Removal of out-of-plane fluorescence for single cell visualization and quantification in cryo-imaging.
We developed a cryo-imaging system, which alternates between sectioning (10-40 microm) and imaging bright field and fluorescence block-face image volumes with micron-scale-resolution. For applications requiring single-cell detection of fluorescently labeled cells anywhere in a mouse, we are developing software for reduction of out-of-plane fluorescence. In mouse experiments, we imaged GFP-labeled cancer and stem cells, and cell-sized fluorescent microspheres. To remove out-of-plane fluorescence, we used a simplified model of light-tissue interaction whereby the next-image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing an objective function on subtracted images. Tissue-specific attenuation parameters [micro(T): heart (267 +/- 47.6 cm(-1)), liver (218 +/- 27.1 cm(-1)), brain (161 +/- 27.4 cm(-1))] were found to be within the range of estimates in the literature. "Next-image" processing removed out-of-plane fluorescence equally well across multiple tissues (brain, kidney, liver, etc.), and analysis of 200 microsphere images gave 97 +/- 2% reduction of out-of-plane fluorescence. Next-image processing greatly improved axial-resolution, enabled high quality 3D volume renderings, and improved automated enumeration of single cells by up to 24%. The method has been used to identify metastatic cancer sites, determine homing of stem cells to injury sites, and show microsphere distribution correlated with blood flow patterns.
Steyer
Grant J
GJ
Department of Biomedical Engineering, Case Western Reserve University, 10900 Euclid Avenue, Wickenden Building, Room 403, Cleveland, OH 44106, USA.
Roy
Debashish
D
Salvado
Olivier
O
Stone
Meredith E
ME
Wilson
David L
DL
D000818:Animals; D018827:Carcinoma, Lewis Lung / Q000473:pathology*; D045744:Cell Line, Tumor; D005615:Freezing; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation* / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D008863:Microspheres; D009362:Neoplasm Metastasis; D013234:Stem Cells / Q000473:pathology*; D013978:Tibial Fractures / Q000473:pathology*