Background suppression with dual modulation by saturated absorption competition microscopy

https://doi.org/10.1016/j.optlaseng.2021.106750Get rights and content

Highlights

  • In this report, we propose a novel approach by simultaneously modulating the two beams of SAC with two distinct frequencies, called dual-modulation SAC (dmSAC).

  • In dmSAC modality, the homodyne detection would not identify the uncorrelated fluorescence, the image background could be free.

  • We have experimentally demonstrated the dependences of the background contribution on the modulation frequencies and spatial resolution as high as one seventh of the illumination wavelength is gained.

  • dmSAC is believed to hold great potential in biological and physical applications and to become another powerful tool for the study of the micro world through the follow-up researches.

Abstract

Optical super-resolution microscopy can break the diffraction limit in far-field imaging to achieve nanoscopic resolution. Nowadays, super-resolution imaging based on saturated excitation exhibits great potentials in extracting high-frequency components. Current saturated absorption competition (SAC) method is based on spatio-temporally modulating the incident laser beams and demodulating the target fluorescence at a particular frequency. However, the uncorrelated background noise spoils the imaging performance and sets an upper limit to the attainable spatial resolution. Here, we propose a novel approach by simultaneously modulating the two beams of SAC with two distinct frequencies, called dual-modulation SAC (dmSAC). In dmSAC, the homodyne detection will not pick up uncorrelated fluorescence signals, therefore, the image is free of background noise. This technique has the potential to enable super-resolution imaging with fluorescent and non-fluorescent probes, which may remain out of reach to fluorescence-based imaging methods.

Introduction

Optical far-field fluorescence microscopy plays a significant role in life science research, as it has several advantages such as non-invasiveness and specificity. However, owing to the diffraction limit, its resolving ability is restricted to approximately half of the illumination wavelength [1]. Over the past two decades, we have witnessed the emergence of super-resolution optical microscopy, which successfully circumvents this diffraction barrier and increases the resolution to the nanoscale, such as stochastic optical reconstruction microscopy (STORM) [2], photo-activated localization microscopy (PALM) [3], structured illumination microscopy (SIM) [4], and stimulated emission depletion microscopy (STED) [5]. To date, super-resolution approaches are restricted in many aspects for use by the biologist community. For example, the STED modality suffers from photobleaching caused by the necessarily ultra-high depletion power [6]. Furthermore, special fluorescent dyes are also required [7]. Recently, saturation effects have been used to achieve super-resolution imaging [8], [9], [10], [11], [12], including saturated excitation microscopy (SAX) [13], excitation state saturation microscopy (ESSat) [14], nonlinear focal modulation microscopy (NFOMM) [15] and so on. Saturated absorption competition microscopy (SAC) is a newly-proposed super-resolution technique under the saturated excitation condition so that nonlinearities that contain high-spatial-frequency components can be extracted using lock-in detection [16]. SAC is advantageous in either fluorescent or non-fluorescent imaging. Only a single laser source is required and other advantages include non-constrain in fluorescent dye selection and easy implementation. Especially, in case of pulsed mode, high transverse spatial resolution improvement is demonstrated in the imaging of nonbleaching inorganic fluorescent probes [17]. Indeed, using differential strategy, the attainable resolution could be further enhanced [18].

In conventional SAC, the fluorescence excited by non-modulated hollow beam is routinely regarded as direct current component to lock-in amplifier (LIA) and consequently filtered out. However, in practical experiment, due to undulation of hardware, like laser diodes or optical modulating device, the direct current and uncorrelated fluorescence signals could not be completely decoupled by LIA [19], [20], [21]. Hence, this DC contribution will be partially extracted and mixed with the effective fluorescence derived from solid beam illumination. The underlying remnant obeys Poisson distribution which cannot be removed with regular approaches. Until now extensive variants have been presented to overcome this background noise [7, [22], [23], [24]], including gated detection [25], temporal modulation [26], and subtraction strategy [27, 28]. Here, we propose a feasible route, called dual-modulation SAC (dmSAC), to diminish the above-mentioned Poisson noise. By applying temporal modulation to both solid and hollow excitation beams with two different frequencies, f1 and f2, the fluorescence introduced by solid beam and hollow beam, respectively, can be unmixed and the speckle-like noise can also be alleviated pronouncedly during lock-in demodulation. We also experimentally investigate the dependences of two types of background noise on f1 and f2. We have experimentally demonstrated that higher modulation frequency is beneficial because low frequency modulation not only increases 1/f noise but also raises the lock-in time constant needed that lengthens the pixel dwell time and the acquisition time in dmSAC imaging. The attainable resolution with an improvement factor of 7.4 is achieved on 40-nm fluorescent microspheres with background-free imaging.

Section snippets

Theory

For organic dye molecules, according to the five-level diagram (Fig. 1a), the rate equation system is established as follows [29]:S0=k01S0+kfS1+kTT1S1=k01S0(k0+k1n+kbS)S1+kn1SnSn=k1nS1(kn1+kbSn)SnT1=kISCS1(kT+kT1n+kbT)T1+kTn1TnTn=kT1nT1(kTn1+kbTn)Tnwhere S0, S1, and Sn represent singlet electronic populations of the ground state, excited state and higher excited state, respectively; T1 and Tn represent populations of triplet state and higher triplet state, respectively. And k() represents

System setup

The experimental scheme of dual-modulation saturated competition microscopy (dmSAC) is depicted in Fig. 2. This architecture is suitable for either continuous or pulsed lasers. For the pulsed laser, the laser repetition rate is 80 MHz. The laser beam of 532 nm wavelength is collimated by a collimation lens (CL1). The incident beam is split into S-polarized and P-polarized beams by polarizing beam splitter (PBS1). A half wave plate (HWP) is used to adjust the intensity ratio between the two

Results and discussions

We first test the system background noise without lock-in detection. Using a time correlated single photon counting module (PicoHarp300, PicoQuant), while no fluorescence excitation occurs, the system noise is still detected. It is found that with the increment of laser beam power, this undesired noise increases linearly (Fig. 3a). The time trace of background photon counts with the increment of laser beam radiation ranging from 100 to 900  μW is indicated in Fig. 3b. Shot noise, read noise,

Conclusion and outlook

In this article, we introduced dual-modulation saturated absorption competition microscopy (dmSAC). As a novel method that can provide relatively high resolution and excellent signal-to-background ratio (SBR). There is no constraint on labeling selection, which makes dmSAC a versatile tool in fluorescent super-resolution microscopy that can resolve signals from either organic dyes or inorganic probes. Compared with conventional SAC implementation, only one set of optical modulator is added.

In

Disclosures

The authors declare that there are no conflicts of interest related to this article.

CRediT authorship contribution statement

Chuankang Li: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. Renjie Zhou: Methodology, Writing – review & editing. Wensheng Wang: Investigation, Software. Zhengyi Zhan: Formal analysis. Zhimin Zhang: Investigation, Software. Yuhang Li: Writing – review & editing. Yuzhu Li: Writing – review & editing. Xiang Hao: Conceptualization, Funding acquisition, Resources, Supervision. Cuifang Kuang: Resources, Supervision. Xu Liu: Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was funded by the National Natural Science Foundation of China (61827825 and 61735017), Major Program of the Natural Science Foundation of Zhejiang Province (LD21F050002), Key Research and Development Program of Zhejiang Province (2020C01116), Fundamental Research Funds for the Central Universities (K20200132), and Zhejiang Lab (2020MC0AE01).

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