Study Case
A 65-year-old female was referred to Tianjin Chest Hospital on Feb 7, 2022 due to flustered and weak for more than one month. Chest enhanced CT showed a mass occupied the lower lobe of the left lung and lung cancer was primarily considered. This patient underwent lobectomy by VATS, the mass was completely removed. The postoperative pathological outcome was adenocarcinoma and the mass invaded the visceral pleura.
Tissue Preparation
A 1 × 1 × 1cm3 specimen was collected within 30 min after the removal of the surgical mass and post-fixed for 24 hours with 4% paraformaldehyde at 4℃. After post-fixation and dehydration, the tissue was immersed in a Lowicryl HM20 series (Ted Pella Inc., Redding, CA, USA), containing 0.2% SBB (70%, 85%, and 100% HM20 for 2h each and 100% HM20 overnight). The sample was then impregnated in a prepolymerization HM20 solution for 3 days at 4℃ and embedded in a vacuum oven at 50℃, 24h.
La Tissue Imaging
LA tissure imaging was performed on f-MOST system (Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College). The prepared-sample was immobilized anterior-posterior in a water bath on a 3D translation stage prior to imaging. The sample was submerged in a solution of propidium iodide (PI) and 0.01 M Na2CO3 to provide a matching refractive index for the objective lens during imaging.
Throughout data collection, the liquid level of the solution was kept above the bottom surface of the objective lens. For wide-field high volume tomography, sectioning was accomplished utilizing a fixed diamond knife and a 3D translation stage. The imaging plane was commonly positioned at 1 µm below the surface to prevent the compromising effects of sectioning markings on the machined surface. Prior to data collection, we concentrated on the sample's top surface and made necessary adjustments to ensure a clear image. We then lowered the sample to position the imaging plane below the specimen's surface. The WVT system automatically carried out the sectioning and imaging after the imaging settings were established in order to finish the tissue-wide data collection. Additionally, we made flexible adjustments to the image parameters, such as the area of interest and exposure time. In order to maintain a flattened section, get rid of the cutting chips, clean the PI solution, and keep a consistent PI concentration, we also used a recirculating filtering equipment.
The objective performed a line-scanning block-face scan of the sample's surface. at the depth of 1 µm. In order to cover the entire coronal plane in each layer imaging, we used a strip-scanning (x axis) model along with a montage in the y axis. The fluorescence was captured with a TDI-CCD camera after being gathered using a microscope objective, passing through a bandpass filter. The diamond knife removed the photographed surface from one surface when it had been finished, exposing the smooth, brand-new surface for imaging. To get the required dataset, we repeated these steps across the entire sample volume.
Last but not least, the fMOST system carried out a number of cycles of automatic sectioning with an axial step size of 1 µm, then imaging at a voxel size of 0.32 x 0.32 x 1.0 µm3 with one channel for PI-stained cytoarchitecture and another channel for Alexa fluor 488, which were simultaneously detected using two cameras. The raw data acquisition lasted continuously for more than two weeks and the raw dataset was larger than 8 terabytes, including 4000 coronal sections for each channel.
The BioMapping7500 system (Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, and Peking Union Medical College), which consists of a 20X Olympus microscope objective, one laser with a wavelength of 561nm, and a TDI-CCD camera, was used to detect the images from fMOST.
Processing Of Images
Preprocessing of Images
Two detection channels performed all image preprocessing of the obtained data. Based on precise spatial orientation and nearby overlap, the stripes of each coronal portion were stitched to generate a full section. Section by section, lateral illumination adjustment was carried out. Calculating the mean intensity along each direction and fitting the relevant polynomial curves led to the determination of the correction coefficient along each direction. By calculating the images’ average grey-scale values, it was possible to adjust for axial lighting by balancing the brightness of the various coronal portions. Finally, using LZW compression, we created a single image at the imaging plane for image storage in a 16-bit depth TIFF format. Matlab and C + + were implemented to preprocess obtained images. On a computer server with 32 cores operating at 2.9 GHz per core, image preprocesses for the LA tissue data set at the voxel resolution of 0.32 x 0.32 x 1.0 µm3 were carried out.
Reconstruction and visualization
To create the charts and movies, we used the Amira software (version 2020.1, FEI, Merignac Cedex, France) and the Imaris software (version 9.7.2, bitplane, Switzerland) to visualize the dataset. Amira software was used to import the preprocessed dataset using a desktop graphical workstation (7920 with one Intel Xeon Gold 6226R CPU, 512 GB memory and an Nvidia GeForce RTX 3090 graphics card, Dell Inc., Round Rock, Texas, USA). We used the OTSU thresholding technique in conjunction with manually corrected parameters on the red channel cytoarchitecture pictures to reconstruct the bronchus. Amira was used to convert the TIFF data format to the native LDA type so that the TB-sized data could be processed on a single workstation. The extraction of the data in the range of interest, sampling or interpolation, reslicing of the images, determination of the maximum intensity projection, volume and surface rendering, and creation of movies using Amira's main module were all steps in the visualization process.
Quality Control
The quality of samples directly determines the success of fMOST imaging, so the sample should be selected strictly. A small piece of tissue was scraped off the surface of the tissue and made into HE sections for pathological examination. The sections were examined back-to-back by two professional and technical personnel of the department of pathology, and the typical stage II lung adenocarcinoma were confirmed as qualified samples.