Two-photon excitation microscopy enables in vivo deep-tissue imaging within organisms. This technique is based on two-photon excitation, a nonlinear optical process that uses near-infrared light for excitation, resulting in high tissue permeability. Notably, two-photon excitation occurs only near the focal plane; therefore, minimally invasive tomographic images can be obtained. Owing to these features, two-photon excitation microscopy is currently widely used in medical and life-science research, particularly in the domain of neuroscience for in vivo visualization of deep tissues. However, the use of long-wavelength excitation light in two-photon excitation microscopy has resulted in a larger focused spot size and relatively low spatial resolution, which is a limitation of this technique for further applications. Recent studies have described super-resolution microscopy techniques applied to two-photon excitation microscopy in an attempt to observe living organisms \"as they are in their natural state\" with high spatial resolution. We have also addressed this topic using an optical approach (two-photon stimulated emission depletion microscopy) and an image analysis approach (two-photon super-resolution radial fluctuation). Here, we describe these approaches together with a discussion of our recent accomplishments.

Two-photon vision enables near-infrared light perception in humans. We investigate the possibility to utilize this phenomenon as an indicator of the location of the outer segments of photoreceptor cells in the OCT images. Since two-photon vision is independent on OCT imaging, it could provide external to OCT reference relative to which positions of retinal layers visible in OCT imaging could be measured. We show coincidence between OCT imaging of outer retinal layers and two-photon light perception. The experiment utilizes an intrinsic nonlinear process in the retina, two-photon absorption of light by visual photopigments, which triggers perception of near-infrared light. By shifting the focus of the imaging/stimulus beam, we link the peak efficiency of two-photon vision with the visibility of outer segments of photoreceptor cells, which can be seen as in vivo identification of a retinal layer containing visual photopigments in OCT images. Determination of the in-focus retinal layer is achieved by analysis of en face OCT image contrast. We discuss experimental methods and experimental factors that may influence two-photon light perception and the accuracy of the results. The limits of resolution are discussed in analysis of the one-photon and two-photon point spread functions.

BACKGROUND: Radiotherapy is essential in the treatment of prostate cancer. An alternative to conventional photon radiotherapy is the application of carbon ions, which provide a superior intratumoral dose distribution and less induced damage to adjacent healthy tissue. A common characteristic of prostate cancer cells is their dependence on androgens which is exploited therapeutically by androgen deprivation therapy in the advanced prostate cancer stage. Here, we aimed to analyze the transcriptomic response of prostate cancer cells to irradiation by photons in comparison to carbon ions, focusing on DNA damage, DNA repair and androgen receptor signaling.
METHODS: Prostate cancer cell lines LNCaP (functional TP53 and androgen receptor signaling) and DU145 (dysfunctional TP53 and androgen receptor signaling) were irradiated by photons or carbon ions and the subsequent DNA damage was assessed by immuno-cytofluorescence. Furthermore, the cells were treated with an androgen-receptor agonist. The effects of irradiation and androgen treatment on the gene regulation and the transcriptome were investigated by RT-qPCR and RNA sequencing, followed by bioinformatic analysis.
RESULTS: Following photon or carbon ion irradiation, both LNCaP and DU145 cells showed a dose-dependent amount of visible DNA damage that decreased over time, indicating occurring DNA repair. In terms of gene regulation, mRNAs involved in the TP53-dependent DNA damage response were significantly upregulated by photons and carbon ions in LNCaP but not in DU145 cells, which generally showed low levels of gene regulation after irradiation. Both LNCaP and DU145 cells responded to photons and carbon ions by downregulation of genes involved in DNA repair and cell cycle, partially resembling the transcriptome response to the applied androgen receptor agonist. Neither photons nor carbon ions significantly affected canonical androgen receptor-dependent gene regulation. Furthermore, certain genes that were specifically regulated by either photon or carbon ion irradiation were identified.
CONCLUSIONS: Photon and carbon ion irradiation showed a significant congruence in terms of induced signaling pathways and transcriptomic responses. These responses were strongly impacted by the TP53 status. Nevertheless, irradiation mode-dependent distinct gene regulations with undefined implication for radiotherapy outcome were revealed. Androgen receptor signaling and irradiations shared regulation of certain genes with respect to DNA-repair and cell-cycle.

Living cells have spontaneous ultraweak photon emission derived from metabolic reactions associated with physiological conditions. The ORCA-Quest CMOS camera (Hamamatsu Photonics, Japan) is a highly sensitive and essential tool for photon detection; its use with a microscope incubator (Olympus) enables the detection of photons emitted by embryos with the exclusion of harmful visible light. With the application of the second law of thermodynamics, the low-entropy energy absorbed and used by embryos can be distinguished from the higher-entropy energy released and detectable in their environment. To evaluate higher-entropy energy data from embryos, we developed a unique algorithm for the calculation of the entropy-weighted spectral fractal dimension, which demonstrates the self-similar structure of the energy (photons) released by embryos. Analyses based on this structure enabled the distinction of living and degenerated mouse embryos, and of frozen and fresh embryos and the background. This novel detection of ultra-weak photon emission from mouse embryos can provide the basis for the development of a photon emission embryo control system. The ultraweak photon emission fingerprints of embryos may be used for the selection of viable specimens in an ideal dark environment.

This is an explanatory paper on Sun Il Kwon et al., Nat. Photon. 15: 914-918, 2021 and some parts of this manuscript are translated from the paper. Medical imaging modalities such as X-ray computed tomography, Magnetic resonance imaging, positron emission tomography (PET), and single photon emission computed tomography, require image reconstruction processes, consequently constraining them to form cylindrical shapes. However, among them, only PET can use additional information, so called time of flight, on an event-by-event basis. If coincidence time resolution (CTR) of PET detectors improved to 30 ps, which corresponds to spatial resolution of 4.5 mm, directly localizing electron-positron annihilation point is possible, allowing us to circumvent image reconstruction processes and free us from the geometric constraint. We call this concept direct positron emission imaging (dPEI). We have developed ultrafast radiation detectors by focusing on Cherenkov photon detection. Furthermore, the CTR of 32 ps being equivalent to 4.8 mm spatial resolution is achieved by combining deep learning-based signal processing with the detectors. In this article, we explain how we developed the detectors and demonstrated the first dPEI using different types of phantoms, how we will tackle limitations to be addressed to make the dPEI more practical, and how dPEI will emerge as an imaging modality in nuclear medicine.

Since the emergence of the first photon-counting computed tomography (PCCT) system in late 2021, its advantages and a wide range of applications in all fields of radiology have been demonstrated. Compared to standard energy-integrating detector-CT, PCCT allows for superior geometric dose efficiency in every examination. While this aspect by itself is groundbreaking, the advantages do not stop there. PCCT facilitates an unprecedented combination of ultra-high-resolution imaging without dose penalty or field-of-view restrictions, detector-based elimination of electronic noise, and ubiquitous multi-energy spectral information. Considering the high demands of orthopedic imaging for the visualization of minuscule details while simultaneously covering large portions of skeletal and soft tissue anatomy, no subspecialty may benefit more from this novel detector technology than musculoskeletal radiology. Deeply rooted in experimental and clinical research, this review article aims to provide an introduction to the cosmos of PCCT, explain its technical basics, and highlight the most promising applications for patient care, while also mentioning current limitations that need to be overcome.

The abnormal deposition of protein in the brain is the central factor in neurodegenerative disorders (NDs). These detrimental aggregates, stemming from the misfolding and subsequent irregular aggregation of α-synuclein protein, are primarily accountable for conditions such as Parkinson\'s disease, Alzheimer\'s disease, and dementia. Two-photon-excited (TPE) probes are a promising tool for the early-stage diagnosis of these pathologies as they provide accurate spatial resolution, minimal intrusion, and the ability for prolonged observation. To identify compounds with the potential to function as diagnostic probes using two-photon techniques, we explore three distinct categories of compounds: Hydroxyl azobenzene (AZO-OH); Dicyano-vinyl bithiophene (DCVBT); and Tetra-amino phthalocyanine (PcZnNH2). The molecules were structurally and optically characterized using a multi-technique approach via UV-vis absorption, Raman spectroscopy, three-dimensional fluorescence mapping (PLE), time-resolved photoluminescence (TRPL), and pump and probe measurements. Furthermore, quantum chemical and molecular docking calculations were performed to provide insights into the photophysical properties of the compounds as well as to assess their affinity with the α-synuclein protein. This innovative approach seeks to enhance the accuracy of in vivo probing, contributing to early Parkinson\'s disease (PD) detection and ultimately allowing for targeted intervention strategies.

Current quantitative gene expression detection in genomic and transcriptomic research heavily relies on quantitative real-time PCR (qPCR). While existing multiplex gene detection techniques offer simultaneous analysis of multiple targets, we present an alternative assay capable of detecting gene expression simultaneously within a single well. This highly sensitive method utilizes πCode MicroDiscs, featuring unique identification patterns and fluorescent detection. Our study compared this multiplex πCode platform with a qPCR platform for profiling cytokine gene expression. The πCode MicroDisc assay successfully demonstrated the expression of polymerization markers for M1- and M2-like macrophages generated from THP-1-derived macrophages in a qualitative assay. Additionally, our findings suggest a pattern agreement between the πCode assay and the qPCR assay, indicating the potential of the πCode technology for comparative gene expression analysis. Regarding the inherent sensitivity and linearity, the developed πCode assay primarily provides qualitative gene expression to discriminate the polarization of macrophages. This remarkable capability presents substantial advantages for researchers, rendering the technology highly suitable for high-throughput applications in clinical diagnosis and disease monitoring.

OBJECTIVE: The goal of this study is to determine the accuracy of the PTW Beamscan program in determining the inflection point from Flattening Filter Free Beam Profile utilizing Multiple Detectors.
METHODS: True Beam Linear Accelerator with 6FFF and 10FFF Photon Energies and 10 cm, 15 cm and 20 cm Field Sizes were used for this study. Profile measurements were taken with PTW\'s 729, 1,500, and 1,600 and the Starcheck system, the Pinpoint 3D with Beamscan system, and Linac\'s EPID. The first-order derivative was utilized in both the Excel spreadsheet and Beamscan software to analyse raw measured data to locate inflection point and the FWHM was calculated. The accuracy of inflection points and FWHM between the Excel sheet calculation and the software program were investigated.
RESULTS: For 10X10 cm2 in the 729 Array, the greatest differences in FWHM were 5.16 mm and 5.04 mm for the X6 FFF and X10 FFF Energies, respectively. The largest difference was 2.26 mm for 1,600 SRS arrays with a 15×15 cm2 field size. The difference in FWHM between Manual and software analysis for 10X10 cm2 and 20X20 cm2 Field Sizes is in decreasing order for detectors from 729, 1,500, 1,600 SRS, Starcheck, Pinpoint 3D, and EPID. In contrast, there is no climbing or declining pattern detected in the difference in Field Width for the 15×15 cm2 Field Size. Similarly, for all detectors except the 1,600 SRS array, the peak of the first-order derivative occurs at the chamber position for a 15X15 cm2 field size.
CONCLUSIONS: The higher resolution of measurement yields more accuracy in inflection point and the FWHM. Irrespective of measurement resolution, the Beamscan software provided the FWHM closer to the respective nominal Field Size. Out of all detectors, results obtained with Excel Starcheck and EPID are good in agreement with values obtained by the software analysis. Thus, it is shown that Beamscan software is so accurate in determining inflection point of a FFF beam profile and used for routine profile analysis.

Background Photon-counting CT (PCCT) represents a recent advancement in CT, offering improved spatial resolution and spectral separability. By using multiple adjustable energy bins, PCCT enables K-edge imaging, allowing mixed contrast agent distinction. Deep-silicon is a new type of photon-counting detector with different characteristics compared with cadmium photon-counting detectors. Purpose To evaluate the performance of a prototype deep-Si PCCT scanner and compare it with that of a state-of-the-art dual-energy energy-integrating detector (EID) scanner in imaging coronary artery plaques enhanced with iodine and K-edge contrast agents. Materials and Methods A series of 10 three-dimensional-printed inserts (diameter, 3.5 mm) was prepared, and materials mimicking soft and calcified plaques were added to simulate stenosed coronary arteries. Inserts filled with an iodine- or gadolinium-based contrast agent (GBCA) were scanned. Virtual monoenergetic images (VMIs) and iodine maps were generated using two- and eight-energy bin data from EID CT and PCCT, respectively. Gadolinium maps were calculated for PCCT. The CT numbers of VMIs and iodine maps were compared. Spatial resolution and blooming artifacts were compared on the 70-keV VMIs in plaque-free and calcified coronary arteries. Results No evidence of a significant difference in the CT number of 70-keV images was found except in inserts containing GBCAs. In the absence of a GBCA, excellent (r > 0.99) agreement for iodine was found. PCCT could quantify the GBCA within 0.2 mg Gd/mL ± 0.8 accuracy of the ground truth, whereas EID CT failed to detect the GBCA. Lumen measurements were more accurate for PCCT than for EID CT, with mean errors of 167 versus 442 µm (P < .001) compared with the 3.5-mm ground truth. Conclusion Deep-Si PCCT demonstrated good accuracy in iodine quantification and could accurately decompose mixtures of two contrast agents. Its improved spatial resolution resulted in sharper images with blooming artifacts reduced by 50% compared with a state-of-the-art dual-energy EID CT scanner. © RSNA, 2024.