Multiresolution nondestructive 3D pathology of whole lymph nodes for breast cancer staging

A Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy

“…Many tissue samples are stored for a long time in formalin or are FFPE to avoid cell and tissue degradation for further analysis at any time. However, the compatibility of 3D histology was demonstrated with both fresh [ 15 ], formalin-fixed [ 16 ], and FFPE tissues [ 17 , 18 , 19 , 20 ]. In particular, iDISCO, or its variants, demonstrated high compatibility with both fresh and FFPE human tissues [ 19 , 21 , 22 , 23 ]; other methodologies working on fresh/formalin-fixed/FFPE include CUBIC [ 17 , 20 ], ACT-PRESTO [ 16 ], BABB [ 24 ], X-CLARITY [ 15 ], PACT [ 25 , 26 ], FACT [ 27 , 28 ], and MASH [ 29 , 30 ].…”

“…A similar approach, with a combination of 4′,6-diamidino-2-phenylindole (DAPI)/Eosin, was provided by Olson et al [ 24 ] to be applied to both fresh and 24 months-fixed samples of 1-mm thickness. Other commonly used nucleic acid stains are SYTO 16 [ 16 , 20 ], SYTOX Green [ 17 ], and TO-PRO-3 [ 17 , 21 , 23 ]. Eosin is the most frequently reported cytoplasmic stain.…”

“…Eosin is the most frequently reported cytoplasmic stain. However, Alexa 647 [ 17 ], Rhodamine [ 18 ], and autofluorescence enhancement with 5-sulfosalicylic acid dihydrate are also suitable for 3D histology [ 19 ].…”

“…Confocal and multiphoton microscopy offer high contrast and resolution [ 16 , 24 ], although the slow acquisition speed represents an intrinsic limitation for the acquisition of large-volume tissues. On the other hand, Light Sheet Microscopy (LSM) allows rapid 3D acquisition of cleared samples, and it is compatible with several H&E-analog staining methods [ 15 , 17 , 21 ]. Once the acquisition is finished, images are generally post-processed to provide 3D volumetric reconstructions and, eventually, pseudo-colored to mimic classical H&E colors [ 17 , 24 ].…”

“…On the other hand, Light Sheet Microscopy (LSM) allows rapid 3D acquisition of cleared samples, and it is compatible with several H&E-analog staining methods [ 15 , 17 , 21 ]. Once the acquisition is finished, images are generally post-processed to provide 3D volumetric reconstructions and, eventually, pseudo-colored to mimic classical H&E colors [ 17 , 24 ].…”