Abstract
Environmental-scanning electron microscopy (ESEM) is routinely applied to various biological samples due to its ability to maintain a wet environment while imaging; moreover, the technique obviates the need for sample coating. However, there is limited research carried out on electron-beam (e-beam) induced tissue damage resulting from using the ESEM. In this paper, we use quantitative second-harmonic generation (SHG) microscopy to examine the effects of e-beam exposure from the ESEM on collagenous tissue samples prepared as either fixed, frozen, wet or dehydrated. Quantitative SHG analysis of tissues, before and after ESEM e-beam exposure in low-vacuum mode, reveals evidence of cross-linking of collagen fibers, however there are no structural differences observed in fixed tissue. Meanwhile wet-mode ESEM appears to radically alter the structure from a regular fibrous arrangement to a more random fiber orientation. We also confirm that ESEM images of collagenous tissues show higher spatial resolution compared to SHG microscopy, but the relative tradeoff with collagen specificity reduces its effectiveness in quantifying collagen fiber organization. Our work provides insight on both the limitations of the ESEM for tissue imaging, and the potential opportunity to use as a complementary technique when imaging fine features in the non-collagenous regions of tissue samples.
Highlights
In scanning electron microscopy (SEM) a raster-scanned beam of focused electrons irradiate a sample, the resulting secondary and backscattered electrons are detected and used to form an image of the sample with nanometer resolution[1,2]
We show that the higher spatial resolution afforded by Environmental-scanning electron microscopy (ESEM), compared to second-harmonic generation (SHG) imaging, permits visualization of fine structural details, but the relatively lower contrast makes it difficult to estimate individual collagen fiber orientation
Low-vacuum mode is intermediate between high-vacuum and wet mode
Summary
In scanning electron microscopy (SEM) a raster-scanned beam of focused electrons irradiate a sample, the resulting secondary and backscattered electrons are detected and used to form an image of the sample with nanometer resolution[1,2]. The standard procedure for sample preparation usually involves initial dehydration followed by coating with an electrically conductive material, typically gold, which makes it challenging to image biological samples in their natural state To mitigate this issue, the environmental-SEM (ESEM) was invented to allow gas inside the sample chamber, through the use of multiple pressure-limiting apertures, while permitting the electron-beam (e-beam) to remain under high vacuum[3,4]. We have previously demonstrated that SHG imaging combined with spatial Fourier analysis (referred to as FT-SHG) provides a simple yet powerful approach to quantifying the collagen architecture This form of quantitative SHG has been applied to the collagenous environments of various samples including tendon[15], bone[16], breast tissue[17] and cervix[18]. We show that the higher spatial resolution afforded by ESEM, compared to SHG imaging, permits visualization of fine structural details, but the relatively lower contrast makes it difficult to estimate individual collagen fiber orientation
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