Specific detection of CA242 by antibody-modified chiral symmetric double "N" metasurface biosensor.

In this work, a biosensor based on Fano resonance metasurface is proposed for the specific detection of CA242 which is a typical marker of pancreatic cancer. The biosensor consists of a chiral symmetric plasma double "N" structure, which utilises coherent coupling of bright and dark modes to generate Fano resonance, achieving suppression of radiation loss, concentrating and storing energy more efficiently in the structure, and contributing to increased sensitivity to changes in ambient refractive index, resulting in a sensitivity of the sensor of up to 842.8 nm /RIU. After a series of antibody functionalization modifications, the metasurface has become an immune biosensor that can specifically detect the tumor marker CA242 of pancreatic cancer. The detection of mixed and single antigen solutions with different concentrations has verified the high sensitivity, high specificity, and high linear relationship of the biosensor to CA242, and the detection limit is as low as 0.0692 ng/mL. It is superior to other common methods and breaks the traditional disadvantages of lower detection accuracy and greater damage in tumour detection methods. The detection of the wavelength shift of localized surface plasmon resonance in plasma metasurface has been successfully applied to the highly sensitive detection of tumor markers. This study demonstrates the sensitivity and maneuverability of the chiral symmetric double "N" plasmonic metasurface biosensor, suggesting the potential application of metamaterials in biosensing based on environmental refractive index changes.

Accurate evaluation of the treatment effects of immunotherapy on subcutaneous ovarian cancer in mice with nonlinear optical imaging and algorithmic analysis.

Immunotherapy and its evaluation have shown great promise for cancer treatment. Here, a mouse subcutaneous transplantable tumor model was applied to testing therapeutic strategies. The mouse model was treated by regulating anti-PD-L1, anti-CTLA-4, cisplatin and their combined therapy. Biochemistry experiments have found that after immunotherapy, mice have more immune responses, which were manifested by an increase in the content of growth factors and the activation of T cells. Meanwhile, multimodal nonlinear optical microscopy imaging combined with algorithms was used to evaluate the treatment's effectiveness. By detecting the metabolism rate and microstructure in tissue, it was proved that combined therapies including immune checkpoint inhibitors do have a better effect on ovarian tumors. Our discovery of valid treatments for mice with ovarian tumor and provides an evaluation tool via nonlinear optics combined with algorithms offers new insights into therapeutic effect.

In vivo two-photon fluorescence lifetime imaging microendoscopy based on fiber-bundle.

Fluorescence lifetime imaging microendoscopy (FLIME) has been reported to investigate the physicochemical microenvironment in biological tissue. In this work, we designed a two-photon (TP) FLIME system based on a fiber-bundle glued with an achromatic mini-objective with 1.4-mm diameter, which was coupled to a standard TP microscope containing a dispersion precompensation module in the laser source. With 840 nm excitation, the field of view and lateral resolution of our system are 390 µm and 1.55 µm, respectively. To examine the capability of imaging in vivo, we obtained Z-stack (0-130 µm) TP-FLIME images from the intestine's surface of a mouse injected with squaraine dye. Further, we utilized the TP-FLIME system to image the kidney, liver, and xenografted tumor at 100-µm depth in vivo with cellular resolution, which features the distribution of cells and tissue structures with better contrast than intensity images. These results demonstrated that the proposed system is capable of measuring fluorescence lifetime in situ and provides a powerful tool to research the deep tissue microenvironment naturally.

Investigating tunneling nanotubes in ovarian cancer based on two-photon excitation FLIM-FRET.

Precise and efficient cell-to-cell communication is critical to the growth and differentiation of organisms, the formation of various organism, the maintenance of tissue function and the coordination of their various physiological activities, especially to the growth and invasion of cancer cells. Tunneling nanotubes (TNTs) were discovered as a new method of cell-to-cell communication in many cell lines. In this paper, we investigated TNTs-like structures in ovarian cancer cells and proved their elements by fluorescent staining, which showed that TNTs are comprised of natural lipid bilayers with microtubules as the skeleton that can transmit ions and organelles between adjacent cells. We then used fluorescence resonance energy transfer (FRET) based on two-photon excitation fluorescence lifetime imaging microscopy (FLIM) (TP-FLIM-FRET) to detect material transport in TNTs. The experimental results showed that the number of TNTs have an impact on the drug treatment of cancer cells, which provided a new perspective for TNTs involvement in cancer treatment. Our results also showed that TP-FLIM-FRET would potentially become a new optical method for TNTs study.

Human serum albumin gradient in serous ovarian cancer cryosections measured by fluorescence lifetime.

Human serum albumin (HSA) is a depot and carrier for many endogenous and exogenous molecules in blood. Many studies have demonstrated that the transport of HSA in tumor microenvironments contributes to tumor development and progression. In this report, we set up a multimodal nonlinear optical microscope system, combining two-photon excitation fluorescence, second harmonic generation, and two-photon fluorescence lifetime imaging microscopy. The fluorescence lifetime of a small squaraine dye (SD) is used to evaluate HSA concentrations in tumor tissue based on specific binding between SD and HSA. We used SD to stain the cryosections from serous ovarian cancer patients in high-grade (HGSOC) and low-grade (LGSOC), respectively, and found a gradient descent of HSA concentration from normal connective tissue to extracellular matrix to tumor masses from 13 to 2 µM for LGSOC patients and from 36 to 12 µM for HGSOC patients. We demonstrated that multimodal nonlinear optical microscopy can obtain similar results as those from traditional histologic staining, thus it is expected to move to clinical applications.

Quantitative laser-induced breakdown spectroscopy for discriminating neoplastic tissues from non-neoplastic ones.

In this paper, we present a method to distinguish neoplastic tissues from non-neoplastic ones using calibration-free laser-induced breakdown spectroscopy (CF-LIBS). For this propose, plasma emission was collected from neoplastic and non-neoplastic tissues taken from the ovarian cancer mice models. Results were obtained by utilizing the characteristic plasma emission lines of different elements that have been confirmed in the investigated samples. From the temporal evolution of plasma emission, the optimum temporal-observation-windows are identified for LIBS investigation. The concentrations of the detected elements in tissues were measured by a calibration-free approach based on data process of plasma parameters at the local thermodynamic equilibrium. The neoplastic specimens provided more energetic plasma than non-neoplastic ones that resulting in higher peaks intensities, electron density and electron temperature especially in the early windows (between 0.1 µs to 0.8 µs). Results demonstrated higher concentrations of major and trace elements such as Mg, Fe, Ca, Na, and K in the neoplastic tissues. Finally, the results using CF-LIBS method were found to be in good agreement with that of Inductive coupled plasma-optical emission spectroscopy (ICP-OES).

Monitoring the extracellular matrix remodeling of high-grade serous ovarian cancer with nonlinear optical microscopy.

The mortality of high-grade serous ovarian cancer (HGSOC) accounts for 70% to 80% of all ovarian cancer deaths and overall mortality rate has not declined in the last decade. Recently, many studies have demonstrated that HGSOC originates from the fallopian tubes. The extracellular matrix (ECM) is present in all tissues, its remodeling and interaction with cells are crucial for regulating cell proliferation, migration, and differentiation. In this paper, we used label-free nonlinear optical microscopy to image tissues of the fallopian tube and ovary. Combining a set of image processing algorithms, we monitored the remodeling of ECM in the fallopian tube and ovary during the invasion of primary serous fallopian tube tumor into the ovary in microscopic dimension. With this approach, we can obtain physiological information of HGSOC at the early stage, which provided useful data for auxiliary clinical diagnosis.

An all-graphene quantum dot Förster resonance energy transfer (FRET) probe for ratiometric detection of HE4 ovarian cancer biomarker.

Ovarian cancer (OVC), the most lethal form of all gynecological cancers, is a big threat to women's health. Late diagnosis at the advanced stages is one of the major reasons for the ovarian cancer-related deaths. Conventionally, the up-regulated proteins CA125 (cancer antigen 125) and HE4 (human epididymis protein 4) are used as biomarkers to diagnose the OVC malignancies. The lack of sensitivity/specificity and the false-positive results create complexity in the diagnostic process. With specificity over 90 %, HE4 is suitable for diagnosing ovarian cancer. Herein, we have developed an ultrasensitive all-graphene quantum dot (GQD) Förster resonance energy transfer (FRET) probe for the ratiometric detection of HE4 biomarker. A set of two GQD samples were solvothermally prepared and then analyzed by the morphological, structural, and photophysical characterization. One GQD sample exhibited a strong green emission, peaked at around 515 nm, while the other GQD sample displayed a strong red emission with maximum at around 615 nm. The good spectral overlap between the emission and excitation spectra of the green and red GQDs, respectively, all allowed us to consider them for the design of FRET-based probe. The green and red-emitting GQDs were conjugated with HE4 antibody and used as donor and acceptor, respectively for the ratiometric sensing of HE4 ovarian cancer biomarker. The all GQD FRET probe was able to detect as low as 4.8 pM, along with a large dynamic detection range up to 300 nM. The selectivity and interference effect of the developed FRET probe was also investigated against different protein combinations.

Rapid and Targeted Photoactivation of Ca2+ Channels Mediated by Squaraine To Regulate Intracellular and Intercellular Signaling Processes.

As an important cellular signal transduction messenger, Ca2+ has the capability to regulate cell function and control many biochemical processes, including metabolism, gene expression, and cell survival and death. Here, we introduce an accessible method for the photoactivation of Ca2+ channels mediated by squaraine (SQ) to rapidly induce cellular Ca2+ release and activate signal transduction. With a short preparation time, the maximum Ca2+ concentration increase could reach approximately 450% in 30 s, resulting from marked Ca2+ release channel opening in the endoplasmic reticulum (ER). This release was enhanced by another target location of SQ, that is, the outer mitochondrial-associated membrane where Ca2+ channels accumulate, and by the consequent large amounts of reactive oxygen species resulting from the respiratory chain activity stimulated by Ca2+ load. We used this method to investigate cellular signal transduction in different cancer cells and revealed rapid intracellular Ca2+ flow, unidirectional intercellular signaling processes, and neuronal signaling activity, which demonstrated the potential and convenience of the method for routine Ca2+ research.

Solo Smart Fluorogenic Probe for Potential Cancer Diagnosis and Tracking in Vivo Tumorous Lymphatic Systems via Distinct Emission Signals.

A versatile twisted-intramolecular-charge-transfer (TICT)-based near-infrared (NIR) fluorescent probe (L) has been judiciously designed and synthesized that could be utilized for potential cancer diagnosis and to track lymph node(s) in mice through distinct emission signals. Essentially, the probe rendered the capability to preferentially recognize the cancer cells over the noncancer cells by polarity-guided lipid droplet specific differential bioimaging (in green emission channel) studies. The probe also exhibited selective turn-on fluorescence response toward HSA/BSA in physiological media (aqueous PBS buffer; pH 7.4) at far-red/NIR regions, because of the 1:1 chelation between the probe and HSA/BSA. Therefore, the fluorescent probe was then maneuvered to track the draining lymphatic system and sentinel lymph node in tumor mice model by fluorescence imaging (NIR/deep-red channel), wherein the accumulated albumin protein in the draining tumor lymphatic system facilitated the in situ formation of the fluorescent albumin-L complex.

Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra.

The spontaneous excitation-emission (ExEm) spectrum is introduced to the quantitative mExEm-spFRET methodology we recently developed as a spectral unmixing component for quantitative fluorescence resonance energy transfer measurement, named as SPEES-FRET method. The spectral fingerprints of both donor and acceptor were measured in HepG2 cells with low autofluorescence separately expressing donor and acceptor, and the spontaneous spectral fingerprint of HEK293 cells with strong autofluoresence was measured from blank cells. SPEES-FRET was performed on improved spectrometer-microscope system to measure the FRET efficiency (E) and concentration ratio (R C ) of acceptor to donor vales of FRET tandem plasmids in HEK293 cells, and obtained stable and consistent results with the expected values. Moreover, SPEES-FRET always obtained stable results for the bright and dim cells coexpressing Cerulean and Venus or Cyan Fluorescent Protein (CFP)-Bax and Yellow fluorescent protein (YFP)-Bax, and the E values between CFP-Bax and YFP-Bax were 0.02 for healthy cells and 0.14 for the staurosporine (STS)-treated apoptotic cells. Collectively, SPEES-FRET has very strong robustness against cellular autofluorescence, and thus is applicable to quantitative evaluation on the protein-protein interaction in living cells with strong autofluoresence.

IIem-spFRET: improved Iem-spFRET method for robust FRET measurement.

We recently developed a quantitative Förster resonance energy transfer (FRET) measurement method based on emission-spectral unmixing (Iem-spFRET). We here developed an improved Iem-spFRET method (termed as IIem-spFRET) for more robust FRET measurement in living cells. First, two background (BG) spectral fingerprints measured from blank living cells are introduced to remove BG and autofluorescence. Second, we introduce a ? factor denoting the ratio of two molar extinction coefficient ratios (?) of acceptor to donor at two excitations into IIem-spFRET for direct measurement of the ? values using a tandem construct with unknown FRET efficiency (E). We performed IIem-spFRET on our microscope­spectrometer platform to measure the ? values of Venus (V) to Cerulean (C) and the E values of C32V, CVC, VCV, and VCVV constructs, respectively, in living Huh7 cells. For the C32V or CVC cells, the Iem-spFRET and IIem-spFRET methods measured consistent E values. However, for the cells especially with low expressing levels of VCV or VCVV, the E values measured by Iem-spFRET showed large deviations and fluctuations, whereas the IIem-spFRET method greatly improved the measured E values. Collectively, IIem-spFRET is a powerful and robust tool for quantitatively measuring FRET signal in living cells.