Significant progress in non-invasive imaging technologies have been made in recent decades, including the development of magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET) (Azaripour et al., 2016). These imaging techniques enable a resolution at the cellular level; however, specific research requires a subcellular resolution. In this context, the interface between established chemical nonlinear optical (NLO) techniques and biological microscopy has led to further advances in imaging capabilities, e.g., the development of multiphoton microscopy (MPM), second harmonic generation (SHG), and third harmonic generation (THG) imaging modalities (Campagnola, 2011).

Among these imaging modalities, MPM is a fast and non-invasive method for probing biological tissues at the subcellular level while maintaining a high resolution (Zipfel et al., 2003; Liang et al., 2017). MPM is known for its ability to generate label-free images using two-photon excited fluorescence (TPF) signals from intrinsic fluorophores, such as flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide (NADH) (Croce and Bottiroli, 2014). This allows researchers to visualize large areas of unstained tissue while simultaneously providing real-time depth analysis. However, the accuracy of MPM imaging can sometimes be affected by sampling limitations (Chen et al., 2021).

On the other hand, SHG works by combining two photons of a lower energy to generate one with twice the frequency of the original light. In his 1986 pioneering work, Freund first used SHG for biological imaging, specifically to study collagen fibers in rat tail tendons (Freund et al., 1986). Due to the reliance on endogenous contrast, SHG microscopy offers several advantages for tissue imaging (e.g., direct visualization of tissue architecture without external dyes (Campagnola and Loew, 2003)). Technically, the SHG signal is generated by nonlinear interactions within the material via induced polarization, which significantly reduces photobleaching and phototoxicity compared to traditional fluorescence techniques. For SHG to occur, the environment must exhibit non-centrosymmetry (Zhuo et al., 2010) to an extent corresponding to the wavelength of the emitted light. This highlights the crucial role of molecular alignment within the medium. By using lasers in the near-infrared range (700–1000 nm), SHG microscopy can provide high-resolution images at depths of several hundred microns, enhancing its utility in biological research (Campagnola, 2011). Within the broad applications of SHG for imaging biological tissues, collagen- and myosin-based animal tissues are among the most extensively studied biological samples in SHG imaging (Chu et al., 2007; Lin et al., 2005; Plotnikov et al., 2006; Tiaho et al., 2007; Williams et al., 2005).

Despite these advances in imaging, light penetration depth is still insufficient to capture complete tissue images (Meador et al., 2020). Tissue opacity is mainly caused by the mismatch in refractive indices (RI) at the interfaces between the lipid and aqueous media (Chung and Deisseroth, 2013) and by the uneven distribution of scatterers such as lipids, collagen fibers and myofibrils (Richardson and Lichtman, 2015). The propagation of light through tissue results in attenuation due to absorption and scattering. This attenuation can be modeled by the Beer-Lambert law using (Equation 1) I z = I 0 e − μ eff z (1) where I 0 is the initial intensity of the light, I z is the light intensity at a depth z, and μ eff is the effective coefficient accounting for both absorption and​ scattering (Plotnikov et al., 2006). An effective method to improve the penetration depth is to homogenize the RI mismatch of the biomolecule through tissue clearing (Costantini et al., 2019; Tuchin, 2005; Zhu et al., 2013). To achieve tissue transparency for imaging purposes while minimizing damage to the tissue, various chemical solutions have been developed, including benzyl alcohol benzyl benzoate (BABB), THF/DBE, Scale, SeeDB, CLARITY, 3DISCO, and CUBIC (Becker et al., 2012; Hama et al., 2011; Ke et al., 2013; Epp et al., 2015; Ertürk et al., 2012; Tainaka et al., 2014).

Overall, ex vivo tissue clearing methods can be divided into three main types based on their clearing principles and the chemical properties of the reagents used: hydrophobic, hydrophilic, and hydrogel-based methods (Ueda et al., 2020). Hydrophobic and hydrophilic clearing methods, also known as solvent-based and aqueous-based techniques, respectively (Zhu et al., 2021), each involve distinct processes with unique advantages and limitations (see Table 1). As discussed in several recent reviews (Richardson and Lichtman, 2015; Costantini et al., 2019; Ueda et al., 2020), the choice of the appropriate clearing agent depends on the specific biological sample and the experimental context. Therefore, no single agent is ideal for all situations. Most of the previously studied methods have been used for central nerve system (CNS) imaging (Yang et al., 2014). However, the research on clearing methods for cardiovascular tissues remains limited (Kolesová et al., 2016; Nehrhoff et al., 2016; Yokoyama et al., 2017), particularly the development of effective clearing protocols for cardiovascular tissue. Despite advances in tissue clearing and imaging, there are few studies on cardiovascular applications. For example, solvent-based approaches that include BABB were first used by Miller et al. (2005) and later refined by Kolesová et al. (2016) and Ivins et al. (2016). FluorClear BABB was introduced by Epah et al. (Epah et al., 2018). In addition, simple immersion techniques (e.g., FRUIT and MACS) have been employed for cardiac tissue by Xu H. et al. (2019); Xu J. et al. (2019) and Zhu et al. (2020).

Calle et al. (2014) combined BABB-based optical clearing with MPM and SHG to achieve subcellular, three-dimensional visualization of tissue-engineered constructs. Based on this pioneering work, Sung et al. (2016) evaluated and compared two optical clearing techniques—BABB (a representative of hydrophobic clearing) and glycerol (GLY, a representative of hydrophilic clearing)—for microstructural analysis of the left anterior descending artery. The previous method in literature minimized light scattering and enabled imaging at depths beyond 1 mm. Their method, which overcomes the spatial limitations of traditional 2D histology and allows researchers to capture intrinsic fluorescence and SHG signals, has been used to reveal the detailed microarchitecture (e.g., axially, helically, and circumferentially aligned collagen fibers in vascular grafts) and provide rapid, high-resolution volumetric data on decellularized and recellularized lung scaffolds. Building on this foundation, we propose in this work to utilize SHG and autofluorescence (AF) imaging to systematically develop an effective and feasible clearing approach for the microstructural analysis of cardiovascular tissue.

Optical tissue clearing is a widely recognized technique that enables quantitative 3D imaging in advanced microscopy by making tissue transparent for detailed imaging. The success of tissue clearing techniques depends not only on the chemical properties of the clearing agents but also on factors such as lipid content, structural components (e.g., extracellular matrix), and tissue fixation (Lee et al., 2016; Seo et al., 2016). However, the clearing of cardiovascular tissue—e.g., the left anterior descending artery, the focus of this study—poses greater challenges than the clearing of tissues such as the brain. Cardiac tissue presents a significant challenge for fluorescent labeling techniques such as immunolabeling and immunohistochemistry due to its dense, fibrous, and muscular structure. This complexity not only complicates tissue clearing in thicker or mature hearts but also limits the diffusion of antibodies, which are critical for effective labeling. Moreover, the high autofluorescence of intrinsic proteins, such as myoglobin and sarcomeric proteins, can obscure specific signals and interfere with fluorescent dyes, often requiring spectral shifting to distinguish true signals from background noise. The extensive vascular network further complicates the process, as residual blood and hemoglobin absorb light and create additional background interference. Moreover, the preservation of fluorescent proteins poses a significant challenge, e.g., BABB-based clearing methods can degrade fluorescence signals within a few days, complicating subsequent analysis (Kolesová et al., 2016). These factors, including poor fluorescent protein preservation, restricted antibody diffusion, and high autofluorescence, have contributed to the paucity of studies in the literature on the clearing method for cardiovascular tissue.

To address this knowledge gap, the present study carefully optimized the clearing protocols for cardiovascular tissue without the use of immunolabeling. The two commonly used clearing reagents—BABB and glycerol—were compared, along with fixation conditions and clearing durations. Both AF and SHG imaging methods evaluated tissue characteristics and quantified image transparency by measuring signal intensities after each protocol. For the tissue clearing method, BABB, a hydrophobic clearing agent (Lu et al., 2021), was chosen due to its suitability for connective tissue, ease of use, and lack of specialized equipment (Azaripour et al., 2016). However, glycerol was also tested as a representative hydrophilic clearing method (Jiang et al., 2008).

Study 1 shows that BABB provided significantly higher AUC results for both AF and SHG signals than glycerol. Glycerol-cleared tissues also showed increased AF signal intensity compared to uncleared tissues; however, no significant SHG signal intensity was observed between GLY and NC. We therefore assume that glycerol cannot outperform BABB in clearing because it relies on a simple immersion method. In particular, the AF signals observed in the emission window (500–550 nm) are most likely attributed to elastin in the LADA tissue (Zoumi et al., 2004). Elastin exhibits strong intrinsic fluorescence due to its unique cross-linked structure (Samouillan et al., 2002). In contrast, collagen, although a less prominent AF source in cardiovascular tissues, is a robust generator of SHG signals due to its highly ordered triple-helical structure (Zoumi et al., 2004; Du et al., 2025), typically detected in the range of 410–420 nm. BABB enhances elastin fluorescence by several mechanisms. First, it removes lipids, which both scatter light and quench fluorescence. Second, its high RI (∼1.55) (Becker et al., 2012) closely matches that of elastin (RI ≈ 1.51) (Costantini et al., 2019; Jacques, 2013), thereby reducing optical scattering and improving image depth (Fujimoto et al., 2000). Third, benzyl alcohol may partially denature proteins (e.g., tryptophan, tyrosine, and cross-linked desmosine) and expose buried fluorescent residuals, thus increasing the fluorescence quantum yield (Thirumangalathu et al., 2006; Singh et al., 2010). In comparison, glycerol improves elastin fluorescence, but to a lower extent. This lower efficacy is likely due to its inability to remove lipids and its lower RI (∼1.44) (Costantini et al., 2019), resulting in less effective optical clearing compared to clearing with BABB (Richardson and Lichtman, 2015). Achieving the necessary RI with highly concentrated glycerol solutions presents a significant challenge due to their high viscosity, which directly influences the diffusion coefficient of the clearing agents. Further, other challenges include precipitation at room temperature and the entrapment of air bubbles in the solution (Richardson and Lichtman, 2015). It has been found that reducing the viscosity of highly viscous fluids, such as glycerol (Azaripour et al., 2016), can improve the penetration of reagents (Yu et al., 2024).

Since our study compared only one representative of each of the solvent- and aqueous-based clearing methods, we compared BABB with alternative clearing agents available in the literature. Compared with Scale (Hama et al., 2011) and CUBIC (Susaki et al., 2014), both of which employ high-concentration urea, BABB delivers a clear advantage in terms of speed and transparency: Scale, for example, requires weeks to months to reach complete transparency, whereas CUBIC typically takes ∼1–2 weeks, and both cause substantial tissue swelling, which distorts microanatomical details (Hama et al., 2011; Susaki et al., 2014). Although CLARITY (Chung et al., 2013) excels at preserving endogenous fluorescence and enabling deep immunolabeling through hydrogel embedding and lipid removal, the original electrophoresis protocol requires 1–2 days of active clearing and specialized equipment, making it less ideal for high-throughput workflows. Although BABB causes moderate isotropic shrinkage, this uniform size reduction allows for the acquisition of 2–3 times larger volumes within the optimal imaging field of the light sheet without sacrificing subcellular resolution (Pan et al., 2016). Collectively, these comparisons suggest that BABB remains the clearing agent of choice when speed, depth, and controlled dimensional changes are critical.

In Study 2, despite some variation in AUC with formalin fixation duration, SHG imaging revealed no statistically significant differences across fixation times, except between the 60- and 120-min groups. In AF imaging, only the 120-min fixation resulted in a significantly lower AUC than all other durations, while no other pairwise comparisons reached statistical significance. Formalin, which contains formaldehyde, is a widely accepted protein fixative (Fox et al., 1985). Formalin-fixation induces covalent cross-links between proteins as well as between proteins and nucleic acids through hydroxymethylene bridges (French and Edsall, 1945; Helander, 1994). Werner et al. (2000) have suggested that such cross-links can mask epitopes by perturbing the tertiary structure of proteins. One possible explanation for the observed AUC reduction after 120 min is that prolonged fixation may temporarily alter the local chemical environment of harmonophores and fluorophores. This reduces their accessibility or alters their conformation, resulting in reduced signal intensity. The lower AUC observed after 120 min could also be due to biological variability: i.e., an intrinsically low autofluorescence signal in the tissue group examined at this timepoint. The latter explanation is supported by the lack of a similar reduction in the 240-min group. However, the exact mechanism is still unclear and is beyond the scope of this work.

In Study 3, we found that the long-term storage of cleared samples in BABB did not cause significant difference in the AUC values—as a measure of signal intensity—during the extended storage in this solution. This suggests that the clearing process is relatively fast, consistent with other reported results where BABB was used in skin or gingiva samples cleared within only 1–4 h (Azaripour et al., 2016). Furthermore, in this study, AUC values did not deteriorate significantly after long-term storage in BABB of up to 14 days (D14) compared to the first day (D0). This suggests that BABB effectively preserves fluorescent structures in LADA tissues, allowing fluorescence signals to remain detectable even after prolonged storage (∼2 weeks). This observation regarding the preservation of fluorescent proteins is consistent with previous findings from studies on skin and gingiva samples (Azaripour et al., 2016), as well as other studies using different protocols (e.g., FluoClearBABB (Schwarz et al., 2015), uDISCO (Pan et al., 2016), and vDISCO (Cai et al., 2019)), all of which used BABB as an RI-matching agent and concluded that tissue remains stable during storage over long periods of time.

Although our study highlights promising outcomes, such as enhanced imaging depth and a relatively fast, minimally invasive clearing protocol, some limitations of this work must also be acknowledged. First, only one representative of each solvent-based and aqueous-based clearing method was selected, and no comparative analysis of the different clearing agents within each method was performed. Second, although the effects of sequential tissue clearing in the four graded solutions (combinations of BABB and ethanol) were evaluated using imaging and intensity data analysis, this pilot comparative study (see Supplementary Appendix SB) was limited to a single region from four distinct specimens. Due to the small sample size, no statistical analysis was performed for this pilot comparative study. Third, we observed a pronounced decrease in autofluorescence after 120 min of formalin fixation and attributed this decrease to two possible hypotheses that require further validation. Fourth, imaging was limited to small strips of the proximal portion of the LADA tissue, which may have influenced the results due to the limited tissue volume. Although two-photon microscopy offers greater penetration depth and is less affected by scattering than confocal microscopy, it has inherent limitations, including the ability to image only one single plane at a time and to capture large volumes of tissue simultaneously (Costa et al., 2019). Therefore, the findings of this study and their implications should be interpreted with caution, particularly given the potential bias due to the limited imaging area. Finally, in this study we utilized endogenous autofluorescence and second harmonic generation to enhance sensitivity to native fluorophores and collagen. This allowed us to bypass the usual blocking and antibody incubation steps. However, these steps remain important considerations for adapting the protocol to broader histochemical assay applications.

To further refine the comparison between aqueous-based and solvent-based clearing methods, future studies should evaluate additional aqueous-based approaches. One promising direction is the investigation of hyperhydration-based clearing (see Table 1). One promising direction in tissue clearing is the use of hyperhydration-based methods (see Table 1). These techniques reduce the tissue’s refractive index (RI) primarily through urea-induced protein dispersion and increased hydration, rather than relying solely on matching the RI with a high-index clearing solution. The Scale technique (Hama et al., 2011), for example, follows this principle by combining urea-driven protein denaturation with hydration in a glycerol-based medium. Urea disrupts hydrogen bonding within proteins, leading to partial denaturation and separation of dense, high-RI protein complexes, thereby lowering the overall RI. Glycerol serves to modulate tissue expansion, mitigate excessive hydration, and preserve structural integrity by reducing fragility (Richardson and Lichtman, 2015).

Additional experiments could be performed for each clearing step of this protocol to enable a more detailed statistical analysis of the different clearing stages. The significant loss of autofluorescence after 2 hours of formalin fixation might also represent an uncharacterized phenomenon that deserves targeted explorations at the molecular level. Future work could also focus on optical clearing analysis of the entire artery or vein in the cardiovascular system to validate the results for tissues with larger volumes. Future studies should also address histochemical analysis in BABB-cleared tissue, considering specific optimal protocols to improve staining quality. For example, extended PBS washes are recommended for effective removal of BABB residuals (Zhao et al., 2020), while detergents combined with prolonged incubation can enhance tissue permeability (Zhao et al., 2020). Antibody penetration and stability may be improved by extended incubations (Pomaville and Wright, 2023), the use of Fab fragments or nanobodies (Ariel, 2017; Li et al., 2015), and neutralization of the pH during rehydration. To preserve the fluorescence signal and minimize the background signal, researchers can further consider the use of solvent-resistant fluorophores such as Alexa Fluor (Panchuk-Voloshina et al., 1999), increased Tween-20 concentrations, and bovine serum albumin (BSA) in antibody buffers (Piña et al., 2022).

In this work, we conducted a comparative study to evaluate the efficacy of ex vivo clearing techniques on representative cardiovascular tissue (LADA) using AF and SHG imaging. We investigated the clearing agents, BABB and glycerol, and assessed their effect with and without formalin fixation, and compared the results with those on uncleared tissue. We further observed that both BABB and glycerol significantly enhance the AF signals compared to NC tissues, with BABB additionally characterized by a significant increase in SHG intensity compared to glycerol-cleared and NC tissues. Formalin fixation showed significant alterations in the AF signal (i.e., a decrease in AUC with BABB and an increase with glycerol), but no notable changes in SHG signals. We also conducted studies on fixation duration and prolonged tissue storage in BABB. The results showed no significant difference in the signal intensity of the AF or SHG signals between 30, 60, and 240 min of fixation. However, a fixation after 120 min resulted in a significantly lower AF signal compared to all other durations and a lower SHG signal compared to a fixation of 60 min. Finally, the suitability of the BABB agent for LADA tissue was demonstrated by its ability to bind fluorescence molecules even after extended storage in the clearing solution.