A simple preparation protocol for shipping and storage of tissue sections for laser ablation-inductively coupled plasma-mass spectrometry imaging

Abstract

A rapid and cost-efficient tissue preparation protocol for laser ablation-inductively coupled plasma-mass spectrometry imaging (LA-ICP-MSI) has been developed within this study as an alternative to the current gold standard using fresh-frozen samples or other preparation techniques such as formalin fixation (FFix) and formalin-fixed paraffin-embedding (FFPE). Samples were vacuum dried at room temperature (RT) and stored in sealed vacuum containers for storage and shipping between collaborating parties. We compared our new protocol to established methods using prostate tissue sections investigating typical endogenous elements such as zinc, iron, and phosphorous with LA-ICP-MSI. The new protocol yielded comparable imaging results as fresh-frozen sections. FFPE sections were also tested due to the wide use and availability of FFPE tissue. However, the FFPE protocol and the FFix alone led to massive washout of the target elements on the sections tested in this work. Therefore, our new protocol presents an easy and rapid alternative for tissue preservation with comparable results to fresh-frozen sections for LA-ICP-MSI. It overcomes washout risks of commonly used tissue fixation techniques and does not require expensive and potentially unstable and time-critical shipping of frozen material on dry ice. Additionally, this protocol is likely applicable for several bioimaging approaches, as the dry condition may act comparable to other dehydrating fixatives, such as acetone or methanol, preventing degradation while avoiding washout effects.

Keywords: LA–ICP–MSI; human prostate; tissue preparation; zinc.

Graphical Abstract

Vacuum dried and sealed samples result in equivalent data quality as fresh frozen samples using LA-ICP-MSI.

Fig. 1

Schematic overview of the prostate tissue sample collection and sectioning. The cylindric sample (3 mm diameter, 2 mm height) was collected from the peripheral zone of a slice of whole-mount human prostate and subsequently cryo-sectioned. The consecutive sectioning order of the three replicates (sets A, B, and C) with randomized section order is shown for each protocol per set (FF = fresh frozen, RTV = room temperature vacuum dried and sealed, FFix = formalin fixed, FFPS = formalin fixed, paraffin sealed).

Fig. 2

Histology of the prostate tissue sample. Bright-field microscopic images of the two hematoxylin, eosin, and saffron (HES) stained prostate tissue sections adjacent to replicate set A (A: HES 1, B: HES 2 as indicated in Fig. 1). Corpora amylacea/small prostatic calculi are marked with asterisks.

Fig. 3

Phosphorous, zinc, and iron distributions after using the four different tissue preparation and storage protocols 1–4 presented in Fig. 1 (replicate set A). Shown are bright-field microscopic images (AD) and pseudo color images of the qualitative phosphorous (A1–D1), iron (A3–D3), and quantitative zinc (A2–D2) distributions as obtained by laser ablation-inductively coupled plasma-mass spectrometry imaging (LA–ICP–MSI). Images are of serial sections of the same tissue sample, which were preserved, stored, and shipped according to the FF (A), RTV (B), FFix (C), and FFPS (D) protocols. Calibration for zinc was performed using matrix-matched standards based on gelatin. Zinc hotspot areas are marked with asterisks in the bright-field microscopic images. (FF = fresh frozen, RTV = room temperature vacuum dried and sealed, FFix = formalin fixed, FFPS = formalin fixed, paraffin sealed).

Fig. 4

Recovery rates of the four tested protocols. Shown are box plots of the recovery rates obtained (n = 3 technical replicates) over the whole sections for the elements phosphorous, zinc, and iron of RTV, FFix, and FFPS relative to FF. (FF = fresh frozen, RTV = room temperature vacuum dried and sealed, FFix = formalin fixed, FFPS = formalin fixed, paraffin sealed).

Fig. 5

Segmentation of MSI data was based on zinc (top left) and phosphorous (top right) data by thresholding and verification using adjacent hematoxylin, eosin, and saffron (HES) stained sections (top middle, features indicated by arrows). As position and morphology slightly changed between the consecutive sections, all MS images were cropped to equally sized smaller images covering roughly the same area of the original sample to increase comparability (indicated with white boxes on Zn and P distribution images and as a dashed green box on the HES image). Cropped images were segmented based on the P contrast and distribution into on tissue/low P = stroma, on tissue/high P = gland epithelium, lumen, and off tissue.

Fig. 6

Analysis of the zinc content in gland epithelium, stroma, and hotspots for all four protocols. (A) Box-and-whisker plot [box: Q1 to Q3 quartile, whisker: 1.5 * IQR (IQR = Q3 − Q1) from edges of the box] of the zinc concentration distribution obtained from image segmentation and pixel classification as indicated in Fig. 5 for each protocol. (B) The relative distributions of zinc between gland epithelium, stroma, and hotspots for each protocol (normalized to respective protocol's total zinc) are shown as stacked boxplots. (FF = fresh frozen, RTV = room temperature vacuum dried and sealed, FFix = formalin fixed, FFPS = formalin fixed, paraffin sealed).