Chrysalis: A new method for high-throughput histo-cytometry analysis of images and movies

Abstract

Advances in imaging have led to the development of powerful multispectral, quantitative imaging techniques, like histo-cytometry. The utility of this approach is limited, however, by the need for time consuming manual image analysis. We therefore developed the software Chrysalis and a group of Imaris Xtensions to automate this process. The resulting automation allowed for high-throughput histo-cytometry analysis of 3D confocal microscopy and two-photon time-lapse images of T cell-dendritic cell interactions in the spleen. It was also applied to epi-fluorescence images to quantify T cell localization within splenic tissue by using a 'signal absorption' strategy that avoids computationally intensive distance measurements. In summary, this image processing and analysis software makes histo-cytometry more useful for immunology applications by automating image analysis.

Image‐Based Cell Profiling Enables Quantitative Tissue Microscopy in Gastroenterology

“…Surprisingly, however, a population of highly juxtaposed, CD11c‐CD3 neighboring cells (i.e., region shown in Fig. 5D) that still identified positive in both gates after bystander removal were identified, indicating an interaction (17) relative to other cells, and suggesting a likelihood of cell–cell communication (Fig. 5F).…”

“…Finally, some of the original, pioneering, work in quantitative, flow cytometry‐type immunofluorescence analysis of tissues (e.g., histocytometry, Refs. 1–3,16,17) relies upon commercial software for implementation, limiting accessibility. Moreover, while the histocytometry approach utilizes both 2D and 3D confocal images, analyses have primarily focused on the spatial relationships of just the CD‐marker delineated cells.…”

“…There are also a number of philosophies as to what constitutes quantitative immunofluorescence microscopy, ranging from basic summation of fluorescence data across a given area, through integration within approximated cell‐objects, to accurate per‐cell identification and quantification (termed “cell segmentation”) (1,2,15). The latter has marked advantage as, within the limits of a microscope's resolution, it permits per‐cell quantification of information in a manner amenable to familiar, flow cytometry‐type gated analyses (1–3,16,17). It also allows distances to be established accurately, meaning that not only can cells be counted, but their content and spatial relationship to other cells or histological features can also be quantified (6).…”

Clinical and research applications of multiplexed immunohistochemistry and in situ hybridization

“…Currently, histocytometry/C e 3D approximates what future tissue‐based testing may look like: single‐platform, high‐plex, multi‐omic characterization of single cells with spatial and temporal context [30]. However, the need for confocal microscopy and computational power required may limit the implementation to smaller studies and specialized research centers.…”

The cyclic dinucleotide 2’3’-cGAMP induces a broad anti-bacterial and anti-viral response in the sea anemone Nematostella vectensis

“…Finally, samples were washed in wash buffer, stained with 1 µg/mL of DAPI for 10 minutes, washed again, and mounted in Vectashield HardSet Mounting medium and imaged on a Zeiss LSM 710 AxioObserver. Imaris 9.2 (Bitplane) was used to create 3D surfaces based on DAPI expression, and surface statistics were exported and analyzed in FlowJo (BD) to quantify nuclear nvNF-kB expression as previously described (63).…”