Impact of glycerol solution on myocardium tissue – Ex vivo deep-UV Raman spectroscopy study

Abstract

Cardiomyopathies are often characterized by significant fibrotic remodelling of the heart, marked by an abnormal accumulation of collagen type I. Label free Raman spectroscopy, a non-invasive diagnostic technique, holds promise for monitoring biochemical changes throughout the initiation and progression of different diseases, including cardiomyopathies. This study demonstrates the effectiveness of 70% glycerol as a hyperosmotic immersion liquid for in-depth controlling the optical properties of ex vivo myocardium tissue during deep-UV Raman spectroscopy with 244 nm excitation. The results revealed a considerable enhancement in the intensities of Raman peak, particularly the amide I region after glycerol treatment. This occurred across all depths (0−120 µm) and glycerol treatment durations (30 and 60 min). A noticeable enhancement of the Raman peak at 1647 cm⁻¹ was also observed that is attributable to structural transformations of the collagen due to the dehydration induced by glycerol. This finding suggest that deep-UV Raman can be employed as a specific probe of the collagen environment. As the amide I region reflects structural changes in collagen type I, these findings propose the potential of deep-UV Raman spectroscopy in combination with glycerol as optical clearing agent for monitoring collagen modifications.

Graphical abstract

Keywords

Amide I / Collagen / Deep-UV Raman spectroscopy / Glycerol / Optical clearing.

1 Introduction

Light-based methods, such as optical imaging and spectroscopy, are getting significant acceptance in revealing the physiologic and pathological alterations in organs, tissues and cells. These methods show the ability to investigate the optical properties in vitro, in vivo, and even in situ [1], making them important tools for clinical applications. Their fast development holds promise for complementing and eventually improving the conventional methods in treatment and diagnosis of cardiovascular disease [2,3]. With nearly 17.9 million mortalities yearly, reporting for almost 30% of all mortalities in 2019, this disease will rise more than 24 million lives by 2030 [4]. Cardiomyopathies are associated to heart fibrotic remodelling, characterized by a rise in collagen type I (COL I) accumulation. It is worth to mention that early-stage detection of tissue fibrosis followed by punctual treatment could potentially permit the inhibition or even reverse organ damage [5], which, practically every tissue and organ in the body can undergo fibrosis [6]. Therefore, developing new approaches for real-time monitoring of fibrosis initiation and progression holds significant value for diagnosis and treatment. The cardiovascular disease, supposed to be promoted by epigenetic and chronic inflammatory factors was studied also using Raman micro-spectroscopy (RMS) [7,8].

RMS appears as a promising method for monitoring and diagnosing different diseases due to its distinctive ability to precisely reveal the pathological alterations in organs, tissues and cells [9,10]. It offers a non-destructive, non-invasive measurement with minimal sample handling preparation [11], making it an alternative or complement to conventional methods, such as biopsy [8,12]. In spite of this, the use of RMS for monitoring and diagnosing cardiovascular diseases stays poorly explored [13]. Conventional RMS faces significant challenges in clinical applications for heart tissue examination due to the weakness of the Raman signal. Previous studies have shown that employing resonant Raman scattering through specific cytochromes significantly enhances both selectivity and sensitivity for assessing myocardial infarcts in rats [14,15]. This approach involves selecting a laser excitation wavelength that can excite the target molecules electronic transitions directly, leading to an enhancement of the Raman signal of these proteins in rat hearts by 10³−10⁵ times compared to the non-resonant case [16,17].

Ultraviolet (UV) excitation can increase the vibrational intensities of specific protein bonds through resonant effects [18,19]. In a UV resonant Raman spectroscopy study using 200 nm excitation a selectively increase of the amide I vibrational intensity by π→ π* transition was observed [20]. In contrast, excitation wavelengths between 230 and 250 nm selectively enhanced features related to the side chains of aromatic amino acids [21]. Ager et al. [22] suggested that deep-UV Raman with 244 nm excitation can strongly enhance the amide I band through resonance. This approach also eliminates the fluorescence background typically observed with visible and NIR excitations [22].

The current diagnosis of fibrosis still depends on the gold-standard histological evaluation using invasive biopsy method. In fibrosis alterations, collagen serves as the main molecular biomarker, which can be identified by RMS, enabling the characterization and monitoring of fibrillogenesis and ischemic tissue remodelling [23–26]. For instance, RMS has been successfully employed to detect fibrotic alterations in collagen type I through its distinct molecular signatures [8]. However, RMS application for diagnosing cardiovascular diseases faces several challenges that must be addressed before can be fully implemented in clinical practice [25]. Although spectral variances in the Raman profile are most pronounced during the early stages of tissue remodeling, the changing collagen composition continues to offer important molecular information for classifying and discriminating myocardium as fully fibrotic intermediate, or non-fibrotic [27]. One of the major challenges in RMS of cardiac tissues is the limited penetration depth due to the strong light scattering of biological objects [25,28,29]. Therefore, further studies are important to development of RMS techniques capable of probing deeper tissue layers.

For deep-tissue optical imaging, light scattering rising from mismatch in the refractive index (RI) poses major challenges in biological applications [30–32]. However, a recent study published in Science [33] by Ou et al. adopted an innovative method by utilizing strongly absorbing molecules (such as food dye tartrazine) in the near UV to blue wavelength range, which can rise the RI of water in the near-IR range. This counterintuitive phenomenon reversibly and effectively made tissue transparent in live mice representing a revolution in the discipline of live tissue optical clearing (TOC). Recent study has investigated tartrazine and 4-aminoantipyrine as strongly absorbing molecules aimed at enhancing the penetration depth of optical imaging [34]. They found that mice treated with 4-aminoantipyrine exhibited substantially enhanced penetration depth compared to mice treated with tartrazine. It is worth to mention that the absorbing molecules were chosen according to analysis of the dielectric properties of materials, utilizing the Kramers–Kronig relation and Lorentz oscillator model. In our case, this applies to UV wavelength. For optical clearing (OC), it is both necessary and possible to utilize a strongly absorbing molecules with a sufficiently steep and narrow absorption band. Due to anomalous dispersion of glycerol [33,35], this method can significantly increase the RI of the interstitial fluid or cytoplasm, thereby improving the match between the RI of the structural elements within the tissue.

TOC has been developed as a more promising path for deep-tissue optical techniques. In recent years, researches on the in vivo OC field has greatly intensified, inventing several in vivo skin OC techniques and development of skull OC windows [36,37]. Over the past three decades, TOC techniques operating in the visible to NIR spectrum have seen rapid advancements, demonstrating their potential for integration with various clinical optical methods [38,39]. However, a crucial question now that demands attention is: can OC be made equally effective in the UV range? Exploring this possibility would initiate a fundamentally new field of UV-based OC for in-depth studies of tissue molecular structures using UV-RMS [40,41]. Moreover, glycerol as an OC agent (OCA) and its aqueous solutions possess cryo-protective capabilities for various types of cells, tissues, and organs [42]. This ability stems from their capacity to disrupt ice crystal formation through hydrogen bonding networks within the solutions. For that reason, the studies of glycerol/water mixtures interaction with myocardium tissue could benefit the field of cryobiology, potentially leading to improved tissue preservation technique, while RMS can serve as a good method for monitoring the persistence of implanted organs during storage.

To investigate the efficacy of UV-OC for studying tissue molecular structures, we evaluated the effect of 70% glycerol as an OCA on ex vivo chicken heart tissue samples using deep-UV Raman spectroscopy in-depth. We focused on differences in Raman amide I region intensities to assess the OCA effectiveness in improving tissue transparency. This study could serve as a basis for developing new tissue optical clearing practices applicable in deep-UV Raman spectroscopy.

2 Materials and method

2.1 Sample preparation

The myocardium, the main component of the heart, consists of specialized striated muscle tissue. Each muscle fiber is surrounded by a cell membrane called the sarcolemma and the intracellular fluid within is called the sarcoplasm. Unique to cardiac muscle, the sarcoplasm contains myoglobin, a protein responsible for its red color. Like skeletal muscle, cardiac muscle sarcoplasm contains myofibrils. These tightly packed and organized myofibrils contribute to the strong light scattering observed in myocardial tissue.

This study utilized eight myocardium samples obtained from two freshly excised chicken hearts purchased from a local slaughterhouse (Jena, Germany). The hearts were transported on ice and stored at −20°C overnight before sample preparation. On the following day, the hearts were sectioned using a manual microtome to obtain six uniform circular samples with a diameter of approximately 1 cm and a thickness of around 700 µm. Prior to each Raman spectroscopic measurement, the samples were thawed at room temperature on microscope slides. For the treatment, the myocardium samples were soaked in a 5 mL bath of 70% glycerol solution for either 30 or 60 min. Untreated samples served as controls. The OCA was gently removed from the sample surface using a paper towel before each Raman measurement. Thickness measurements were taken before and after treatment using a calliper at five designated points on each sample (placed between two microscope slides), with the average value recorded.

2.2 Optical clearing agent

Glycerol (Gly) was chosen as OCA for several reasons: its widespread use in biological applications attributable to its affordability, biocompatibility, and pharmacokinetics [43]. The high RI of Gly closely matches that of the biological tissue, and its unique property of significantly increasing RI with decreasing wavelength down to 120 nm (see Fig. 1) [44]. This latter characteristic is particularly advantageous in the UV range, as recent studies suggest that RI matching is more effective and can even create new transparency windows in tissues during UV-based OC [45] due to Gly optical dispersion properties, which calculated from the absorption coefficient through Kramers−Kronig relations [33,35,46]. Moreover, it has no absorption from 150 nm to the NIR wavelengths (see Fig. 1b). This experiment employed 70%-Gly with a measured RI of 1.429 at 589 nm using a Carl Zeiss refractometer (Jena, Germany) and RI recalculated using optical dispersion, presented in Fig. 1a.

2.3 Raman spectroscopy

Raman spectra were recorded in the deep-UV range employing a Raman micro- spectrometer (HR800, Horiba/Jobin-Yvon, Bensheim, Germany). The device is supplied with a 244 nm frequency-doubled Argon-ion laser (Innova 300, FReD, Coherent, Dieburg, Germany) delivering 0.4 mW power before the objective. The excitation laser was focused onto the samples using a 15 × objective and the collected Raman signal was redirected via a 400 µm entrance slit onto a 2400 lines/mm grating (see Fig. 2). The dispersed light was captured by a nitrogen-cooled CCD camera with 2 cm⁻¹ spectral resolution. An xyz-stage (manual-controlled) with micrometer resolution allowed for vertical movement of the sample, facilitating acquisition of an in-depth profile (Z) by collecting Raman spectra at each depth with a 30 s exposure time (later averaged). All Raman spectra were collected and analyzed in the fingerprint region (400−1900 cm⁻¹).

2.4 Data analysis

The Spectragryph software was used to process and analyze the Raman spectra collected from the samples. All Raman spectra were averaged, smoothed using a Savitzky−Golay filter (3rd-order polynomial, 13 intervals), and then subjected to advanced baseline subtraction employing an adaptive algorithm [47]. This background signal is likely due to sample fluorescence at wavelengths outside the recorded spectra. Although the grating diffracts this light away from the detector, residual diffuse light within the spectrometer contributes to the background signal.

The deconvolution of Raman spectra was performed with the Renishaw WiRE software. The spectral deconvolution was utilized to calculate the area under the curve (AUC) of the substructural peaks of COL I region between 1500 and 1750 cm⁻¹. Figure 3 illustrates the fitting with five Gaussians located at 1547 ± 2, 1584 ± 2, 1608 ± 2, 1647 ± 2, and 1678 ± 2 cm⁻¹. The assignments of the five peaks are given in Table 1. To obtain biochemically and reproducible reasonable outcomes, the full width at half maximum of the Gaussian components was constrained to be vary within 50 cm⁻¹.

Statistical evaluation of the data was performed utilizing the IBM Statistical Package for the Social Sciences (SPSS). Paired-sample t-tests were used to assess differences in mean values between untreated and 70%-Gly treated myocardium samples [48]. To conclude whether there was a statistically significant difference between the mean values of control and 70%-Gly-treated myocardium samples, a paired Student’s t-test was implemented. Differences with p < 0.01 were considered highly significant (*) and those with p < 0.05 were considered statistically significant (**). The data are presented as mean ± standard error of the mean (SEM).

3 Results and discussion

Figure 4 visually demonstrates the OC effect of 70%-Gly treatment on myocardium samples. Figures 4a−c depict the samples before and after 30 and 60 min of treatment. The figure clearly displays the OC effect of the investigated tissues, i.e., the light transmission increases in the visible range after OC.

Moreover, Table 2 summarizes the changes in myocardium sample thickness after treatment with 70%-Gly for 30 and 60 min. According to the data in Table 2, the myocardium tissue is shrinking after the OC treatment, which is an indication of the Gly-induced dehydration [29,49]. Tissue dehydration is recognized as one of the processes of OC besides RI matching and reversible dissociation of protein [38].

Cardiomyopathies often involve significant fibrotic remodelling of the heart's connective tissue, characterized by an abnormal accumulation of COL I. Recent studies utilizing RMS and spectral deconvolution of the COL I (amide I region) demonstrated the potential for detecting fibrotic collagen alterations in various human tissues [7,8]. The amide I region, ranging from 1550 to 1720 cm⁻¹ in proteins, reflects modification in their secondary structure. The secondary structure in COL I is characterized primarily by α-helices, β-sheets, β-turns, and random coils [50].

Raman spectra of the surface (0 µm depth) of untreated (control) ex vivo myocardium sample and the 70%-Gly (OCA) by using deep-UV excitation at 244 nm are shown in Figs. 5a and b. The Raman peaks located at 1003, 1460, 1547, 1584, 1608, 1647, and 1678 cm⁻¹ [7,8,21] and for Gly located at 484, 676, and 1054 cm⁻¹ [43] are clearly observed in Figs. 5a and b, respectively.

To assess the impact of the 70%-Gly treatment on the ex vivo myocardium tissue, the Raman spectra were acquired from both untreated and treated samples after 30 and 60 min treatment time from the surface 0 to 120 µm depths (in 20 µm increments). Figure 6 illustrates the depth-dependent averaged Raman spectra of myocardium tissue for 30 and 60 min treatments in the fingerprint region. Figure 6a shows the impact of OC on depths at 0 and 120 μm, which clearly indicate the enhancement of Raman spectra after OC (before they are normalized to the Raman peak at 1003 cm⁻¹).

For consistent comparison across different depths and treatment times, the spectra were normalized to the area of Raman peak at 1003 cm⁻¹ corresponding to phenylalanine/urea in the range from 975 to 1025 cm⁻¹, which is not influenced by Gly (see Fig. 5). Figures 6b−d illustrate depth-dependent averaged Raman spectra before and after 30 and 60 min treatment, respectively.

Figure 6 clearly shows that the Raman intensities are increased in all depths after OC treatment. This enhancement likely due to two mechanisms associated with OC: improved RI matching and tissue dehydration [29], which leads to a reduced light scattering and more condense tissue molecular structure. Previous research has established that light scattering can be significantly decreased in the UV range during OC treatment [45,51]. Moreover, other research has indicated that the efficiency of transparency is significantly higher in the deep-UV compared to the visible-NIR range [52,53].

Comparison of the shapes of the Raman spectra in Fig. 6 clearly shows that, compared to the untreated sample, the relative Raman peak intensity at 1647 cm⁻¹ increases across all depths and treatment times, indicating structural changes in the collagen during OC. Since the first mechanism described above (RI matching), results in reduced light scattering, but cannot affect the collagen structure, the observed changes in the Raman spectra can be attributed to tissue dehydration. These will be discussed in detail later.

Figure 7 presents a quantitative analysis of the depth-dependent impact of OC on the intensity of Raman peaks at 1608 and 1647 cm⁻¹, as a function of different treatment (30 and 60 min). It is clearly seen that both Raman peaks increase in intensity after 30 min of treatment, followed by some decrease after 60 min, indicating that the prolonged OC leads to decay in Raman signal intensity [54], which suggests an optimal OC treatment time about 30 min (Tissue should be not transparent but semi-transparent, otherwise no sufficient backscattering ability to detect signal, see Fig. 4).

The increase is more pronounced for the Raman peak at 1647 cm⁻¹, potentially due to the dehydration effect [21]. Moreover, deep-UV Raman studies have reported an increased intensity of the amide I band at 1650 cm⁻¹, which indicating changes in collagen quality due to factors like dehydration/hydration [21], aging [22], or radiation damage [55]. Additionally, RMS focusing on the amide I region, has shown promise in discriminating between non-fibrotic (healthy) and fibrotic COL I fibers in different organs [7,8]. Specifically, the peak at 1608 cm⁻¹ serves as a reliable endogenous marker for structural modifications in COL I conformation, allowing researchers to distinguish between fibrotic and non-fibrotic fiber structures.

The multi-peak fitting of the amid I region of the Raman spectra allows to study the in-depth and OC-treatment related changes in the secondary structure of collagen. The spectra were fitted with 5 Gaussians as described in the 2.4 Data analysis section. It can be seen that two of the five peaks are related to CH₂ wag /tyrosine (around 1547 and 1608 cm⁻¹), one to the Fermi doublet of the aromatic ring (around 1584 cm⁻¹) and two to the amide I of the collagen (around 1647 and 1678 cm⁻¹). The main difference between the latter two is in the secondary structure of the collagen: the first peak is characteristic for α-helices, while the second peak for β-sheets. As Fig. 6 shows, the intensity of the α-helix band significantly changes during the OC treatment.

To study the changes in the secondary structure of the collagen, the AUCs of the two amide I peaks were normalized to the AUC of the tyrosine peak at 1608 cm⁻¹, similarly to the method used in Ref. [56]. The change of the peak area ratios with depth and OC treatment are shown in Fig. 8.

The data in Fig. 8 reveal the changes of α-helices and β-sheets relative to the tyrosine content of the tissue. In the control sample the ratio of the α-helices Raman peak increases with depth, from 0.75 at the surface to 0.92 at 120 μm. At the same time, after some initial rise from 0.53 at the surface to 0.63 at 20 μm, the ratio of the β-sheet band slightly decreases to 0.57 at 120 μm. So, it can be concluded that, based on the UV-excited Raman spectra, in the control myocardium tissue the amount of collagens organized into β-sheets decreases, while those in α-helices increases with depth.

Interestingly, the OC treatment with the 70%-Gly OCA changes this behavior. In general, the intensity of both α-helix and β-sheet Raman peaks is much larger than that in the control sample, which proves the efficiency of the optical clearing. The dehydration causes the contraction of the tissue (see the thickness measurements later), which increases the relative concentration of the collagen molecules in the excitation volume, leading to a stronger Raman signal. In addition, the α-helix content is the highest at the surface and the lowest at 120 μm depth, while the amount of β-sheets increases with depth. This is the reason of the higher intensity in the region of the 1647 cm⁻¹ Raman band in the treated samples (see Fig. 6).

It has been shown earlier [56] that the α-helix and β-sheet amide I peaks have similar Raman cross sections. This means that, if the collagen content of the tissue does not change, the combined AUC of the two peaks should be constant, and the relative AUCs of the α-helix and β-sheet amide I Raman peaks can be used as semiquantitative measures to estimate the ratio of the two collagen forms in the tissue. Figure 9 shows the change of the combined AUC of the two Raman peaks with depth. It can be seen that for the control sample, except the surface, its value is between 1.45 and 1.50 for the entire investigated depth range. The sample with 60 min OC treatment behaves similarly, with AUC values around 2.00 (except the surface of the tissue). The 30 min sample has similar values up to 80 microns depth, but then it decreases remarkably. This could be due to incomplete OC effect in the deeper regions of the tissue for the 30 min treatment.

4 Conclusions

This study is the first use of label free deep-UV Raman spectroscopy to comprehensively examine the effects of 70%-glycerol on chicken myocardial tissue ex vivo. Treatment with 70%-glycerol resulted in increased intensities across all depths and treatment time, particularly for the amide I peak. The increase of the relative intensity of the 1647 cm⁻¹ Raman band indicated structural changes of collagen caused by the dehydration induced by the OCA. These changes were attributed to the transformation of α-helices to β-sheets. Treatment with 70%-glycerol led to increased intensity, reflecting enhanced focused light penetration deeper into the myocardial tissue. This observation, combined with the enhanced amide I band in the Raman spectra after 70%-glycerol treatment, suggests the potential of deep-UV Raman spectroscopy in conjunction with optical clearing agents like glycerol for detecting structural changes in collagen I fibers. However, further research is necessary to draw definitive conclusions on the feasibility and efficacy of this approach for heart-related optical researches.

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