Stimulated emission double depletion nanoscopy with background correction at the single-pixel level.

Fluorescence microscopy images are inevitably tainted by background contributions including emission from out-of-focus planes, scattered light, and detector noise. In stimulated emission depletion (STED) nanoscopy, an additional, method-specific background arises from incomplete depletion and re-excitation by the depletion beam. Various approaches have been proposed to remove the background from a STED image, some of which rely on the acquisition of a separate background image that is subtracted from the STED image with a weighting factor. Using stimulated emission double depletion (STEDD) nanoscopy, we observed that the weighting factor varies locally in densely labeled samples, so that background removal with a single (global) weighting factor generates local image artifacts due to incorrect background subtraction. Here we present an algorithm that computes the optimal weighting factor at the single-pixel level, yielding a difference image with excellent suppression of low-frequency background.

Label-free and dynamic monitoring of cell evolutions using wavelength-multiplexing surface plasmon resonance holographic microscopy.

Dynamic characterizations of intracellular variations and cell-substrate interactions under different external environments are critical to study cell behaviors and exploring biological applications. However, techniques that are capable of dynamically and simultaneously measuring multiple parameters of living cells in a wide-field manner have rarely been reported. Here, we present a wavelength-multiplexing surface plasmon resonance holographic microscopy which allows wide-field, simultaneous, and dynamic measurements of cell parameters, including cell-substrate distance and cytoplasm refractive index (RI). We use two lasers of 632.8 nm and 690 nm as light sources. Two beam splitters are employed in the optical setup to separately adjust the incident angle of two light beams. Then, surface plasmon resonance (SPR) can be excited for each wavelength under SPR angles. We demonstrate the advances of the proposed apparatus by systematically studying the cell responses to osmotic pressure stimuli from the environmental medium at the cell-substrate interface. The SPR phase distributions of the cell are firstly mapped at two wavelengths, then the cell-substrate distance and cytoplasm RI are retrieved using a demodulation method. Based on phase response differences between two wavelengths and monotonic changes of SPR phase with cell parameters, cell-substrate distance, and cytoplasm RI can be determined simultaneously using an inverse algorithm. This work affords a new optical measurement technique to dynamically characterize cell evolutions and investigate cell properties in various cellular activities. It may become a useful tool in the bio-medical and bio-monitoring areas.

Soy Protein Amyloid Fibril Scaffold for Cultivated Meat Application.

It is important to have sustainable and edible scaffolds to produce cultivated meat. In this research, three-dimensional (3D) porous scaffolds were developed by soy protein amyloid fibrils for cultivated meat applications. Food-safe biological and physical cross-linking methods using microbial transglutaminase and temperature-controlled water vapor annealing technique were employed to crosslink soy protein amyloid fibrils, resulting in the production of 3D scaffolds. The generated 3D scaffolds had pores with sizes ranging from 50 to 250 µm, porosities of 72-83%, and compressive moduli of 3.8-4.2 kPa, depending on the type of soy protein used in the process (ß-conglycinin (7S), glycinin (11S) and soy protein isolate (SPI)). When present with pepsin, these scaffolds can degrade within an hour but remain stable in phosphate-buffered saline for at least 30 days. The soy protein amyloid fibril scaffolds enabled C2C12 mouse skeletal myoblasts proliferate and differentiate without adding cell adhesive proteins or other coatings. The results demonstrate the potential of abundant and inexpensive soy protein amyloid fibrils to be utilized as scaffold materials for cultivated meat in the food industry.

Dual-wavelength surface plasmon resonance holographic microscopy for simultaneous measurements of cell-substrate distance and cytoplasm refractive index.

Studying the basic characteristics of living cells is of great significance in biological research. Bio-physical parameters, including cell-substrate distance and cytoplasm refractive index (RI), can be used to reveal cellular properties. In this Letter, we propose a dual-wavelength surface plasmon resonance holographic microscopy (SPRHM) to simultaneously measure the cell-substrate distance and cytoplasm RI of live cells in a wide-field and non-intrusive manner. Phase-contrast surface plasmon resonance (SPR) images of individual cells at wavelengths of 632.8 nm and 690 nm are obtained using an optical system. The two-dimensional distributions of cell-substrate distance and cytoplasm RI are then demodulated from the phase-contrast SPR images of the cells. MDA-MB-231 cells and IDG-SW3 cells are experimentally measured to verify the feasibility of this approach. Our method provides a useful tool in biological fields for dual-parameter detection and characterization of live cells.

Optical tweezers integrated surface plasmon resonance holographic microscopy for characterizing cell-substrate interactions under noninvasive optical force stimuli.

The rapid development of bio-mechanical research increases the significance of studying cell behaviors near the substrate under the force stimuli in a real-time manner. Here, we present an optical tweezers (OT) integrated surface plasmon resonance holographic microscopy (SPRHM) to realize the dynamical and in-situ characterizations of cell-substrate interactions with noninvasive optical force stimulations. Using the OT integrated SPRHM (OT-SPRHM), we dynamically manipulate the living cells by OT, and simultaneously, the phase-contrast surface plasmon resonance images of the living cells are obtained and the cell-substrate distance is determined via SPRHM. We show that OT-SPRHM has the advanced capabilities of measuring the optical force and its tiny variations applied to the K562 cells near the substrate. Also, we for the first time reveal the manipulation of the MC3T3-E1 cells by OT. Demonstrating its robustness, this technique provides a powerful tool to explore the responses of various biological specimens to the force stimuli along the cell-substrate interface in the bio-sensing area.

Dual-channel illumination surface plasmon resonance holographic microscopy for resolution improvement.

Surface plasmon resonance holographic microscopy (SPRHM), combining digital holographic microscopy with surface plasmon resonance (SPR), can simultaneously obtain the amplitude and phase distributions of the reflected beam carrying specimen information in SPR. Due to the decaying length of the surface plasmon wave as large as tens of micrometers, the spatial resolution of SPRHM is lower than that of ordinary optical microscopes. In this work, we propose a scheme to improve the spatial resolution of SPRHM by applying dual-channel SPR excitations. Through the polarization multiplexing technique, two holograms carrying the information of SPR excited in orthogonal directions are simultaneously acquired. Via a numerical reconstruction and filtering algorithm for holograms, the lateral spatial resolution of SPRHM can be effectively enhanced to reach nearly 1 µm at a wavelength of 632.8 nm. This is comparable to the resolution of traditional optical microscopes, while possessing the advantages of wide-field imaging and high measurement sensitivity of SPR.

Real-time and wide-field mapping of cell-substrate adhesion gap and its evolution via surface plasmon resonance holographic microscopy.

As one of the most common biological phenomena, cell adhesion plays a vital role in the cellular activities such as the growth and apoptosis, attracting tremendous research interests over the past decades. Taking the cell evolution under drug injection as an example, the dynamics of cell-substrate adhesion gap can provide valuable information in the fundamental research of cell contacts. A robust technique of monitoring the cell adhesion gap and its evolution in real time is highly desired. Herein, we develop a surface plasmon resonance holographic microscopy to achieve the novel functionality of real-time and wide-field mapping of the cell-substrate adhesion gap and its evolution in situ. The cell adhesion gap images of mouse osteoblast cells and human breast cancer cells have been effectively extracted in a dynamic and label-free manner. The proposed technique opens up a new avenue of revealing the cell-substrate interaction mechanism and renders the wide applications in the biosensing area.

Complex refractive index measurement for atomic-layer materials via surface plasmon resonance holographic microscopy.

The optical characterization of atomic-layer materials is significant for the clarification of fundamental physical properties of newly emerging nanomaterials. Here we propose to utilize the surface plasmon resonance (SPR) holographic microscopy to measure the complex refractive index (RI) of atomic-layer materials (i.e., graphene). We unambiguously determine the complex RI of single-layer graphene and few-layer graphene by fitting the measured reflection phase shift difference with theoretical values under the five-layer SPR model. The measurement results of the graphene layer grown by chemical vapor deposition at the visible range agree with the previous reports. Our method offers a cost-effective and robust avenue to characterize the complex RI of atomic-layer materials with distinct optical absorption, particularly the two-dimensional materials.

Measurement of thermal effect in laser pumped silicon employing infrared digital holographic interferometry.

We present a short-coherence infrared digital holographic interferometry (IRDHI) to quantitatively measure the weak thermal effect in silicon wafer under visible laser pumping. In IRDHI, a superluminescent diode and a narrow-band filter are introduced to eliminate the self-interference fringes and suppress the noise. The effect of coherence length of the detection light source is analyzed and the optimal coherence length range in the proposed configuration is given. Meanwhile, we measure the weak thermal effect in silicon pumped by two different approaches of a continuous visible laser with different powers. The proposed configuration, which shows high stability and sensitivity, can be easily adapted and improved to measure the variation of thermal effect or refractive index in other near infrared transparent materials.

Sb2Te3 topological insulator: surface plasmon resonance and application in refractive index monitoring.

Topological insulators as new emerging building blocks in electronics and photonics present promising prospects for exciting surface plasmons and enhancing light-matter interaction. Thus, exploring the visible-range plasmonic response of topological insulators is significant to reveal their optical characteristics and broaden their applications at high frequencies. Herein, we report the experimental demonstration of a visible-range surface plasmon resonance (SPR) effect on an antimony telluride (Sb2Te3) topological insulator film. The results show that the SPR can be excited with a relatively small incident angle in the Kretschmann configuration based on the Sb2Te3 film. Especially, we develop an impactful digital holographic imaging system based on the topological insulator SPR and realize the dynamic monitoring of refractive index variation. Compared with the traditional SPR, the Sb2Te3-based SPR possesses a broader measurement range. Our findings open a new avenue for exploring the optical physics and practical applications of topological insulators, such as environmental and biochemical sensing.

Integrated digital holographic microscopy based on surface plasmon resonance.

We propose a novel digital holographic microscopy (DHM) by integrating surface plasmon holographic microscopy (SPHM) with reflection DHM based on the angular and polarization multiplexing techniques. Taking advantages of the high sensitivity of surface plasmon resonance (SPR) and the high reflectivity of gold film, the tiny variations of specimen's refractive index (RI) can be measured by using SPHM, and meanwhile, the thickness changes of the specimen can be determined by means of reflection DHM. We experimentally monitor the volatilization process of an alcohol-water mixture droplet to verify the validity of the integrated DHM. The proposed microscopy is very promising in the objective-coupling SPR microscopy for multi-information measurements of diverse specimens with low-contrast RI distributions (biomolecules, nanofluids, etc.) in a dynamic and nondestructive way.

Measurement of ultrafast combustion process of premixed ethylene/oxygen flames in narrow channel with digital holographic interferometry.

The premixed ethylene and oxygen flame that is burning in a narrow channel is investigated with digital holographic interferometry (DHI). Combustion in either a narrow tube or channel is quite different. This is caused by the significant effects of the boundary layer. The flame's acceleration rate will be enhanced as the tube diameter decreases. Usually, flame and shock wave propagation, which occurs during the premixed ethylene/oxygen flame combustion in the measurement area, is less than few milliseconds, so that general camera can rarely capture this fast event. This paper demonstrates that, by introducing the high-speed camera to DHI, the propagation of weak compression wave, flame, and shock wave generated in the narrow channel is successfully measured with a temporal resolution of 10 µs. The ultrafast processes of the flame front changing, as well as the shock wave coupling and separating, are clearly shown from the reconstructed phase distributions of the recorded holograms; corresponding density variations are simultaneously calculated. The results could provide references for the micro-scale propulsion and power devices design and use, and this proposed configuration can also easily adapt to other kinds of ultrafast processes in fluids.

Wavelength-multiplexing surface plasmon holographic microscopy.

Surface plasmon holographic microscopy (SPHM), which combines surface plasmon microscopy with digital holographic microscopy, can be applied for amplitude- and phase-contrast surface plasmon resonance (SPR) imaging. In this paper, we propose an improved SPHM with the wavelength multiplexing technique based on two laser sources and a common-path hologram recording configuration. Through recording and reconstructing the SPR images at two wavelengths simultaneously employing the improved SPHM, tiny variation of dielectric refractive index in near field is quantitatively monitored with an extended measurement range while maintaining the high sensitivity. Moreover, imaging onion tissues is performed to demonstrate that the detection sensitivities of two wavelengths can compensate for each other in SPR imaging. The proposed wavelength-multiplexing SPHM presents simple structure, high temporal stability and inherent capability of phase curvature compensation, as well as shows great potentials for further applications in monitoring diverse dynamic processes related with refractive index variations and imaging biological tissues with low-contrast refractive index distributions in the near field.

Compact surface plasmon holographic microscopy for near-field film mapping.

We develop a compact objective-coupling surface plasmon holographic microscopy with a common-path configuration by introducing a Wollaston prism. Through off-axis hologram recording and numerical reconstruction, amplitude- and phase-contrast surface plasmon resonance (SPR) images can be obtained simultaneously. Based on the four-layer SPR model, the thin film thickness distribution in near field can be mapped unambiguously using a novel demodulation method without a priori knowledge. The technique demonstrates nondestructive and full-field measurement capabilities with sub-nanometer resolution theoretically. Furthermore, owing to the high temporal stability, the recommended system shows great potential for dynamic measurement of near-field tiny refractive index or thickness variation in fields such as chemistry and biomedicine, etc.

Common-path digital holographic microscopy for near-field phase imaging based on surface plasmon resonance.

We develop a common-path digital holographic microscopy based on prism-coupling surface plasmon resonance (SPR) for near-field phase imaging. A single beam splitter with specific configuration is introduced in an SPR imaging system to realize off-axis holographic recording. By measuring the phase shift difference of the reflected light at SPR exploiting the proposed holographic microscopy with high temporal stability, near-field characteristic measurement can be realized. With its simplicity, vibration isolation, and inherent capability of phase curvature compensation, the recommended system shows advanced performance in monitoring tiny refractive index variations and imaging biological tissues.

Transmission and total internal reflection integrated digital holographic microscopy.

We develop a transmission and total internal reflection (TIR) integrated digital holographic microscopy (DHM) by introducing a home-made Dove prism with a polished short side. With the help of angular and polarization multiplexing techniques, the 2D refractive index distribution of a specimen adhered on the prism surface is determined using TIR-DHM. Meanwhile, the thickness profile is unambiguously calculated from the phase information using transmission DHM. This integrated microscopy is nondestructive and dynamic and can be used to simultaneously measure the index distribution and thickness profile of transparent or semi-transparent liquid or solid samples.