s diffraction limit. To be more convincing, we counted a number of beads to estimate their average FWHM as shown in Fig. 8(e). The black dots give the statistical average FWHM of 161 nm for PSFc-sp excited by RP beam and the blue dots for FED yield statistical average FWHM of 147 nm. It is clearly seen from the normalized profiles of PSF_c-SP under RP, CP illumination and FED technique [Fig. 8(d)], FED technique enhances the lateral resolution of C-SPCEM after carefully optimizing subtraction coefficient. We have to point out that the subtractive factor used here is a little smaller than that used in simulation (Fig. 6) to avoid over-subtraction [31].

Fig. 8 Images of a 150 nm fluorescent bead with confocal aperture diameter of 100 µm. (a) and (b) are RP and CP illuminations, respectively. (c) is the subtracted result of (a) and (b). (d) The intensity profiles of (a), (b) and (c). (e) The statistical FWHM for PSFc-sp excited by RP beam and for FED. Scale bar: 500 nm.

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3.3 Resolution enhancement of C-SPCEM with fluorescence emission difference

The super-resolution capability of C-SPCEM combined with the FED technique is further demonstrated by comparing the confocal images of sparsely distributed fluorescence beads obtained respectively by C-SPCEM with only RP illumination and with combination of FED technique. Figure 9(a) presents the image achieved by C-SPCEM with RP illumination and image of the same sample region is also obtained by using FED technique with γ = 0.2 [Fig. 9(b)]. The dashed rectangles in the pictures indicate fluorescence bead pairs consisted with two beads closely separated. The center-to-center distance is 220 nm for the left-up pair and 240 nm for right-down pair. According to Rayleigh resolution limit, both pairs are undistinguishable because the separations are smaller than the diffraction limit (244 nm). From the normalized intensity profiles inserted in the pictures, it is clearly seen that the bead pairs are hardly distinguishable by only C-SPCEM, but are distinctly distinguished in the confocal image after FED technique is introduced.

Fig. 9 Images of two FPSs aggregate together obtained by C-SCPEM with RP illumination (a) and with FED technique with γ = 0.2 (b). Two different sets of normalized intensity profiles along lines indicated by white arrows are shown in (a) and by yellow arrows are shown in (b). The black lines and blue lines represent the normalized intensity profiles obtained by C-SPCEM with RP illumination and with FED technique, respectively. Scale bar: 1µm.

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4. Conclusion

In conclusion, we proposed and demonstrated a novel method to realize super-resolution scanning confocal microscopy by taking advantage of surface plasmon-coupled fluorescence emission and FED technique. Because of the unique interaction between fluorescent emitter and surface plasmon polaritons, the longitudinal component of the focal field in the vicinity of metal surface dominates the PSF for C-SPCEM, which makes it possible to realize solid PSF by radially-polarized illumination and hollow PSF by circularly-polarized illumination. Therefore, FED technique is feasible to be applied into C-SPCEM by controlling the polarization of illumination. Resolution of λ/4 is experimentally demonstrated, which is unambiguously beyond Rayleigh’s diffraction limit. We expect that the method reported here will be valuable and widely applied in high-resolution imaging for biological specimens and nanomaterials.

Funding

National Key Basic Research Program of China (2012CB921900); National Natural Science Foundation of China (NSFC) (Grant Nos.11274293, 61377053, 11574293); Natural Science Foundation of Anhui Province (1408085MKL01).

Acknowledgments

This work was partially carried out at the USTC Center for Micro and Nanoscale Research and Fabrication.

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Fluorescence emission difference with surface plasmon-coupled emission applied in confocal microscopy

Abstract

1. Introduction

2. Theory

2.1 Focal field of oil-immersed lens through silver layer

2.2 Radiation pattern of dipole emitter located near the silver film surface

2.3 PSF of confocal surface plasmon-coupled emission microscopy (C-SPCEM)

2.4 Principle of Fluorescence emission difference with C-SPCEM

3. Experimental results and discussions

3.1 Experiment preparation and C-SPCEM configuration

3.2 Characterization of PSFs for C-SPCEM

3.3 Resolution enhancement of C-SPCEM with fluorescence emission difference

4. Conclusion

Funding

Acknowledgments

References and links

Cited By

Figures (9)

Equations (12)

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