Background

Skeletal muscles are heterogeneous, allowing them to engage in a spectrum of activities, from continuous low-intensity activity, such as breathing and posture control, to repeated submaximal or maximal contractions required for sprinting or lifting heavy objects [1]. Historically, muscle fibers have been categorized based on their morphological (red vs. white), biochemical (oxidative capacity, as determined by measuring succinate dehydrogenase, nicotinamide adenine dinucleotide dehydrogenase reductase, or ATP synthase activities), or molecular characteristics [2–6]. Currently, fiber types are distinguished based on the expression of specific myosin heavy chain (MyHC) isoforms. In adult rodent muscles, four major isoforms are detected, including type 1, 2A, 2X, and 2B (or a combination of these in hybrid fibers) corresponding to MYH7, MYH2, MYH1, and MYH4, respectively [1,3,7,8]. Type 1 fibers (slow twitch) have high mitochondrial content, oxidative metabolism, and slow contraction speed, making them well-suited for sustained endurance activities. Type 2A fibers (fast twitch oxidative) can generate greater power while maintaining endurance capacity. Type 2X and type 2B fibers (fast twitch glycolytic) contract the fastest and are adapted for short bursts of power. Noticeably, metabolic and contractile properties are highly adaptable and context-dependent, influenced by muscle function, species, and environmental factors rather than strictly by MyHC expression alone [3].

The orofacial muscles constitute a subset of skeletal muscle originating from the branchial arches, a distinct origin from muscles of the trunk and limbs [9]. The masseter is the dominant jaw-closing muscle in rodents, accounting for approximately 50% of the masticatory muscle mass and contributing to mastication, biting, and swallowing [10]. The adult muscle masseter consists of three anatomical partitions: superficial, intermediate, and deep [10–12]. Fiber orientation shifts from vertical in the anterior regions to an oblique orientation in the posterior aspect of the superficial and intermediate layers [10]. Notably, the insertion area for the superficial and deep masseter is vast, extending from the mandible angle to the first molar position for the superficial masseter and almost the entire length of the jugal bone for the deep masseter [10]. Given the complex architecture of the masseter, high-resolution imaging techniques are essential to capture its detailed structure [13]. However, the phenotypic characterization of the masseter remains limited [14,15].

Determining MyHC isoforms by immunofluorescence for muscle fiber typing has become widespread, in part because it also provides information about fiber cross-sectional areas [1,2,16]. Further, simultaneous labeling of muscle fibers is possible thanks to mouse monoclonal anti-MyHC antibodies with isotype-specific secondary antibodies [17]. Fluorescence microscopy often uses optical filters to separate emission from excitation light, accommodating several filter cubes, each one directing a specific predetermined wavelength [18]. Although only a single filter cube may be engaged at a time, there is potential for spectral overlap between fluorophores, limiting the number of compatible markers in a single experiment. Additionally, fluorescence microscopy captures light from all focal planes, limiting the signal-to-noise ratio and image resolution [19].

To overcome the limitations of widefield microscopy, Marvin Minsky developed the confocal microscope in 1957 [20]. Confocal microscopy provides better resolution than widefield fluorescent microscopes, yet its main advantage is the ability to acquire high-contrast images by minimizing out-of-focus light using a pinhole [18,21]. Additionally, confocal microscopy can capture multiple optical planes, making it ideal for analyzing compartmentalization and colocalization of specific molecules with high accuracy, as well as enabling the creation of 3D reconstruction of the imaged tissue [21]. Newer systems, such as the Stellaris 5 (Leica Microsystems, Durham, NC), feature next-generation white light lasers (WLL), which allow for the simultaneous excitation and detection of multiple fluorophores. The fine-tuning of excitation wavelengths enables highly efficient spectral unmixing, overcoming the limitations of traditional systems. This technology expands the range of usable fluorophores, including those in the near-infrared spectrum, and supports the use of multiple labels in parallel [22,23].

The objective of this study is to demonstrate the effective use of confocal microscopy in obtaining high-resolution, quantifiable images for muscle fiber typing from cryosections. We aim to show that this approach offers excellent resolution and contrast, even in applications where confocal capabilities are typically deemed unnecessary. Multiple fluorescent channels allow for the simultaneous visualization of several molecular markers in addition to the corresponding MyHC labels. This flexibility provides detailed insights into muscle fiber architecture and is particularly advantageous for reducing background noise and imaging alongside other protein targets that require precise 3D localization.