Dark pixel intensity determination and its applications in normalizing different exposure time and autofluorescence removal
- 22 December 2011
- journal article
- Published by Wiley in Journal of Microscopy
- Vol. 246 (1), 1-10
- https://doi.org/10.1111/j.1365-2818.2011.03581.x
Abstract
The purpose of this study is to investigate how to scale pixel intensity acquired from one exposure time to another. This is required when comparing grayscale images acquired at different exposure times and other image processing such as autofluorescence removal. Pixel intensity is linear to exposure time as long as images are acquired at the linear range of a camera, but importantly there exists an intercept, which is set by the camera. We termed this intercept as dark pixel intensity, as it is the pixel intensity under conditions of no light and zero exposure time. Dark pixel intensity is determined by camera's readout noise (electron/pixel), gain, and DC offset. Knowing dark pixel intensity, image acquired from one exposure time can be linearly scaled to an image at a different exposure time. Dark pixel intensity can be directly measured by obtaining an image at no light and zero (or minimum) exposure time. It can be also indirectly calculated by capturing images at a series of exposure times. Finally, the prestained and poststained images were acquired at their optimal exposures and autofluorescence was completely removed by normalizing images with the exposure time ratio and dark pixel intensity followed by subtraction.Keywords
This publication has 17 references indexed in Scilit:
- Metadata matters: access to image data in the real worldThe Journal of cell biology, 2010
- Quantitative analysis of estrogen receptor heterogeneity in breast cancerLaboratory Investigation, 2007
- Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imagingJournal of Biomedical Optics, 2005
- Psoriatic Plaques Exhibit Red Autofluorescence that is Due to Protoporphyrin IXJournal of Investigative Dermatology, 1998
- A pyramid approach to subpixel registration based on intensityIEEE Transactions on Image Processing, 1998
- Advanced Maillard Reaction and Crosslinking of Corneal Collagen in DiabetesBiochemical and Biophysical Research Communications, 1995
- Elimination of autofluorescence in immunofluorescence microscopy with digital image processing.Journal of Histochemistry & Cytochemistry, 1995
- Symmetric phase-only matched filtering of Fourier-Mellin transforms for image registration and recognitionIEEE Transactions on Pattern Analysis and Machine Intelligence, 1994
- Argon ion laser-excited autofluorescence in normal and atherosclerotic aorta and coronary arteries: Morphologic studiesAmerican Heart Journal, 1989
- Autofluorescence of viable cultured mammalian cells.Journal of Histochemistry & Cytochemistry, 1979