Intraoperative microscopic autofluorescence detection and characterization in brain tumors using stimulated Raman histology and two-photon fluorescence.

Introduction: The intrinsic autofluorescence of biological tissues interferes with the detection of fluorophores administered for fluorescence guidance, an emerging auxiliary technique in oncological surgery. Yet, autofluorescence of the human brain and its neoplasia is sparsely examined. This study aims to assess autofluorescence of the brain and its neoplasia on a microscopic level by stimulated Raman histology (SRH) combined with two-photon fluorescence. Methods: With this experimentally established label-free microscopy technique unprocessed tissue can be imaged and analyzed within minutes and the process is easily incorporated in the surgical workflow. In a prospective observational study, we analyzed 397 SRH and corresponding autofluorescence images of 162 samples from 81 consecutive patients that underwent brain tumor surgery. Small tissue samples were squashed on a slide for imaging. SRH and fluorescence images were acquired with a dual wavelength laser (790 nm and 1020 nm) for excitation. In these images tumor and non-tumor regions were identified by a convolutional neural network that reliably differentiates between tumor, healthy brain tissue and low quality SRH images. The identified areas were used to define regions.of- interests (ROIs) and the mean fluorescence intensity was measured. Results: In healthy brain tissue, we found an increased mean autofluorescence signal in the gray (11.86, SD 2.61, n=29) compared to the white matter (5.99, SD 5.14, n=11, p<0.01) and in the cerebrum (11.83, SD 3.29, n=33) versus the cerebellum (2.82, SD 0.93, n=7, p<0.001), respectively. The signal of carcinoma metastases, meningiomas, gliomas and pituitary adenomas was significantly lower (each p<0.05) compared to the autofluorescence in the cerebrum and dura, and significantly higher (each p<0.05) compared to the cerebellum. Melanoma metastases were found to have a higher fluorescent signal (p<0.01) compared to cerebrum and cerebellum. Discussion: In conclusion we found that autofluorescence in the brain varies depending on the tissue type and localization and differs significantly among various brain tumors. This needs to be considered for interpreting photon signal during fluorescence-guided brain tumor surgery.

Proton beam range verification by means of ionoacoustic measurements at clinically relevant doses using a correlation-based evaluation.

Purpose: The Bragg peak located at the end of the ion beam range is one of the main advantages of ion beam therapy compared to X-Ray radiotherapy. However, verifying the exact position of the Bragg peak within the patient online is a major challenge. The goal of this work was to achieve submillimeter proton beam range verification for pulsed proton beams of an energy of up to 220 MeV using ionoacoustics for a clinically relevant dose deposition of typically 2 Gy per fraction by i) using optimal proton beam characteristics for ionoacoustic signal generation and ii) improved signal detection by correlating the signal with simulated filter templates. Methods: A water tank was irradiated with a preclinical 20 MeV proton beam using different pulse durations ranging from 50 ns up to 1 µs in order to maximise the signal-to-noise ratio (SNR) of ionoacoustic signals. The ionoacoustic signals were measured using a piezo-electric ultrasound transducer in the MHz frequency range. The signals were filtered using a cross correlation-based signal processing algorithm utilizing simulated templates, which enhances the SNR of the recorded signals. The range of the protons is evaluated by extracting the time of flight (ToF) of the ionoacoustic signals and compared to simulations from a Monte Carlo dose engine (FLUKA). Results: Optimised SNR of 28.0 ± 10.6 is obtained at a beam current of 4.5 µA and a pulse duration of 130 ns at a total peak dose deposition of 0.5 Gy. Evaluated ranges coincide with Monte Carlo simulations better than 0.1 mm at an absolute range of 4.21 mm. Higher beam energies require longer proton pulse durations for optimised signal generation. Using the correlation-based post-processing filter a SNR of 17.8 ± 5.5 is obtained for 220 MeV protons at a total peak dose deposition of 1.3 Gy. For this clinically relevant dose deposition and proton beam energy, submillimeter range verification was achieved at an absolute range of 303 mm in water. Conclusion: Optimal proton pulse durations ensure an ideal trade-off between maximising the ionoacoustic amplitude and minimising dose deposition. In combination with a correlation-based post-processing evaluation algorithm, a reasonable SNR can be achieved at low dose levels putting clinical applications for online proton or ion beam range verification into reach.

Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging.

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

Multifunctional Magneto-Plasmonic Fe3O4/Au Nanocomposites: Approaching Magnetophoretically-Enhanced Photothermal Therapy.

Magneto-plasmonic nanocomposites can possess properties inherent to both individual components (iron oxide and gold nanoparticles) and are reported to demonstrate high potential in targeted drug delivery and therapy. Herein, we report on Fe3O4/Au magneto-plasmonic nanocomposites (MPNC) synthesized with the use of amino acid tryptophan via chemical and photochemical reduction of Au ions in the presence of nanosized magnetite. The magnetic field (MF) induced aggregation was accompanied by an increase in the absorption in the near-infrared (NIR) spectral region, which was demonstrated to provide an enhanced photothermal (PT) effect under NIR laser irradiation (at 808 nm). A possibility for therapeutic application of the MPNC was illustrated using cancer cells in vitro. Cultured HeLa cells were treated by MPNC in the presence of MF and without it, following laser irradiation and imaging using confocal laser scanning microscopy. After scanning laser irradiation of the MPNC/MF treated cells, a formation and rise of photothermally-induced microbubbles on the cell surfaces was observed, leading to a damage of the cell membrane and cell destruction. We conclude that the synthesized magneto-plasmonic Fe3O4/Au nanosystems exhibit magnetic field-induced reversible aggregation accompanied by an increase in NIR absorption, allowing for an opportunity to magnetophoretically control and locally enhance a NIR light-induced thermal effect, which holds high promise for the application in photothermal therapy.

Homogentisic acid-derived pigment as a biocompatible label for optoacoustic imaging of macrophages.

Macrophages are one of the most functionally-diverse cell types with roles in innate immunity, homeostasis and disease making them attractive targets for diagnostics and therapy. Photo- or optoacoustics could provide non-invasive, deep tissue imaging with high resolution and allow to visualize the spatiotemporal distribution of macrophages in vivo. However, present macrophage labels focus on synthetic nanomaterials, frequently limiting their ability to combine both host cell viability and functionality with strong signal generation. Here, we present a homogentisic acid-derived pigment (HDP) for biocompatible intracellular labeling of macrophages with strong optoacoustic contrast efficient enough to resolve single cells against a strong blood background. We study pigment formation during macrophage differentiation and activation, and utilize this labeling method to track migration of pro-inflammatory macrophages in vivo with whole-body imaging. We expand the sparse palette of macrophage labels for in vivo optoacoustic imaging and facilitate research on macrophage functionality and behavior.