Tissue clearing techniques enable 3D imaging of whole tissues and organs (Ueda et al., 2020). Unlike traditional histology, these techniques reduce light scattering and absorption, enhancing depth imaging capabilities and achieving single-cell resolution in deep samples (Susaki et al., 2014). These approaches are now widely used in neurobiology, developmental biology, immunology, and oncology. To achieve deep imaging of fully cleared tissues, various optical sectioning techniques for three-dimensional imaging have rapidly developed, including light sheet microscopy, two-photon microscopy, and confocal microscopy (Ueda et al., 2020).

For effective 3D imaging of cleared tissues, it is crucial to utilize microscopes equipped with objectives that offer both low to medium magnification and long working distances (Jonkman et al., 2020). Objectives with low numerical aperture (NA) are often used to achieve these long working distances, but they face significant challenges in maintaining high axial resolution for deep tissue imaging (Birk et al., 2017). The main issue arises from light scattering and reflection at the interfaces within the sample and between the sample and the lens, leading to image distortion and blurring. This phenomenon, known as spherical aberration, occurs when light rays entering the periphery of the lens focus at a different point on the optical axis compared to those entering near the center, leading to a significant degradation in fluorescence signal intensity and image resolution (Diel et al., 2020). Using dry objectives with high-refractive-index mounting media or cleared tissues can exacerbate this issue, as the sample itself contributes to the aberration (Gibson and Lanni, 1992). Therefore, matching the refractive index (RI) of the objective’s immersion medium to that of the sample’s immersion medium is essential to mitigate axial distortion caused by spherical aberration.

Many immersion objectives feature correction collars that can be adjusted to compensate for RI mismatches. Achieving an exact match between the refractive indices of the immersion media and the sample medium is often challenging. Although dipping caps with glass windows can transform air objectives into immersion lenses by enabling the front lens to be directly inserted into the sample media, this approach helps to avoid axial deviation during imaging. However, immersion objectives on inverted microscopes encounter limitations (Dodt et al., 2015). These objectives cannot be inserted into sample chambers and must move up and down outside the chamber, which restricts the imaging depth.

To overcome these limitations, several alternative methods have been developed. One such approach involves moving the sample within a chamber filled with mounting media, while the imaging objective remains fixed outside the chamber (Engelbrecht and Stelzer, 2006). Another technique utilizes adaptive optics, such as deformable mirrors or spatial light modulators, to correct aberrations across the field of view (Ji, 2017). Additionally, radially symmetric phase masks are used to balance spherical aberration at various depths within the sample, thereby simplifying the image restoration process through post-capture deconvolution (Patwary et al., 2016).

However, inverted confocal microscopes encounter issues due to the gravitational dispersion of the RI buffer between the objective and the confocal dish, resulting in RI mismatches with air and complicating high-resolution imaging. Additionally, the use of long-working-distance air objectives causes severe spherical aberrations due to RI mismatches, further limiting imaging depth and resolution.

Here, we developed the Refractive Index Matching-Deep (RIM-Deep) system to address these challenges, which includes an immersion chamber designed around the optical components of the objective lens and a specimen holder integrated with a motorized x–y–z stage. This design effectively stabilizes the refractive index between the objective and the sample media. When paired with an immersion objective, the RIM-Deep system enables high-resolution, deep imaging, significantly enhancing the capabilities of inverted confocal microscopy. This innovation provides a practical solution for achieving high-quality 3D imaging of cleared tissues, paving the way for more detailed and accurate biological studies.

Official adapters for 10 X immersion objectives in Nikon inverted confocal microscopy significantly enhance cleared tissue imaging, but RI buffer leakage still occurs, causing RI mismatch and limiting imaging depth (Figure 1A, Figure 1—figure supplement 1A). In response to this challenge, the RIM-Deep was designed based on a light sheet microscopy imaging chamber. It features a media reservoir affixed to the objective and filled with RI buffer, enabling 3D imaging of the sample within an imaging buffer. To further optimize imaging performance, a support bracket integrated into the motorized stages elevates the imaging platform and centrally positions a specimen holder (Figure 1B, Figure 1—figure supplement 1B and Figure 1—figure supplement 2).

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Figure 1—source data 1

Source data for FWHM axial and lateral resolution comparison between RIM-Deep and Standard adapter across different focal depths.

https://cdn.elifesciences.org/articles/101143/elife-101143-fig1-data1-v1.csv

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To demonstrate the effectiveness of the RIM-Deep, imaging resolution was characterized by comparing 3 μm diameter fluorescence beads using standard adapter versus the RIM-Deep in 10 X immersion objective (Figure 1A and B). Within the 2 mm imaging limit, 10 X immersion objectives with the standard adapter maintain both optical axial (XZ) and lateral (XY) resolution (Figure 1C and E). As expected, the RIM-Deep also maintained optimal axial resolution across 5 mm (Figure 1D and E). However, there was no significant difference in lateral resolution between the two types of chambers (Figure 1C–E).

Subsequently, the high-depth imaging performance of the RIM-Deep was evaluated and compared to the standard adapter. Imaging was performed on a Macaca fascicularis brain section cleared using the iDISCO, CUBIC, and MACS methods (Figure 1F). Gravity affected the refractive index matching fluid at the tip of the objective, limiting the imaging depth to less than 2 mm (Figure 1G–I2, Figure 1—videos 1–3). As observed, with the RIM-Deep, the specimen holder was fully immersed in the RI liquid, the depth of images is obviously enhanced to about 5 mm, and the vessel structures could thereby be observed clearly (Figure 1J–L4, Figure 1—video 4, Figure 1—video 5 and Figure 1—video 6).

The performance of the RIM-Deep method was also evaluated using a Leica inverted confocal microscope. Notably, high-resolution imaging of multiple cross-sections was successfully achieved for a Macaca fascicularis brain vasculature at depths of up to 5 mm, demonstrating consistent results across different imaging platforms (Figure 1—figure supplement 3, Figure 1—video 7).

To further assess the resolution and depth robustness of RIM-Deep in neuronal imaging, experiments were conducted on Thy1-EGFP mouse brains cleared with the CUBIC method (Figure 2A). The use of the RIM-Deep also significantly extended the imaging depth to approximately 5 mm (Figure 2B, Figure 2—video 1). This extension enabled the clear visualization and reconstruction of neuronal soma and axon structures in the hippocampus and thalamus (Figure 2C, E, F, G, I), maintaining resolution at the micron level (Figure 2D and H).

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A 3×3 stitching mode with multi-layer Z-axis scanning was used to examine a cleared brain segment (Figure 3A and B, Figure 3—video 1). This method enabled visualization of neuronal cell bodies and axons up to nearly 5 mm in X-Y optical slices (Figure 3C). For large tissue imaging, the RIM-Deep was used on a cleared half-brain, achieving comprehensive imaging (Figure 3D). Its stable and precise positioning ensured accurate alignment across all optical slices, facilitating detailed and thorough imaging (Figure 3E–I).

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Stroke, caused by disrupted brain blood supply, induces cell death and significant vascular changes (Sekerdag et al., 2018). Generally, confocal microscopy is limited to thin sections and cannot fully study vascular networks. However, with the assistance of the RIM-Deep, complete observation of vascular damage is possible. Therefore, mouse brains with induced cortical ischemia were examined to thoroughly investigate this process. Using VALID labeling and MACS tissue clearing techniques, the cerebral vasculature was reconstructed, revealing a significantly reduced vessel density in the ischemic cortex (Figure 3J–L, Figure 3—video 2, Figure 3—video 3, Figure 3—video 4). These findings demonstrate the effectiveness of the RIM-Deep in visualizing disease-related changes in vascular structures, making it a valuable tool in neuroscience research.

Inverted confocal microscopy often faces challenges such as limited axial resolution and spherical aberration when imaging cleared tissues. The RIM-Deep, featuring a media reservoir filled with RI buffer, was designed to address these issues. Experimental validations showed that the RIM deep maintained optimal axial resolution when imaging fluorescent beads. Using the three different tissue cleaning method on Macaca fascicularis brain tissues, imaging depths reached up to 5 mm with clear visualization of vascular structures. Similarly, in Thy1-eGFP mouse brains, the RIM deep extended imaging depth to approximately 5 mm, allowing clear visualization of neuronal and axonal structures. The RIM deep also effectively visualized vascular damage in mouse brains with cortical ischemia, proving valuable in neuroscience research. The RIM-Deep method also demonstrates versatility across different imaging platforms, ensuring consistent performance in various experimental setups.

The development of tissue clearing has revolutionized neuroscientific research. However, two-photon and light sheet microscopes are prohibitively expensive and their operation and maintenance costs are high, limiting their application scenarios. Many hospitals and laboratories, although capable of performing tissue clearing independently, are unable to conduct imaging. The inverted point-scanning confocal microscope, preferred for live cell and tissue slice imaging, offers advantages in deep imaging of cleared tissues over upright microscopes, including reduced sample contact and buffer use, lowering the risk of damage and cost. However, the use of long-working-distance air objectives introduces severe spherical aberrations due to RI mismatch, limiting imaging depth and resolution. Our RIM-Deep addresses these issues, extending imaging depth without compromising the microscope’s normal functionality.

Wide-field microscopes, although efficient for rapid imaging, often face limitations in depth and resolution when applied to cleared tissues (Shaw, 2006). The RIM-Deep can also be employed in these cases to enhance imaging performance. When using wide-field microscopy with the RIM-Deep, it is necessary to apply deconvolution algorithms or other techniques to eliminate out-of-focus signals (Shaw, 2006). However, it is important to note that this system may reduce fluorescence intensity in deeper regions of the sample.

Figure 1—source data 1 contains the numerical data used to generate the figures. All data generated or analysed during this study are included in the manuscript and videos; source data files have been provided for Figure 1 E.

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Author details

1. Yisi Liu

   Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China

   Contribution

   Conceptualization, Formal analysis, Visualization, Methodology, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2121-8557

2. Pu Wang

   Nikon Precision Corporation, Guangzhou , China, Shanghai, China

   Contribution

   Conceptualization, Visualization, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

3. Junjie Zou

   Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, The National Key Clinical Specialty, Guangzhou, China

   Contribution

   Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Hongwei Zhou

   1. Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
   2. State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – review and editing

   For correspondence

   biodegradation@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2472-8541

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