scholarly journals Optimization of immunofluorescence methods by quantitative image analysis.

1996 ◽  
Vol 44 (9) ◽  
pp. 1043-1050 ◽  
Author(s):  
D E Mosedale ◽  
J C Metcalfe ◽  
D J Grainger
Keyword(s):  
Image Analysis ◽  
Immunofluorescent Staining ◽  
Image Analysis System ◽  
Antigen Concentration ◽  
Specific Staining ◽  
Quantitative Image ◽  
Nonspecific Staining ◽  
Analysis System ◽  
Purified Antigen ◽  
Analysis Protocol

There is a growing trend towards the objective quantification of immunohistochemical staining. However, quantification has not been used previously to optimize the original published immunohistochemical methods. We present a quantitative method for analyzing immunofluorescence staining employing the Applied Imaging MAGISCAN image analysis system, which has then been used to optimize major aspects of the standard immunofluorescent staining protocols. The optimization process resulted in a method that increased specific staining up to fivefold over typical published protocols, with no increase in nonspecific staining. The method is extremely reproducible. For slides stained by a single experimenter in one batch on one day, the coefficient of variation between replicate means is 1.2%. The image analysis protocol gave a linear response with increasing antigen concentration, as determined by using purified antigen dried onto slides. The revisions to the standard protocol presented here can also be applied to nonquantitative staining. It will help users of immunofluorescence to maximize their staining and may enable the detection of previously undetected antigens.

scholarly journals Quantitative immunocytochemistry of hypothalamic and pituitary hormones: validation of an automated, computerized image analysis system.

1985 ◽  
Vol 33 (1) ◽  
pp. 11-20 ◽  
Author(s):  
D S Gross ◽  
J M Rothfeld
Keyword(s):  
Image Analysis ◽  
Detection Threshold ◽  
Limiting Factor ◽  
Pituitary Hormone ◽  
Image Analysis System ◽  
Specific Staining ◽  
Computerized Image Analysis ◽  
Hormone Levels ◽  
Quantitative Immunocytochemistry ◽  
Analysis System

A limiting factor in the use of immunocytochemistry in experimental endocrine studies has been the lack of a suitable procedure for quantification of immunoreactive hormones. The objective of the present study was the development of an automated, computerized image analysis system adapted to the quantitative analysis of light microscopic immunocytochemical reaction product. Reaction conditions that result in optimum, standardized, and quantitatively linear development of reaction deposit are described for H2O2 and diaminobenzidine concentrations, antiserum dilutions, and substrate incubation times. In addition, evaluation techniques, including the use of a standard control section to monitor variance and incorporate it into the statistical analysis of the results are documented. For each of the reaction variables, the immunostaining was linear over the range of specific staining. When the optimum conditions were exceeded, marked over-estimations of hormone levels occurred due to the detection of nonspecific background features reaching the detection threshold. Application of this quantitative immunocytochemical (QICC) method to the analysis of variations in hypothalamic and pituitary hormone levels was validated by comparing values obtained with QICC to those with radioimmunoassay (RIA). The relative changes in both hypothalamic gonadotropin-releasing hormone and pituitary luteinizing hormone induced by manipulation of gonadal steroid levels, as measured by RIA and QICC, were highly correlated. Two-way analysis of variance revealed that the two techniques were not significantly different in their detection of changes in either hormone. Thus, under optimally defined conditions, quantitative immunocytochemistry using computerized image analysis has been validated for the accurate measurement of pituitary and brain hormones in precise regions.

Chemical Classification and Particle Sizing of Foundry Sands

1985 ◽  
Vol 43 ◽  
pp. 388-389
Author(s):  
D.S. DeMiglio
Keyword(s):  
Image Analysis ◽  
Energy Dispersive Spectrometer ◽  
Image Analysis System ◽  
Representative Sampling ◽  
Foundry Sands ◽  
Large Atomic Number ◽  
Backscattered Electron ◽  
Sand Particles ◽  
Analysis System ◽  
Reclamation System

Much progress has been made in recent years towards the development of closed-loop foundry sand reclamation systems. However, virtually all work to date has determined the effectiveness of these systems to remove surface clay and metal oxide scales by a qualitative inspection of a representative sampling of sand particles. In this investigation, particles from a series of foundry sands were sized and chemically classified by a Lemont image analysis system (which was interfaced with an SEM and an X-ray energy dispersive spectrometer) in order to statistically document the effectiveness of a reclamation system developed by The Pangborn Company - a subsidiary of SOHIO.The following samples were submitted: unreclaimed sand; calcined sand; calcined & mechanically scrubbed sand and unused sand. Prior to analysis, each sample was sprinkled onto a carbon mount and coated with an evaporated film of carbon. A backscattered electron photomicrograph of a field of scale-covered particles is shown in Figure 1. Due to a large atomic number difference between sand particles and the carbon mount, the backscattered electron signal was used for image analysis since it had a uniform contrast over the shape of each particle.

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