analysis of phase-contrast images
GE Healthcare Life Sciences Application Note 28-9899-71AA
IN Cell Analyzer 2000
Monitoring label-free neurite growth in live cells: analysis of phase-contrast images Transmitted light imaging provides a label-free and non-toxic alternative to the use of fluorescent stains for monitoring live-cell differentiation over several days. In this study, IN Cell Analyzer 2000 was used to analyze brightfield and phasecontrast images for cell proliferation and neurite outgrowth in live mouse Neuro-2a cells treated with retinoic acid over a 48 h time course. The data show that IN Cell Analyzer 2000 enables live-cell imaging and analysis of neuronal cell differentiation and demonstrate the potential for its utility in drug discovery and understanding the mechanisms underlying neurite initiation and extension.
Introduction Neurite outgrowth plays a fundamental role in embryonic development, neuronal differentiation and nervous system function. The process is also critical in some neuropathological disorders as well as neuronal injury and regeneration. Transmitted light imaging allows label-free monitoring of cell health, movement, morphology, and growth and can be particularly beneficial for extended time-course studies where fluorescent markers could have a toxic effect. IN Cell Analyzer 2000 incorporates a range of modes to obtain high quality images, including brightfield, differential interface contrast (DIC), and phase-contrast. In this study, IN Cell Analyzer 2000 was used to capture transmitted light images of live mouse Neuro-2a cells differentiating on exposure to increasing concentrations of retinoic acid. Plates were incubated for 48 h with environmental control (37ºC, 5% CO2, and relative humidity), without removal from the instrument. The imaging field was set to the same location in the well for each image captured over the two-day time course. Phase-contrast images were also optimized with the phase-contrast and DIC transformation feature within IN Cell Investigator, and further analyzed to quantitate the number and length of neurites.
Methods Cell culture and treatment To induce neurite outgrowth, neuroblastoma Neuro-2a cells (ATCC) were incubated in 2% serum media containing retinoic acid* (Sigma) at concentrations ranging from 2 to 15 μM. Seeding density was ~ 2500 cells/well in μClear™ 96-well plates (Greiner Bio-One).
Neurite growth and image capture Plates were incubated on the IN Cell Analyzer 2000 for 48 h with full environmental control and sealed with a Breathe-Easy™ membrane (Fig 1). A Nikon™ 20×/0.45 NA objective was selected, along with the brightfield filter set Brightfield_DAPI 350/50x and 455/50m. A large-chip CCD camera (2048 × 2048 pixel array) was used, which captures approximately four-times the FOV of a standard chip camera. This allows more cells to be viewed in a single image, giving more statistically robust results in a single pass, as well as artifact-free detection and quantitation of large structures or rare events. * Retinoic acid is classified as harmful. Handle in accordance with MSDS and local laboratory safety guidelines.
focus plane is 4, a defocus distance of 2 means that images from planes 2 and 6 are selected as input images for transformation. Therefore a brightfield image stack with at least three z-sections must be acquired as an input for phasecontrast transformation. A new image is derived from these three input images and a new image stack generated.
Analysis protocol The strategy for analysis is to identify neurites by subtracting a binary image of cell bodies from a binary image of the entire cell, including neurites. IN Cell Developer Toolbox can be used to create an analysis routine based on this strategy using phase-contrast images. Macro functions are used to invert the phase-contrast image and subtract the segmentation overlays to generate the neurite-only segmentation. Segmentation bitmap overlays of Figure 2B images are shown in Figure 3 A.
Fig 1. Differentiating Neuro-2a cells imaged at 20× in brightfield using IN Cell Analyzer 2000. Mouse Neuro-2a neuroblastoma cells were seeded in a 96-well plate at 2500 cells per well in DMEM-F12/10% FBS (Invitrogen) and incubated overnight. Retinoic acid was added to 10 µM final concentration in low-serum medium (2%), and the plate incubated for two days on the IN Cell Analyzer 2000. The images show partial FOV in brightfield at time-points zero to 44 h after compound addition
Phase transformation Phase-contrast and DIC images are derived from a series of three brightfield images, one in-focus, one over-focus and one under-focus. They can be generated during image acquisition on the IN Cell Analyzer 2000, or for more flexibility, phase-contrast and DIC images can also be generated post-acquisition with the IN Cell Developer Toolbox phase-contrast and DIC transformation component. Example parameters and images are shown in Figure 2. The high pass filter and in-focus brightfield plane can be varied to generate optimum phase-contrast images. The high pass filter controls the amount of detail that is filtered from the image: as the filter setting increases, the lowest spatial frequencies are removed and the image becomes less quantitative and composed of more boundary or edge details. Defocus distance is the distance from the in-focus brightfield image and is given as the number of z-planes or sections. Using this parameter, images over-focus and under-focus are selected from the image stack and processed by the phase-contrast and DIC transformation algorithm. Values must be taken at equal distances from the in-focus image, for example, if the brightfield 2
28-9899-71 AA 01/2011
B.
Fig 2. (A) Phase-contrast and DIC transformation component of IN Cell Investigator Developer Toolbox, showing parameters for optimization. (B) Transmitted light images generated. ‘Phase-contrast‘ and ‘Common parameters‘ settings used: ‘Focus plane’ = 4, ‘Defocus distance’ = 1, and ‘High pass filter’ = 5; the output phase-contrast and DIC images are therefore generated from brightfield z-sections 3, 4, and 5.
Fig 3. Segmentation of phase-contrast images showing cell body and neurite bitmap overlays (both red). Neurites with length > 20 µm are shown in green, those in yellow are excluded from the data output
Data management
A.
The image stacks and analysis data files were imported into the IN Cell Miner software package, and each well annotated with compound and concentration (Fig 4). Sub-selections of data can be quickly and easily sorted, through filters and guided query options, before directly exporting to Spotfire™ DecisionSite™ analytics software for graphical output (Figs 5 and 6). A.
B.
B.
Fig 4. Plate maps from IN Cell Miner showing (A) Annotation of wells with compound concentration, and (B) selection of subset of wells treated with 10 μM retinoic acid, overlay values show number of cells with neurites for each well.
Fig 5. 3D scatter plot showing cell proliferation over a 48 h time-course and the percentage of cells with neurite growth. Each square represents sixteen replicates and the size of the square is related to the percentage of cells with neurite growth.
C.
Fig 6. Total neurite length plotted against time. (A) 48 h time-course with varying concentrations of retinoic acid from 0 to 15 µM. Each square represents sixteen replicate wells for each concentration of retinoic acid. (B) Total neurite length plotted against time, on a well-by-well basis for 10 µM retinoic acid treatment. Each square represents one well, with sixteen replicate wells per time-point. (C) Plot of individual well treated with 10 μM retinoic acid.
28-9899-71 AA 01/2011
3
Results
Ordering information
Using IN Cell Analyzer 2000, images were acquired from two neurite outgrowth assay plates and incubated on the IN Cell Analyzer 2000 with environmental control or a standard cell culture incubator. Cell and neurite growth were monitored for two days, analyzed with IN Cell Investigator, and the data output plotted in Spotfire DecisionSite analytics software. Although a decrease in the total number of cells was seen with increasing retinoic acid concentrations, a greater proportion of those cells produced neurites (Fig 5).
Product
In addition to the percentage of cells that have neurites, the number and length of neurites can also be quantitated with treatment. Figure 6 shows total neurite length in relation to time, with neurite length increasing with increasing concentrations of retinoic acid. These results show feasibility for monitoring livecell differentiation over several days, using analysis of brightfield and phase-contrast images. Data can be obtained for neurite outgrowth without the use of fluorescent stains. It demonstrates use of the optional environmental chamber module for the IN Cell Analyzer 2000, allowing the user to keep the plate in the instrument between image time points.
Quantity
Code No.
IN Cell Analyzer 2000 with large chip CCD camera
1
28-9535-10
IN Cell Analyzer 2000 with large chip CCD camera (United States only)
1
28-9672-09
IN Cell Investigator 1 Seat, Web download IN Cell Investigator Label-Free Plug-in
1 1
28-4089-71 28-9779-73
References 1.
2.
3. 4.
Graves, R., Benefits of using IN Cell Analyzer 2000 with a large CCD detector and image deconvolution: whole-well imaging capability and improved assay precision. Poster, CHI High-Content Analysis, Jan 12–15. 2010. Cuy, J.L., et al., High-Content Screening Assay for Quantitation of Neurite Outgrowth and Neurotoxicity. Poster presentation at CHI High-Content Analysis, Sept 21–23, 2009. Application note: Neurite outgrowth cell-by-cell analysis using the IN Cell Developer Toolbox, GE Healthcare, 14-0005-35, Edition AA (2005). Voigt, A. and Zintl, F. Effects of retinoic acid on proliferation, apoptosis, cytotoxicity, migration, and invasion of neuroblastoma cells. Medical and Pediatric Oncology 4, 205–213 (2003).
Summary and conclusions The study of neurite outgrowth is relevant to both drug discovery and elucidation of the mechanisms underlying neurite initiation and extension. IN Cell Analyzer 2000 provides a robust platform for highthroughput imaging of live neurite outgrowth assays. The methods are suitable for functional studies as well as screening applications. The resultant image stacks are fully compatible with IN Cell Investigator image analysis software and IN Cell Miner data management software. Image resolution with IN Cell Analyzer 2000 is of sufficient quality to allow live-cell imaging of neuronal cell differentiation and shows the potential for using brightfield image capture and analysis.
For local office contact information, visit www.gelifesciences.com/contact GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway, NJ 08855-1327 USA www.gelifesciences.com/incell
GE, imagination at work, and GE monogram are trademarks of General Electric Company. All third party trademarks are the property of their respective owners. The IN Cell Analyzer 2000 system is for research purposes only. It is not approved for diagnosis of disease in humans or animals. The IN Cell Analyzer 2000 and associated analysis modules are sold under use licenses from Cellomics Inc. under US patent numbers US 5989835, 6416959, 6573039, 6620591, 6671624, 6716588, 6727071, 6759206, 6875578, 6902883, 6917884, 6970789, 6986993, 7060445, 7085765, 7117098; Canadian patent numbers CA 2282658, 2328194, 2362117, 2381334; Australian patent number AU 730100; European patent numbers EP 0983498, 1095277, 1155304, 1203214, 1348124, 1368689; Japanese patent numbers JP 3466568, 3576491, 3683591 and equivalent patents and patent applications in other countries. © 2011 General Electric Company—All rights reserved. First published Jan. 2011 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare that supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. Any use of this software is subject to GE Healthcare Standard Software End-User License Agreement for Life Sciences Software Products. A copy of this Standard Software End-User License Agreement is available on request. Notice to purchaser The IN Cell Investigator/IN Cell Miner HCM are sold for use in a variety of research applications. The purchase of this product does not include a license under any patent or intellectual property to use IN Cell Investigator/IN Cell Miner HCM in any particular application. It is strongly recommended that the purchaser consider the need for a license to the intellectual property of others that may cover an intended use. By using this software, the purchaser acknowledges the above referenced license constraints and accepts responsibility for all patents that may apply in using IN Cell Investigator/IN Cell Miner HCM in any particular application. GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences AB, Björkgatan 30 751 84, Uppsala, Sweden GE Healthcare Europe GmbH, Munzinger Strasse 5, D-79111 Freiburg, Germany GE Healthcare Japan Corporation, Sanken Bldg. 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan 28-9899-71 AA 02/2011
IN Cell Analyzer 2000
Monitoring label-free neurite growth in live cells: analysis of phase-contrast images Transmitted light imaging provides a label-free and non-toxic alternative to the use of fluorescent stains for monitoring live-cell differentiation over several days. In this study, IN Cell Analyzer 2000 was used to analyze brightfield and phasecontrast images for cell proliferation and neurite outgrowth in live mouse Neuro-2a cells treated with retinoic acid over a 48 h time course. The data show that IN Cell Analyzer 2000 enables live-cell imaging and analysis of neuronal cell differentiation and demonstrate the potential for its utility in drug discovery and understanding the mechanisms underlying neurite initiation and extension.
Introduction Neurite outgrowth plays a fundamental role in embryonic development, neuronal differentiation and nervous system function. The process is also critical in some neuropathological disorders as well as neuronal injury and regeneration. Transmitted light imaging allows label-free monitoring of cell health, movement, morphology, and growth and can be particularly beneficial for extended time-course studies where fluorescent markers could have a toxic effect. IN Cell Analyzer 2000 incorporates a range of modes to obtain high quality images, including brightfield, differential interface contrast (DIC), and phase-contrast. In this study, IN Cell Analyzer 2000 was used to capture transmitted light images of live mouse Neuro-2a cells differentiating on exposure to increasing concentrations of retinoic acid. Plates were incubated for 48 h with environmental control (37ºC, 5% CO2, and relative humidity), without removal from the instrument. The imaging field was set to the same location in the well for each image captured over the two-day time course. Phase-contrast images were also optimized with the phase-contrast and DIC transformation feature within IN Cell Investigator, and further analyzed to quantitate the number and length of neurites.
Methods Cell culture and treatment To induce neurite outgrowth, neuroblastoma Neuro-2a cells (ATCC) were incubated in 2% serum media containing retinoic acid* (Sigma) at concentrations ranging from 2 to 15 μM. Seeding density was ~ 2500 cells/well in μClear™ 96-well plates (Greiner Bio-One).
Neurite growth and image capture Plates were incubated on the IN Cell Analyzer 2000 for 48 h with full environmental control and sealed with a Breathe-Easy™ membrane (Fig 1). A Nikon™ 20×/0.45 NA objective was selected, along with the brightfield filter set Brightfield_DAPI 350/50x and 455/50m. A large-chip CCD camera (2048 × 2048 pixel array) was used, which captures approximately four-times the FOV of a standard chip camera. This allows more cells to be viewed in a single image, giving more statistically robust results in a single pass, as well as artifact-free detection and quantitation of large structures or rare events. * Retinoic acid is classified as harmful. Handle in accordance with MSDS and local laboratory safety guidelines.
focus plane is 4, a defocus distance of 2 means that images from planes 2 and 6 are selected as input images for transformation. Therefore a brightfield image stack with at least three z-sections must be acquired as an input for phasecontrast transformation. A new image is derived from these three input images and a new image stack generated.
Analysis protocol The strategy for analysis is to identify neurites by subtracting a binary image of cell bodies from a binary image of the entire cell, including neurites. IN Cell Developer Toolbox can be used to create an analysis routine based on this strategy using phase-contrast images. Macro functions are used to invert the phase-contrast image and subtract the segmentation overlays to generate the neurite-only segmentation. Segmentation bitmap overlays of Figure 2B images are shown in Figure 3 A.
Fig 1. Differentiating Neuro-2a cells imaged at 20× in brightfield using IN Cell Analyzer 2000. Mouse Neuro-2a neuroblastoma cells were seeded in a 96-well plate at 2500 cells per well in DMEM-F12/10% FBS (Invitrogen) and incubated overnight. Retinoic acid was added to 10 µM final concentration in low-serum medium (2%), and the plate incubated for two days on the IN Cell Analyzer 2000. The images show partial FOV in brightfield at time-points zero to 44 h after compound addition
Phase transformation Phase-contrast and DIC images are derived from a series of three brightfield images, one in-focus, one over-focus and one under-focus. They can be generated during image acquisition on the IN Cell Analyzer 2000, or for more flexibility, phase-contrast and DIC images can also be generated post-acquisition with the IN Cell Developer Toolbox phase-contrast and DIC transformation component. Example parameters and images are shown in Figure 2. The high pass filter and in-focus brightfield plane can be varied to generate optimum phase-contrast images. The high pass filter controls the amount of detail that is filtered from the image: as the filter setting increases, the lowest spatial frequencies are removed and the image becomes less quantitative and composed of more boundary or edge details. Defocus distance is the distance from the in-focus brightfield image and is given as the number of z-planes or sections. Using this parameter, images over-focus and under-focus are selected from the image stack and processed by the phase-contrast and DIC transformation algorithm. Values must be taken at equal distances from the in-focus image, for example, if the brightfield 2
28-9899-71 AA 01/2011
B.
Fig 2. (A) Phase-contrast and DIC transformation component of IN Cell Investigator Developer Toolbox, showing parameters for optimization. (B) Transmitted light images generated. ‘Phase-contrast‘ and ‘Common parameters‘ settings used: ‘Focus plane’ = 4, ‘Defocus distance’ = 1, and ‘High pass filter’ = 5; the output phase-contrast and DIC images are therefore generated from brightfield z-sections 3, 4, and 5.
Fig 3. Segmentation of phase-contrast images showing cell body and neurite bitmap overlays (both red). Neurites with length > 20 µm are shown in green, those in yellow are excluded from the data output
Data management
A.
The image stacks and analysis data files were imported into the IN Cell Miner software package, and each well annotated with compound and concentration (Fig 4). Sub-selections of data can be quickly and easily sorted, through filters and guided query options, before directly exporting to Spotfire™ DecisionSite™ analytics software for graphical output (Figs 5 and 6). A.
B.
B.
Fig 4. Plate maps from IN Cell Miner showing (A) Annotation of wells with compound concentration, and (B) selection of subset of wells treated with 10 μM retinoic acid, overlay values show number of cells with neurites for each well.
Fig 5. 3D scatter plot showing cell proliferation over a 48 h time-course and the percentage of cells with neurite growth. Each square represents sixteen replicates and the size of the square is related to the percentage of cells with neurite growth.
C.
Fig 6. Total neurite length plotted against time. (A) 48 h time-course with varying concentrations of retinoic acid from 0 to 15 µM. Each square represents sixteen replicate wells for each concentration of retinoic acid. (B) Total neurite length plotted against time, on a well-by-well basis for 10 µM retinoic acid treatment. Each square represents one well, with sixteen replicate wells per time-point. (C) Plot of individual well treated with 10 μM retinoic acid.
28-9899-71 AA 01/2011
3
Results
Ordering information
Using IN Cell Analyzer 2000, images were acquired from two neurite outgrowth assay plates and incubated on the IN Cell Analyzer 2000 with environmental control or a standard cell culture incubator. Cell and neurite growth were monitored for two days, analyzed with IN Cell Investigator, and the data output plotted in Spotfire DecisionSite analytics software. Although a decrease in the total number of cells was seen with increasing retinoic acid concentrations, a greater proportion of those cells produced neurites (Fig 5).
Product
In addition to the percentage of cells that have neurites, the number and length of neurites can also be quantitated with treatment. Figure 6 shows total neurite length in relation to time, with neurite length increasing with increasing concentrations of retinoic acid. These results show feasibility for monitoring livecell differentiation over several days, using analysis of brightfield and phase-contrast images. Data can be obtained for neurite outgrowth without the use of fluorescent stains. It demonstrates use of the optional environmental chamber module for the IN Cell Analyzer 2000, allowing the user to keep the plate in the instrument between image time points.
Quantity
Code No.
IN Cell Analyzer 2000 with large chip CCD camera
1
28-9535-10
IN Cell Analyzer 2000 with large chip CCD camera (United States only)
1
28-9672-09
IN Cell Investigator 1 Seat, Web download IN Cell Investigator Label-Free Plug-in
1 1
28-4089-71 28-9779-73
References 1.
2.
3. 4.
Graves, R., Benefits of using IN Cell Analyzer 2000 with a large CCD detector and image deconvolution: whole-well imaging capability and improved assay precision. Poster, CHI High-Content Analysis, Jan 12–15. 2010. Cuy, J.L., et al., High-Content Screening Assay for Quantitation of Neurite Outgrowth and Neurotoxicity. Poster presentation at CHI High-Content Analysis, Sept 21–23, 2009. Application note: Neurite outgrowth cell-by-cell analysis using the IN Cell Developer Toolbox, GE Healthcare, 14-0005-35, Edition AA (2005). Voigt, A. and Zintl, F. Effects of retinoic acid on proliferation, apoptosis, cytotoxicity, migration, and invasion of neuroblastoma cells. Medical and Pediatric Oncology 4, 205–213 (2003).
Summary and conclusions The study of neurite outgrowth is relevant to both drug discovery and elucidation of the mechanisms underlying neurite initiation and extension. IN Cell Analyzer 2000 provides a robust platform for highthroughput imaging of live neurite outgrowth assays. The methods are suitable for functional studies as well as screening applications. The resultant image stacks are fully compatible with IN Cell Investigator image analysis software and IN Cell Miner data management software. Image resolution with IN Cell Analyzer 2000 is of sufficient quality to allow live-cell imaging of neuronal cell differentiation and shows the potential for using brightfield image capture and analysis.
For local office contact information, visit www.gelifesciences.com/contact GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway, NJ 08855-1327 USA www.gelifesciences.com/incell
GE, imagination at work, and GE monogram are trademarks of General Electric Company. All third party trademarks are the property of their respective owners. The IN Cell Analyzer 2000 system is for research purposes only. It is not approved for diagnosis of disease in humans or animals. The IN Cell Analyzer 2000 and associated analysis modules are sold under use licenses from Cellomics Inc. under US patent numbers US 5989835, 6416959, 6573039, 6620591, 6671624, 6716588, 6727071, 6759206, 6875578, 6902883, 6917884, 6970789, 6986993, 7060445, 7085765, 7117098; Canadian patent numbers CA 2282658, 2328194, 2362117, 2381334; Australian patent number AU 730100; European patent numbers EP 0983498, 1095277, 1155304, 1203214, 1348124, 1368689; Japanese patent numbers JP 3466568, 3576491, 3683591 and equivalent patents and patent applications in other countries. © 2011 General Electric Company—All rights reserved. First published Jan. 2011 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare that supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. Any use of this software is subject to GE Healthcare Standard Software End-User License Agreement for Life Sciences Software Products. A copy of this Standard Software End-User License Agreement is available on request. Notice to purchaser The IN Cell Investigator/IN Cell Miner HCM are sold for use in a variety of research applications. The purchase of this product does not include a license under any patent or intellectual property to use IN Cell Investigator/IN Cell Miner HCM in any particular application. It is strongly recommended that the purchaser consider the need for a license to the intellectual property of others that may cover an intended use. By using this software, the purchaser acknowledges the above referenced license constraints and accepts responsibility for all patents that may apply in using IN Cell Investigator/IN Cell Miner HCM in any particular application. GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences AB, Björkgatan 30 751 84, Uppsala, Sweden GE Healthcare Europe GmbH, Munzinger Strasse 5, D-79111 Freiburg, Germany GE Healthcare Japan Corporation, Sanken Bldg. 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan 28-9899-71 AA 02/2011
