Calibration of wide-field deconvolution microscopy for quantitative fluorescence imaging

Lee, Ji-Sook, Wee, Tse-Luen Erika, Brown, Claire M (April 2014) Calibration of wide-field deconvolution microscopy for quantitative fluorescence imaging. Journal of Biomolecular Techniques, 25 (1). pp. 31-40. ISSN 1524-0215

[thumbnail of Calibration of wide-field deconvolution microscopy for quantitative fluorescence imaging.pdf] PDF
Calibration of wide-field deconvolution microscopy for quantitative fluorescence imaging.pdf - Published Version
Available under License Creative Commons Attribution Non-commercial.

Download (722kB)
DOI: 10.7171/jbt.14-2501-002


Deconvolution enhances contrast in fluorescence microscopy images, especially in low-contrast, high-background wide-field microscope images, improving characterization of features within the sample. Deconvolution can also be combined with other imaging modalities, such as confocal microscopy, and most software programs seek to improve resolution as well as contrast. Quantitative image analyses require instrument calibration and with deconvolution, necessitate that this process itself preserves the relative quantitative relationships between fluorescence intensities. To ensure that the quantitative nature of the data remains unaltered, deconvolution algorithms need to be tested thoroughly. This study investigated whether the deconvolution algorithms in AutoQuant X3 preserve relative quantitative intensity data. InSpeck Green calibration microspheres were prepared for imaging, z-stacks were collected using a wide-field microscope, and the images were deconvolved using the iterative deconvolution algorithms with default settings. Afterwards, the mean intensities and volumes of microspheres in the original and the deconvolved images were measured. Deconvolved data sets showed higher average microsphere intensities and smaller volumes than the original wide-field data sets. In original and deconvolved data sets, intensity means showed linear relationships with the relative microsphere intensities given by the manufacturer. Importantly, upon normalization, the trend lines were found to have similar slopes. In original and deconvolved images, the volumes of the microspheres were quite uniform for all relative microsphere intensities. We were able to show that AutoQuant X3 deconvolution software data are quantitative. In general, the protocol presented can be used to calibrate any fluorescence microscope or image processing and analysis procedure.

Item Type: Paper
Subjects: Investigative techniques and equipment
Investigative techniques and equipment > microscopy > flourescence microscopy
Investigative techniques and equipment > microscopy
CSHL Authors:
Communities: CSHL labs > Spector lab
SWORD Depositor: CSHL Elements
Depositing User: CSHL Elements
Date: April 2014
Date Deposited: 04 Jan 2024 20:55
Last Modified: 04 Jan 2024 20:55
PMCID: PMC3942261
Related URLs:

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving