Dataset Information

Phase contrast reflectance confocal brain imaging at 1650 nm.

ABSTRACT:

Significance

The imaging depth of microscopy techniques is limited by the ability of light to penetrate biological tissue. Recent research has addressed this limitation by combining a reflectance confocal microscope with the NIR-II (or shortwave infrared) spectrum. This approach offers significant imaging depth, is straightforward in design, and remains cost-effective. However, the imaging system, which relies on intrinsic signals, could benefit from adjustments in its optical design and post-processing methods to differentiate cortical cells, such as neurons and small blood vessels.

Aim

We implemented a phase contrast detection scheme to a reflectance confocal microscope using NIR-II spectral range as illumination.

Approach

We analyzed the features retrieved in the images while testing the imaging depth. Moreover, we introduce an acquisition method for distinguishing dynamic signals from the background, allowing the creation of vascular maps similar to those produced by optical coherence tomography.

Results

The phase contrast implementation is successful to retrieve deep images in the cortex up to 800 μm using a cranial window. Vascular maps were retrieved at similar cortical depth and the possibility of combining multiple images can provide a vessel network.

Conclusions

Phase contrast reflectance confocal microscopy can improve the outlining of cortical cell bodies. With the presented framework, angiograms can be retrieved from the dynamic signal in the biological tissue. Our work presents an optical implementation and analysis techniques from a former microscope design.

SUBMITTER: Martel PD

PROVIDER: S-EPMC10898133 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Publications

Phase contrast reflectance confocal brain imaging at 1650 nm.

Martel Patrick Delafontaine PD Zhang Cong C Linninger Andreas A AA Lesage Frédéric F

Journal of biomedical optics 20240227 2

<h4>Significance</h4>The imaging depth of microscopy techniques is limited by the ability of light to penetrate biological tissue. Recent research has addressed this limitation by combining a reflectance confocal microscope with the NIR-II (or shortwave infrared) spectrum. This approach offers significant imaging depth, is straightforward in design, and remains cost-effective. However, the imaging system, which relies on intrinsic signals, could benefit from adjustments in its optical design and ...[more]

PMID: 38414657

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Project description:The potential for human exposure to manufactured nanoparticles (NPs) has increased in recent years, in part through the incorporation of engineered particles into a wide range of commercial goods and medical applications. NP are ideal candidates for use as therapeutic and diagnostic tools within biomedicine, however concern exists regarding their efficacy and safety. Thus, developing techniques for the investigation of NP uptake into cells is critically important. Current intracellular NP investigations rely on the use of either Transmission Electron Microscopy (TEM), which provides ultrahigh resolution, but involves cumbersome sample preparation rendering the technique incompatible with live cell imaging, or fluorescent labelling, which suffers from photobleaching, poor bioconjugation and, often, alteration of NP surface properties. Reflected light imaging provides an alternative non-destructive label free technique well suited, but not limited to, the visualisation of NP uptake within model systems, such as cells. Confocal reflectance microscopy provides optical sectioning and live imaging capabilities, with little sample preparation. However confocal microscopy is diffraction limited, thus the X-Y resolution is restricted to ~250 nm, substantially larger than the <100 nm size of NPs. Techniques such as super-resolution light microscopy overcome this fundamental limitation, providing increased X-Y resolution. The use of Reflectance SIM (R-SIM) for NP imaging has previously only been demonstrated on custom built microscopes, restricting the widespread use and limiting NP investigations. This paper demonstrates the use of a commercial SIM microscope for the acquisition of super-resolution reflectance data with X-Y resolution of 115 nm, a greater than two-fold increase compared to that attainable with RCM. This increase in resolution is advantageous for visualising small closely spaced structures, such as NP clusters, previously unresolvable by RCM. This is advantageous when investigating the subcellular trafficking of NP within fluorescently labelled cellular compartments. NP signal can be observed using RCM, R-SIM and TEM and a direct comparison is presented. Each of these techniques has its own benefits and limitations; RCM and R-SIM provide novel complementary information while the combination of modalities provides a unique opportunity to gain additional information regarding NP uptake. The use of multiple imaging methods therefore greatly enhances the range of NPs that can be studied under label-free conditions.

Dataset Information

Phase contrast reflectance confocal brain imaging at 1650 nm.

Significance

Aim

Approach

Results

Conclusions

Publications

Phase contrast reflectance confocal brain imaging at 1650 nm.

Similar Datasets

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