PNAS Reports Ultra-Precise Detection of Free Radicals in Mouse Liver Sections Using Diamond Quantum Sensing
Product Introduction
Equipment Parameters
Core Technical Solutions and Technological Innovation
(1) Core Technical Solution for the Experiment
- Sample Preparation: Precision-Cut Liver Slices (PCLS) Preparation and Construction of a Fluorescent Nanodiamond (FNDs) Tissue Uptake System
- Imaging and Fixation Techniques: Two-Photon Microscopy, Cryosection Immunostaining, Transmission Electron Microscopy, and a Stainless Steel Anchoring Fixation Device for Tissue Sections
- Quantum sensing and detection: Nanodiamond Relaxation (T1) Measurement System , Pulsed laser excitation and signal acquisition, rolling-window data processing
- Activity and Quantitative Methods: Assessment of tissue viability using the ATP-to-protein ratio; quantification of FND uptake by cells via flow cytometry.
- Condition optimization: a 5% CO₂ environment maintains tissue viability, and a 650 nm long-pass filter enhances the signal-to-noise ratio. Real-time recording of T1 changes, combined with assessments of tissue viability and imaging, validates the reliability of the assay.
(2)
Nanodiamond Relaxometry (T1) Demonstrates significant innovation in liver tissue detection applications:
Relying on the principles of quantum sensing, it exhibits highly specific responses to free radicals containing unpaired electrons, such as ·OH and ·O₂⁻. Can effectively avoid interference from non-radical species such as hydrogen peroxide. ; At the signal dynamics level, this method Enables real-time, reversible tracking of the processes of free radical level elevation and reduction. , thereby overcoming the limitations of irreversible reactions inherent in traditional probes. Furthermore, by employing single-particle tracking and filter optimization techniques, it It enables stable imaging in liver tissue with high autofluorescence background while also allowing differentiation of intercellular individual variations. , providing a highly precise quantitative assay for oxidative stress research at the cellular level.
Key Research Findings
Nanodiamond relaxation method is suitable for in vivo liver tissue detection.
Based on diamond NV-center T1 relaxation measurement technology, through 650 nm Longpass Filter After optimization, spontaneous autofluorescence interference in liver tissue is effectively suppressed, enhancing the signal-to-noise ratio of measurements without altering T1 values and enabling stable detection of free-radical signals in tissue samples.
Establishing a quantitative method for dynamic detection of free radicals at the organizational level
Using finely sectioned liver slices as a model, combined with Stainless Steel Anchoring Fixtures with 5% CO₂ culture environment By maintaining tissue viability, this approach enables real-time recording of continuous changes in T1 values under ethanol induction and antioxidant intervention, thereby facilitating dynamic monitoring of the rise and fall of free radical levels in liver tissue.
The Applicability of Validation Techniques in Research on Organizational Stress and Aging
This method can distinguish free radical levels across different regions and depths of tissue, with no significant positional dependence in the detection results; at the same time, it enables quantitative identification of The hepatic free radical burden in aged mice is significantly higher than that in young mice. , providing a stable nanoscale detection method for research on oxidative stress and aging.
Illustrated Guide
Figure 1. Principle and Procedure of Diamond Relaxation Method for Detecting Free Radicals in Mouse Liver Sections
Diamond relaxation measurements are used to detect free radicals in mouse liver slices. Precisely sectioned mouse liver slices preserve the diversity and characteristic features of the organ’s multicellular components. During a 24-hour incubation, free-radical precursors (FNDs) are incorporated into the liver slices. In diamond relaxation measurements, a series of green laser pulses is applied to excite the FNDs, followed by optical readout. After data processing, the trend of changes in the T1 value reflects the dynamic behavior of free radicals under different experimental conditions. An increase in free-radical levels leads to a decrease in the T1 value, and vice versa.
Figure 2. Two-Photon, Immunofluorescence, and Electron Microscopy Characterization of Nanodiamonds in Precisely Cut Mouse Liver Sections
Precision cutting of nanodiamonds in mouse liver sections.
(A) Two-photon microscopy image of a mouse liver section. The image was acquired in λ-mode, with each color representing a different emission wavelength range from 410 nm (purple) to 690 nm (red). The red signal corresponds to FNDs, while the green signal represents endogenous tissue autofluorescence.
(B) Immunostaining results of 4-μm frozen sections. Gray: bright-field image; green: Kupffer cells labeled with anti-CD68 antibody or endothelial cells labeled with anti-CD31 antibody; red: FNDs (some FNDs are circled); blue: nuclei stained with DAPI.
(C) Electron micrographs of FNDs in the tissue: tomographic slices (left) and a 3D-rendered volume of the FNDs (right).
Figure 3. Quantitative Comparison of Tissue Viability in Mouse Liver Sections under Different Conditions
Tissue viability of mouse liver slices under different conditions. PCLS were co-incubated with or without FNDs for 24 hours at 37°C under 80% and 5% CO₂ conditions. Subsequently, various methods were employed to maintain tissue viability within 0.5 hours after the slices were removed from the incubator.
The first method is to transfer the PCLS to a 5% CO₂ incubator and culture it for 0.5 hours.
The second method is to place the PCLS in WEGG containing 10 mM HEPES buffer and store it at 37°C.
The third approach combines the two methods. All data are standardized using the FND group as the reference. A unpaired t-test is used to compare differences between the two groups. Error bars represent mean ± standard deviation; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 4. Summary Table of the Advantages and Disadvantages of Different Fixation Methods for Liver Sections
Figure 5. The Influence of Filter Wavelength on Image Quality, Signal-to-Noise Ratio, and T1 Value
The impact of filter wavelength on image and signal quality.
(A) Liver tissue sections containing internalized FNDs were imaged in the same region using different long-pass filters (600 nm, 650 nm, or 700 nm). As the overall signal intensity decreased, the internalized FNDs (green circles) became more prominent. The calibration bar indicates the fluorescence intensity in arbitrary units of photon counts per millisecond of integration time.
(B) Comparison of fluorescence intensity, tissue autofluorescence, and recorded T1 values for three distinct FNDs under different filter wavelengths. FND #1 was derived from a batch of small FNDs with an average hydrodynamic diameter of 70 nm, whereas FND #2 and FND #3 had a size of 120 nm, consistent with the FND specifications used in the remaining experiments of this study. Red bars and yellow bars represent FND fluorescence intensity, while black bars indicate the mean intensity of tissue autofluorescence. The green curve depicts the signal-to-noise ratio (SNR), defined here as the ratio of FND fluorescence intensity to tissue autofluorescence intensity. The blue curve shows the T1 values measured for the same FND under different filter settings. Error bars represent the 95% confidence interval of the calculated T1 values.
Table 1. 95% Confidence Intervals for T1 Values under Different Filters
Figure 6. Consistency of T1 Measurements at Different Locations and Depths in a Single Liver Section
T1 measurement results at different locations within a single liver section.
(A) A total of 42 measurements were performed on different FNDs within three randomly selected 50×50 μm regions, and the results showed no statistically significant differences among the measurement locations.
(B) Linear regression analysis revealed no significant correlation between T1 values and liver slice depth (measured relative to the base of the slice). The P-value indicates the level of significance at which the slope deviates from zero. Slice-depth data were recorded from the surface, and consistency was maintained across the three measurement locations.
Figure 7. Dynamic Changes in T1 Values of Liver Slices Under Ethanol and Ascorbic Acid Treatment
(A) Twenty-five millimolar ethanol (red) induces a rapid decrease in the T1 values recorded by FNDs in liver slices, whereas 0.6 millimolar L-ascorbic acid (green) produces the opposite effect. The baseline time point is labeled as “−1” minute. All T1 values are normalized to the corresponding baseline T1 value from the same FND recording. Data points represent the median of five independent measurements across different liver slices, and error bars indicate the interquartile range.
(B) Paired comparisons were conducted between baseline T1 values (“pre-treatment”) and final T1 values (“post-treatment”) in each experimental group. Ethanol treatment resulted in a decrease in T1 values after 33 minutes of incubation, whereas L-ascorbic acid treatment generally produced higher T1 values, though this difference did not reach statistical significance. The culture medium (negative control) had no significant effect on the recorded T1 values.
Figure 8. Comparison of Free Radical Load in Liver Sections from Young and Aged Mice
The free-radical burden data for young and old mice were compared by measuring the T1 values in PCLS from 12-week-old and 80-week-old mice.
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