Detection of nuclei in 4D Nomarski DIC microscope images of early Caenorhabditis elegans embryos using local image entropy and object tracking

Abstract

BackgroundThe ability to detect nuclei in embryos is essential for studying the development of multicellular organisms. A system of automated nuclear detection has already been tested on a set of four-dimensional (4D) Nomarski differential interference contrast (DIC) microscope images of Caenorhabditis elegans embryos. However, the system needed laborious hand-tuning of its parameters every time a new image set was used. It could not detect nuclei in the process of cell division, and could detect nuclei only from the two- to eight-cell stages.ResultsWe developed a system that automates the detection of nuclei in a set of 4D DIC microscope images of C. elegans embryos. Local image entropy is used to produce regions of the images that have the image texture of the nucleus. From these regions, those that actually detect nuclei are manually selected at the first and last time points of the image set, and an object-tracking algorithm then selects regions that detect nuclei in between the first and last time points. The use of local image entropy makes the system applicable to multiple image sets without the need to change its parameter values. The use of an object-tracking algorithm enables the system to detect nuclei in the process of cell division. The system detected nuclei with high sensitivity and specificity from the one- to 24-cell stages.ConclusionA combination of local image entropy and an object-tracking algorithm enabled highly objective and productive detection of nuclei in a set of 4D DIC microscope images of C. elegans embryos. The system will facilitate genomic and computational analyses of C. elegans embryos.

Label-Free Dynamic Imaging of Chromatin in Live Cell Nuclei by High-Speed Scattering-Based Interference Microscopy

“…To confirm whether the V map provides useful information on nuclear organization, we imaged the nucleus with a conventional differential interference contrast (DIC) microscope that is often used to visualize the nucleolus (Figure 2d). 54 In addition, we Our data show that the dark spots in the V map with very low variance intensities match the areas of nucleoli. The nucleolus provides a low V intensity because it is tightly packed with proteins, DNA, and RNA, resulting in a small magnitude of molecular fluctuation.…”

“…The full V map without nuclear segmentation is displayed in Figure S2. To confirm whether the V map provides useful information on nuclear organization, we imaged the nucleus with a conventional differential interference contrast (DIC) microscope that is often used to visualize the nucleolus (Figure d) . In addition, we fluorescently labeled the DNA (with Hoechst) and nucleolus (with SYTO RNASelect) and imaged the nucleus under a confocal fluorescence microscope for comparison (Figure e,f).…”

Label-Free Dynamic Imaging of Chromatin in Live Cell Nuclei by High-Speed Scattering-Based Interference Microscopy

“…To confirm whether the V map provides useful information on nuclear organization, we imaged the nucleus with a conventional differential interference contrast (DIC) microscope that is often used to visualize the nucleolus (Figure 2d). 54 In addition, we Our data show that the dark spots in the V map with very low variance intensities match the areas of nucleoli. The nucleolus provides a low V intensity because it is tightly packed with proteins, DNA, and RNA, resulting in a small magnitude of molecular fluctuation.…”

“…The full V map without nuclear segmentation is displayed in Figure S2. To confirm whether the V map provides useful information on nuclear organization, we imaged the nucleus with a conventional differential interference contrast (DIC) microscope that is often used to visualize the nucleolus (Figure d) . In addition, we fluorescently labeled the DNA (with Hoechst) and nucleolus (with SYTO RNASelect) and imaged the nucleus under a confocal fluorescence microscope for comparison (Figure e,f).…”

Local cortical pulling-force repression switches centrosomal centration and posterior displacement in C. elegans

“…To clarify whether the polarized cortical pulling mechanism was active in the establishment stage of centering, we quantified the centering migration of the centrosomes. We used image processing that automatically recognizes the pronucleus in Nomarski differential interference contrast images of C. elegans embryos ( Hamahashi et al, 2005 ; Kimura and Onami, 2005 ) because the centrosomes associate with the male pronucleus at this stage. We found that centering migration of the pronucleus–centrosome complex, starting from the posterior pole, was faster in embryos in which the polarized cortical pulling was inactivated through RNAi of the goa-1 and gpa-16 genes ( G α (RNAi) ; Gotta and Ahringer, 2001 ) than in wild-type (WT) embryos (P = 0.004; Fig.…”

A hybrid blob-slice model for accurate and efficient detection of fluorescence labeled nuclei in 3D

“…As 3D maxima typically provide >95% accuracy in nuclear detection, they provide a powerful guide for a greedy slice grouping approach. The strategy of segmenting individual slices of volume data is not unknown [ 22 , 36 , 37 ], but our method is unique in its computational strategy and in being general, fully automatic and capable of good results in the crowded images typical of late embryogenesis. The algorithm remains efficient and fast enough for a typical computer to achieve real-time analysis of in vivo imaging of metazoan embryogenesis, in models ranging from C. elegans to mouse.…”

Label-Free Dynamic Imaging of Chromatin in Live Cell Nuclei by High-Speed Scattering-Based Interference Microscopy

“…To confirm whether the V map provides useful information on nuclear organization, we imaged the nucleus with a conventional differential interference contrast (DIC) microscope that is often used to visualize the nucleolus (Figure 2d). 54 In addition, we Our data show that the dark spots in the V map with very low variance intensities match the areas of nucleoli. The nucleolus provides a low V intensity because it is tightly packed with proteins, DNA, and RNA, resulting in a small magnitude of molecular fluctuation.…”

“…The full V map without nuclear segmentation is displayed in Figure S2. To confirm whether the V map provides useful information on nuclear organization, we imaged the nucleus with a conventional differential interference contrast (DIC) microscope that is often used to visualize the nucleolus (Figure d) . In addition, we fluorescently labeled the DNA (with Hoechst) and nucleolus (with SYTO RNASelect) and imaged the nucleus under a confocal fluorescence microscope for comparison (Figure e,f).…”

Local cortical pulling-force repression switches centrosomal centration and posterior displacement in C. elegans

“…To clarify whether the polarized cortical pulling mechanism was active in the establishment stage of centering, we quantified the centering migration of the centrosomes. We used image processing that automatically recognizes the pronucleus in Nomarski differential interference contrast images of C. elegans embryos ( Hamahashi et al, 2005 ; Kimura and Onami, 2005 ) because the centrosomes associate with the male pronucleus at this stage. We found that centering migration of the pronucleus–centrosome complex, starting from the posterior pole, was faster in embryos in which the polarized cortical pulling was inactivated through RNAi of the goa-1 and gpa-16 genes ( G α (RNAi) ; Gotta and Ahringer, 2001 ) than in wild-type (WT) embryos (P = 0.004; Fig.…”

A hybrid blob-slice model for accurate and efficient detection of fluorescence labeled nuclei in 3D

“…As 3D maxima typically provide >95% accuracy in nuclear detection, they provide a powerful guide for a greedy slice grouping approach. The strategy of segmenting individual slices of volume data is not unknown [ 22 , 36 , 37 ], but our method is unique in its computational strategy and in being general, fully automatic and capable of good results in the crowded images typical of late embryogenesis. The algorithm remains efficient and fast enough for a typical computer to achieve real-time analysis of in vivo imaging of metazoan embryogenesis, in models ranging from C. elegans to mouse.…”

Label-Free Dynamic Imaging of Chromatin in Live Cell Nuclei by High-Speed Scattering-Based Interference Microscopy

“…To confirm whether the V map provides useful information on nuclear organization, we imaged the nucleus with a conventional differential interference contrast (DIC) microscope that is often used to visualize the nucleolus (Figure 2d). 54 In addition, we Our data show that the dark spots in the V map with very low variance intensities match the areas of nucleoli. The nucleolus provides a low V intensity because it is tightly packed with proteins, DNA, and RNA, resulting in a small magnitude of molecular fluctuation.…”

“…The full V map without nuclear segmentation is displayed in Figure S2. To confirm whether the V map provides useful information on nuclear organization, we imaged the nucleus with a conventional differential interference contrast (DIC) microscope that is often used to visualize the nucleolus (Figure d) . In addition, we fluorescently labeled the DNA (with Hoechst) and nucleolus (with SYTO RNASelect) and imaged the nucleus under a confocal fluorescence microscope for comparison (Figure e,f).…”

Local cortical pulling-force repression switches centrosomal centration and posterior displacement in C. elegans

“…To clarify whether the polarized cortical pulling mechanism was active in the establishment stage of centering, we quantified the centering migration of the centrosomes. We used image processing that automatically recognizes the pronucleus in Nomarski differential interference contrast images of C. elegans embryos ( Hamahashi et al, 2005 ; Kimura and Onami, 2005 ) because the centrosomes associate with the male pronucleus at this stage. We found that centering migration of the pronucleus–centrosome complex, starting from the posterior pole, was faster in embryos in which the polarized cortical pulling was inactivated through RNAi of the goa-1 and gpa-16 genes ( G α (RNAi) ; Gotta and Ahringer, 2001 ) than in wild-type (WT) embryos (P = 0.004; Fig.…”

A hybrid blob-slice model for accurate and efficient detection of fluorescence labeled nuclei in 3D

“…As 3D maxima typically provide >95% accuracy in nuclear detection, they provide a powerful guide for a greedy slice grouping approach. The strategy of segmenting individual slices of volume data is not unknown [ 22 , 36 , 37 ], but our method is unique in its computational strategy and in being general, fully automatic and capable of good results in the crowded images typical of late embryogenesis. The algorithm remains efficient and fast enough for a typical computer to achieve real-time analysis of in vivo imaging of metazoan embryogenesis, in models ranging from C. elegans to mouse.…”