Enhanced Visualization of Hematoxylin and Eosin Stained Pathological Characteristics by Phasor Approach

Abstract

The phasor approach to fluorescence lifetime imaging microscopy (FLIM) is used to identify different types of tissues from hematoxylin and eosin (H&E) stained basal cell carcinoma (BCC) sections. The results suggest that working directly on the phasor space with the clustering assignment achieves immunofluorescence like simultaneous five or six-color imaging by using multiplexed fluorescence lifetimes of H&E. The phase approach is of particular effectiveness for enhanced visualization of the abnormal morphology of a suspected nidus. Moreover, the phasor approach to H&E FLIM data can determine the actual paths or the infiltrating trajectories of basophils and immune cells associated with the preneoplastic or neoplastic skin lesions. The integration of the phasor approach with routine histology proved its available value for skin cancer prevention and early detection. We therefore believe that the phasor analysis of H&E tissue sections is an enhanced visualization tool with the potential to simplify the preparation process of special staining and serve as color contrast aided imaging in clinical pathological examination.

A simple method based on confocal microscopy and thick sections recognizes seven subphases in growth plate chondrocytes

“…Eosin has quite good penetration into thick tissue sections of growth plate cartilage and stains the cytoplasm of the chondrocytes providing a strong fluorescent signal that resisted solvent dehydration, epoxy resin embedding and heat polymerization. Eosin is usually not regarded as a fluorochrome but results in the present work shows its utility for fluorescence labelling of chondrocytes, and this agrees with some previous studies in which eosin staining and confocal microscopy were used to analyze the three-dimensional distribution of cytoplasmic constituents of protein nature 9-11 and also for tissue pathology diagnosis 12,13 . Although eosin possesses only 20% the fluorescence quantum yield of more conventional fluorophores, such as fluorescein and rhodamine, eosin conjugated to either an immunoglobulin G (IgG) or steptavidin has been also proposed as an alternative to fluorescein in immunofluorescence 14,15 .…”

Biophotonics in China

“…3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Furthermore, they developed a rapid fluorescence lifetime imaging system (AOD-FLIM) that is based on a pair of acousto-optic deflectors (AOD) for the random regions of interest (ROI) study in the sample [28]. Recently, they used phasor-FLIM to directly measure fluorescence lifetimes from H&E-stained sections and proposed for the first time to develop a further utilization of eosin for fluorescence lifetime encoded multitarget imaging [29,30]. They also proposed to use the coherent optical adaptive technique (COAT) to correct the distorted wavefront of a Stimulated Emission Depletion STED beam in a STED microscope so that the imaging performance of STED for thick biological samples has been improved [31].…”

Multispectral near infrared absorption imaging for histology of skin cancer

“…Our results show how to setup a multispectral NIR absorption imaging setup and use it for histology of skin cancer samples. In comparison to other methods our approach can be easily implemented into an existing microscope setup . Multispectral imaging results in large amounts of data and we show that even the simplest approach to use the spectral information (variance images) enabled us to distinguish tumor from nontumor regions.…”