Two-photon Excited Fluorescence Research Articles

Multimodal 2D and 3D microscopic mapping of growth cartilage by computational imaging techniques - a short review including new research.

Being able to image the microstructure of growth cartilage is important for understanding the onset and progression of diseases such as osteochondrosis and osteoarthritis, as well as for developing new treatments and implants. Studies of cartilage using conventional optical brightfield microscopy rely heavily on histological staining, where the added chemicals provide tissue-specific colours. Other microscopy contrast mechanisms include polarization, phase- and scattering contrast, enabling non-stained or "label-free" imaging that significantly simplifies the sample preparation, thereby also reducing the risk of artefacts. Traditional high-performance microscopes tend to be both bulky and expensive. Computational imaging denotes a range of techniques where computers with dedicated algorithms are used as an integral part of the image formation process. Computational imaging offers many advantages like 3D measurements, aberration correction and quantitative phase contrast, often combined with comparably cheap and compact hardware. X-ray microscopy is also progressing rapidly, in certain ways trailing the development of optical microscopy. In this study, we first briefly review the structures of growth cartilage and relevant microscopy characterization techniques with an emphasis on FPM and advanced X-ray microscopies. We next demonstrate with our own results computational imaging through Fourier ptychographic microscopy (FPM) and compare the images with hematoxylin eosin and saffron (HES)-stained histology. Zernike phase contrast, and the nonlinear optical microscopy techniques of second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are reported. X-ray attenuation-, phase- and diffraction-contrast computed tomography (CT) images of the very same sample are also presented for comparisons. Future perspectives on the links to artificial intelligence, dynamic studies and in vivo possibilities conclude the article.

Highly-selective optical filter for NADH fluorescence detection in multiphoton microscopy

Colorectal cancer (CRC) is a pressing global health concern, emphasizing the need for early detection tools. In this study an optical filter for precise detection of nicotinamide adenine dinucleotide (NADH) fluorescence via two-photon excitation fluorescence (TPEF) was developed. Fabricated with silicon dioxide and titanium dioxide thin films in a Fabry-Perot structure, the filter achieved a peak transmittance of about 95% at 483 nm, with a 12 nm full-width at half maximum. TPEF measurements using a tailored setup and NADH liquid phantoms underscored the filter's significance in selectively capturing NADH fluorescence while mitigating interference from other fluorophores. This work marks a substantial stride towards integrating multiphoton microscopy into conventional colonoscopy, enabling non-invasive, objective optical biopsy for colorectal tissue analysis. Further refinements of the experimental setup are imperative to advance tissue differentiation and enhance CRC diagnosis.

Analysis of age-related changes in the left ventricular myocardium with multiphoton microscopy

Aging induces cardiac remodeling, resulting in an increase in the risk of suffering heart diseases, including heart failure. Collagen deposition increases with age and, together with sarcomeric changes in cardiomyocytes, may lead to ventricular stiffness. Multiphoton (MP) microscopy is a useful technique to visualize and detect variations in cardiac structures in a label free fashion. Here, we propose a method based on MP imaging (both two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) modalities) to explore and objectively quantify age-related structural differences in various components of cardiac tissues. Results in transmural porcine left ventricle (LV) sections reveal significant differences when comparing samples from young and old animals. Collagen and myosin SHG signals in old specimens are respectively 3.8x and >6-fold larger than in young ones. Differences in TPEF signals from cardiomyocyte were ∼3x. Moreover, the increased amount of collagen in old specimens results in a more organized pattern when compared to young LV tissues. Since changes in collagen and myosin are associated with cardiac dysfunction, the technique used herein might be a useful tool to accurately predict and measure changes associated with age-related myocardium fibrosis, tissue remodeling and sarcomeric alterations, with potential implications in preventing heart disease.

Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores.

Single-molecule localization microscopy (SMLM) based on temporal-focusing multiphoton excitation (TFMPE) and single-wavelength excitation is used to visualize the three-dimensional (3D) distribution of spontaneously blinking fluorophore-labeled subcellular structures in a thick specimen with a nanoscale-level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out-of-focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single-wavelength TFMPE achieves wide-field and axially confined two-photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two-dimensional TFMPE-SMLM image of the microtubules in cancer cells with a localization precision of 18 ± 6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE-SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid-beta peptide deposits.

293 The Impact of Tumor Mutational Status on Protoporphyrin IX Accumulation in Tumor-Associated Macrophages in Glioblastoma Patients

INTRODUCTION: Immunotherapies provide promising new treatment options for lung cancer and melanoma, however success in glioblastoma (GBM) patients has been much rarer and uneven. Identifying candidates with GBM that are most likely to benefit from immunotherapy remains a challenge. The abundance of tumor-associated macrophages (TAMs) correlates response to immunotherapy in GBM patients. Here we leverage a new imaging technique for quantifying TAMs via tandem stimulated Raman histology (SRH) and two-photon excitation fluorescence microscopy (TPEF) to assess the impact of genetic classification on the abundance of TAMs. METHODS: We conducted tandem SRH and TPEF on specimens from 79 patients undergoing high-grade glioma resection with 5-ALA. We trained a MaskR-CNN deep neural network to identify TAMs and compute their density in each specimen. Corresponding clinical, demographic, genomic and methylomic data were collected for each patient. Independent sample t-tests were used to identify significant correlations between genetic markers, treatment, and TAM density. RESULTS: We analyzed 3,121,524 300 x 300 micron fields of view, from 151 fresh specimens, enabling segmentation of 611,789 TAMs. IDH-mutant tumors had significantly lower TAM density than IDH-wildtype tumors (p < 0.01). The absence of MGMT promoter hypermethylation was associated with significantly higher TAM density (p = 0.03). Tumors with EGFR amplification averaged significantly lower TAM density than EGFR wildtype specimens (p < 0.01). Recurrent tumors possessed 31.5% lower TAM density than primary resections. CONCLUSIONS: Tandem SRH and TPEF can estimate variations in TAM density in human GBMs. We found TAM density to be associated with IDH status, MGMT promoter methylation, and EGFR amplification. This imaging technique creates a new avenue for forecasting response to immunotherapy in GBM patients.

Collagen modifications predictive of lymph node metastasis in dogs with carcinoma in mixed tumours.

Mixed tumours in the canine mammary gland are the most common histological type in routine diagnosis. In general, these neoplasms have a favourable prognosis that does not evolve into metastatic disease. However, some cases develop into lymph node metastases and are associated with worse patient survival rates. Here is a retrospective study of 46 samples of primary mixed tumours of the canine mammary gland: 15 cases of benign mixed tumours (BMT), 16 cases of carcinoma in mixed tumours without lymph node metastasis (CMT), and 15 cases of carcinomas in mixed tumours with lymph node metastasis (CMTM). In addition, we selected 23 cases of normal mammary glands (NMT) for comparison. The samples were collected from biopsies performed during nodulectomy, simple mastectomy, regional mastectomy, or unilateral/bilateral radical mastectomy. We used multiphoton microscopy, second harmonic generation, and two-photon excited fluorescence, to evaluate the characteristics of collagen fibres and cellular components in biopsies stained with haematoxylin and eosin. We performed Ki67, ER, PR, and HER-2 immunostaining to define the immunophenotype and COX-2. We showed that carcinomas that evolved into metastatic disease (CMTM) present shorter and wavier collagen fibres as compared to CMT. When compared to NMT and BMT the carcinomas present a smaller area of fibre coverage, a larger area of cellular coverage, and a larger number of individual fibres. Furthermore, we observed a correlation between the strong expression of COX-2 and a high rate of cell proliferation in carcinomas with a smaller area covered by cell fibres and a larger number of individual fibres. These findings highlight the fundamental role of collagen during tumour progression, especially in invasion and metastatic dissemination.

Differences in Lipid Order and Dynamics in Plasma Membranes Assessed by Nonlinear Optical Microscopy.

When amphiphilic polar dyes were added to the cells, they intercalated predominantly in the outer leaf of the plasma membrane, making them active for second harmonic generation (SHG). The fluorescence of the dye enabled simultaneous 3D imaging of SHG and two-photon excited fluorescence (TPF). Because SHG intensity is sensitive to the alignment of the dyes, which reflects lipid ordering in the plasma membrane, we assessed the difference in lipid ordering by comparing the SHG intensity normalized to the TPF intensity. Together with an enzyme release assay that detects pore formation in the plasma membrane, our SHG assay revealed how polycations affect lipid ordering at low concentrations, where membrane damage has not yet been examined. By scaling the results of the assays with the charge concentration of the two polycations, polyethylenimine (PEI) and poly-l-lysine (PLL), we found that PEI reduced the lipid order more than PLL, and PLL formed more pores than PEI. A comparison of the SHG and TPF images of the wounded cells revealed that one of the lipid dynamics (flip-flop) was significantly enhanced in the bleb membrane. Moreover, the SHG assay indicated that the biocompatible polymer, poly(N-(2-hydroxypropyl)methacrylamide), did not affect the lipid order. Thus, our technique allows the assessment of the plasma membrane structure at the molecular level.

A 20 MHz Repetition Rate, Sub-Picosecond Ti-Sapphire Laser for Fiber Delivery in Nonlinear Microscopy of the Skin.

Nonlinear microscopy (NM) enables us to investigate the morphology or monitor the physiological processes of the skin through the use of ultrafast lasers. Fiber (or fiber-coupled) lasers are of great interest because they can easily be combined with a handheld, scanning nonlinear microscope. This latter feature greatly increases the utility of NM for pre-clinical applications and in vivo tissue imaging. Here, we present a fiber-coupled, sub-ps Ti-sapphire laser system being optimized for in vivo, stain-free, 3D imaging of skin alterations with a low thermal load of the skin. The laser is pumped by a low-cost, 2.1 W, 532 nm pump laser and delivers 0.5-1 ps, high-peak-power pulses at a ~20 MHz repetition rate. The spectral bandwidth of the laser is below 2 nm, which results in a low sensitivity for dispersion during fiber delivery. The reduction in the peak intensity due to the increased pulse duration is compensated by the lower repetition rate of our laser. In our proof-of-concept imaging experiments, a ~1.8 m long, commercial hollow-core photonic bandgap fiber was used for fiber delivery. Fresh and frozen skin biopsies of different skin alterations (e.g., adult hemangioma, basal cell cancer) and an unaffected control were used for high-quality, two-photon excitation fluorescence microscopy (2PEF) and second-harmonic generation (SHG) z-stack (3D) imaging.

Improving the diagnosis of ductal carcinoma in situ with microinvasion without immunohistochemistry: An innovative method with H&E-stained and multiphoton microscopy images.

Ductal carcinoma in situ with microinvasion (DCISM) is a challenging subtype of breast cancer with controversial invasiveness and prognosis. Accurate diagnosis of DCISM from ductal carcinoma in situ (DCIS) is crucial for optimal treatment and improved clinical outcomes. However, there are often some suspicious small cancer nests in DCIS, and it is difficult to diagnose the presence of intact myoepithelium by conventional hematoxylin and eosin (H&E) stained images. Although a variety of biomarkers are available for immunohistochemical (IHC) staining of myoepithelial cells, no single biomarker is consistently sensitive to all tumor lesions. Here, we introduced a new diagnostic method that provides rapid and accurate diagnosis of DCISM using multiphoton microscopy (MPM). Suspicious foci in H&E-stained images were labeled as regions of interest (ROIs), and the nuclei within these ROIs were segmented using a deep learning model. MPM was used to capture images of the ROIs in H&E-stained sections. The intensity of two-photon excitation fluorescence (TPEF) in the myoepithelium was significantly different from that in tumor parenchyma and tumor stroma. Through the use of MPM, the myoepithelium and basement membrane can be easily observed via TPEF and second-harmonic generation (SHG), respectively. By fusing the nuclei in H&E-stained images with MPM images, DCISM can be differentiated from suspicious small cancer clusters in DCIS. The proposed method demonstrated good consistency with the cytokeratin 5/6 (CK5/6) myoepithelial staining method (kappa coefficient = 0.818).

Label-free assessment of pathological changes in pancreatic intraepithelial neoplasia by biomedical multiphoton microscopy.

Pancreatic intraepithelial neoplasia (PanIN) is the most common precursor lesion that has the potential to progress to invasive pancreatic cancer, and early and rapid detection may offer patients a chance for treatment before the development of invasive carcinoma. Therefore, the identification of PanIN holds significant clinical importance. In this study, we first used multiphoton microscopy (MPM) combining two-photon excitation fluorescence and second-harmonic generation imaging to label-free detect PanIN and attempted to differentiate between normal pancreatic ducts and different grades of PanIN. Then, we also developed an automatic image processing strategy to extract eight morphological features of collagen fibers from MPM images to quantify the changes in collagen fibers surrounding the ducts. Experimental results demonstrate that the combination of MPM and quantitative information can accurately identify normal pancreatic ducts and different grades of PanIN. This study may contribute to the rapid diagnosis of pancreatic diseases and may lay the foundation for further clinical application of MPM.

Computer-Aided Multiphoton Microscopy Diagnosis of 5 Different Primary Architecture Subtypes of Meningiomas

Meningiomas rank among the most common intracranial tumors, and surgery stands as the primary treatment modality for meningiomas. The precise subtyping and diagnosis of meningiomas, both before and during surgery, play a pivotal role in enabling neurosurgeons choose the optimal surgical program. In this study, we utilized multiphoton microscopy (MPM) based on 2-photon excited fluorescence and second-harmonic generation to identify 5 common meningioma subtypes. The morphological features of these subtypes were depicted using the MPM multichannel mode. Additionally, we developed 2 distinct programs to quantify collagen content and blood vessel density. Furthermore, the lambda mode of the MPM characterized architectural and spectral features, from which 3 quantitative indicators were extracted. Moreover, we employed machine learning to differentiate meningioma subtypes automatically, achieving high classification accuracy. These findings demonstrate the potential of MPM as a noninvasive diagnostic tool for meningioma subtyping and diagnosis, offering improved accuracy and resolution compared with traditional methods.

Two-photon excitation fluorescence microspectroscopy protocols for examining fluorophores in fossil plants

Fluorescence emission is common in plants. While fluorescence microscopy has been widely used to study living plants, its application in quantifying the fluorescence of fossil plants has been limited. Fossil plant fluorescence, from original fluorophores or formed during fossilization, can offer valuable insights into fluorescence in ancient plants and fossilization processes. In this work, we utilize two-photon fluorescence microspectroscopy to spatially and spectrally resolve the fluorescence emitted by amber-embedded plants, leaf compressions, and silicified wood. The advanced micro-spectroscope utilized, with its pixel-level spectral resolution and line-scan excitation capabilities, allows us to collect comprehensive excitation and emission spectra with high sensitivity and minimal laser damage to the specimens. By applying linear spectral unmixing to the spectrally resolved fluorescence images, we can differentiate between (a) the matrix and (b) the materials that comprise the fossil. Our analysis suggests that the latter correspond to durable tissues such as lignin and cellulose. Additionally, we observe potential signals from chlorophyll derivatives/tannins, although minerals may have contributed to this. This research opens doors to exploring ancient ecosystems and understanding the ecological roles of fluorescence in plants throughout time. Furthermore, the protocols developed herein can also be applied to analyze non-plant fossils and biological specimens.

Two-photon excitation fluorescence in ophthalmology: safety and improved imaging for functional diagnostics.

Two-photon excitation fluorescence (TPEF) is emerging as a powerful imaging technique with superior penetration power in scattering media, allowing for functional imaging of biological tissues at a subcellular level. TPEF is commonly used in cancer diagnostics, as it enables the direct observation of metabolism within living cells. The technique is now widely used in various medical fields, including ophthalmology. The eye is a complex and delicate organ with multiple layers of different cell types and tissues. Although this structure is ideal for visual perception, it generates aberrations in TPEF eye imaging. However, adaptive optics can now compensate for these aberrations, allowing for improved imaging of the eyes of animal models for human diseases. The eye is naturally built to filter out harmful wavelengths, but these wavelengths can be mimicked and thereby utilized in diagnostics via two-photon (2Ph) excitation. Recent advances in laser-source manufacturing have made it possible to minimize the exposure of in vivo measurements within safety, while achieving sufficient signals to detect for functional images, making TPEF a viable option for human application. This review explores recent advances in wavefront-distortion correction in animal models and the safety of use of TPEF on human subjects, both of which make TPEF a potentially powerful tool for ophthalmological diagnostics.

Automated quantitative assay of fibrosis characteristics in tuberculosis granulomas.

Granulomas, the pathological hallmark of Mycobacterium tuberculosis (Mtb) infection, are formed by different cell populations. Across various stages of tuberculosis conditions, most granulomas are classical caseous granulomas. They are composed of a necrotic center surrounded by multilayers of histocytes, with the outermost layer encircled by fibrosis. Although fibrosis characterizes the architecture of granulomas, little is known about the detailed parameters of fibrosis during this process. In this study, samples were collected from patients with tuberculosis (spanning 16 organ types), and Mtb-infected marmosets and fibrotic collagen were characterized by second harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy using a stain-free, fully automated analysis program. Histopathological examination revealed that most granulomas share common features, including necrosis, solitary and compact structure, and especially the presence of multinuclear giant cells. Masson's trichrome staining showed that different granuloma types have varying degrees of fibrosis. SHG imaging uncovered a higher proportion (4%~13%) of aggregated collagens than of disseminated type collagens (2%~5%) in granulomas from matched tissues. Furthermore, most of the aggregated collagen presented as short and thick clusters (200~620 µm), unlike the long and thick (200~300 µm) disseminated collagens within the matched tissues. Matrix metalloproteinase-9, which is involved in fibrosis and granuloma formation, was strongly expressed in the granulomas in different tissues. Our data illustrated that different tuberculosis granulomas have some degree of fibrosis in which collagen strings are short and thick. Moreover, this study revealed that the SHG imaging program could contribute to uncovering the fibrosis characteristics of tuberculosis granulomas.

Numerical study of a convective cooling strategy for increasing safe power levels in two-photon brain imaging.

Two-photon excitation fluorescence microscopy has become an effective tool for tracking neural activity in the brain at high resolutions thanks to its intrinsic optical sectioning and deep penetration capabilities. However, advanced two-photon microscopy modalities enabling high-speed and/or deep-tissue imaging necessitate high average laser powers, thus increasing the susceptibility of tissue heating due to out-of-focus absorption. Despite cooling the cranial window by maintaining the objective at a fixed temperature, average laser powers exceeding 100-200 mW have been shown to exhibit the potential for altering physiological responses of the brain. This paper proposes an enhanced cooling technique for inducing a laminar flow to the objective immersion layer while implementing duty cycles. Through a numerical study, we analyze the efficacy of heat dissipation of the proposed method and compare it with that of the conventional, fixed-temperature objective cooling technique. The results show that improved cooling could be achieved by choosing appropriate flow rates and physiologically relevant immersion cooling temperatures, potentially increasing safe laser power levels by up to three times (3×). The proposed active cooling method can provide an opportunity for faster scan speeds and enhanced signals in nonlinear deep brain imaging.

Rigidify styryl-pyridinium dyes to benzo[h]coumarin-based bright two-photon fluorescent probes for cellular bioimaging.

Fluorescence imaging of organelles at the cellular level is important for studying biological processes. The development of a highly emissive fluorescent probe that operates under a suitable excitation light source is a key step in high-quality fluorescence imaging. For long-term, high-fidelity fluorescence imaging of mitochondria-related cellular processes using two-photon microscopy and stimulated emission depletion microscopy, we developed a new benzocoumarin-based cationic fluorescent probe (BS-CN) that is far-red emitting, water-soluble, photostable, and very bright in cells. BS-CN showed a remarkably high quantum yield of 0.35 and a large two-photon excited fluorescence action cross-section of 76 GM, enabling the long-term tracking of mitochondria in live cells. In addition, BS-CN exhibited a certain affinity for RNA and stained nucleoli in fixed cells. A comparative assessment of the photophysical properties and bioimaging performance of benzo[h]coumarin-pyridinium and the structurally similar styryl-pyridinium (BS-MN) clearly indicated the importance of structural rigidity for fluorescence efficiency.

Revealing two chemical strategies to tune bright one- and two-photon excited fluorescence of carbon nanodots

The internal structure (oxygenous groups and conjugated aromatic domains) and the hydrogen-bonding network with solvent molecules are two ways to tune the fluorescence of carbon nanodots upon one- and two-photon excitation.

Development of hydrogel-based standards and phantoms for non-linear imaging at depth.

Rapid advances in medical imaging technology, particularly the development of optical systems with non-linear imaging modalities, are boosting deep tissue imaging. The development of reliable standards and phantoms is critical for validation and optimization of these cutting-edge imaging techniques. We aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth. Standards were made using a robust double-network hydrogel matrix consisting of agarose and polyacrylamide. The materials generated ranged from single layers to more complex constructs consisting of up to seven layers, with modality-specific markers embedded between the layers. These standards proved useful in the determination of the axial scaling factor for light microscopy and allowed for depth evaluation for different imaging modalities (conventional one-photon excitation fluorescence imaging, two-photon excitation fluorescence imaging, second harmonic generation imaging, and coherent anti-Stokes Raman scattering) achieving actual depths of 1550, 1550, 1240, and , respectively. Once fabricated, the phantoms were found to be stable for many months. The ability to image at depth, the phantom's robustness and flexible layered structure, and the ready incorporation of "optical markers" make these ideal depth standards for the validation of a variety of imaging modalities.

Restoration of metabolic functional metrics from label-free, two-photon human tissue images using multiscale deep-learning-based denoising algorithms.

Label-free, two-photon excited fluorescence (TPEF) imaging captures morphological and functional metabolic tissue changes and enables enhanced understanding of numerous diseases. However, noise and other artifacts present in these images severely complicate the extraction of biologically useful information. We aim to employ deep neural architectures in the synthesis of a multiscale denoising algorithm optimized for restoring metrics of metabolic activity from low-signal-to-noise ratio (SNR), TPEF images. TPEF images of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavoproteins (FAD) from freshly excised human cervical tissues are used to assess the impact of various denoising models, preprocessing methods, and data on metrics of image quality and the recovery of six metrics of metabolic function from the images relative to ground truth images. Optimized recovery of the redox ratio and mitochondrial organization is achieved using a novel algorithm based on deep denoising in the wavelet transform domain. This algorithm also leads to significant improvements in peak-SNR (PSNR) and structural similarity index measure (SSIM) for all images. Interestingly, other models yield even higher PSNR and SSIM improvements, but they are not optimal for recovery of metabolic function metrics. Denoising algorithms can recover diagnostically useful information from low SNR label-free TPEF images and will be useful for the clinical translation of such imaging.

Crystallography, Charge Transfer, and Two-Photon Absorption Relations in Molecular Cocrystals for Two-Photon Excited Fluorescence Imaging.

Two-photon excited fluorescence imaging requires high-performance two-photon absorption (TPA) active materials, which are commonly intramolecular charge transfer systems prepared by traditional chemical synthesis. However, this typically needs harsh conditions and new methods are becoming crucial. In this work, based on a collaborative intermolecular charge transfer (inter-CT) strategy, three centimeter-sized organic TPA cocrystals are successfully obtained. All three cocrystals exhibit a mixed stacking arrangement, which can effectively generate inter-CT between the donor and acceptor. The ground and excited state characterizations compare their inter-CT ability: 1,2-BTC > 2D-BTC > 1D-BTC. Transient absorption spectroscopy detects TCNB•-, indicating that the TPA mechanism arises from molecular polarization caused by inter-CT. Meanwhile, 1,2-BTC exhibits the highest excited-state absorption and the longest excited-state lifetime, suggesting a stronger TPA response. First-principles calculations also confirm the presence of inter-CT interactions, and the significant parameter Δµ which can assess the TPA capability indicates that inter-CT enhances the TPA response. Besides, cocrystals also demonstrate excellent water solubility and two-photon excited fluorescence imaging capabilities. This research not only provides an effective method for synthesizing TPA crystal materials and elucidates the connection between inter-CT ability and TPA property but also successfully applies them in the fields of multi-photon fluorescence bioimaging.