Image overlap and stitching using the IN Cell Analyzer 1000 and IN Cell Investigator

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1 GE Healthcare Application Note AA IN Cell Analyzer 1000 Image overlap and stitching using the IN Cell Analyzer 1000 and IN Cell Investigator Key Words: IN Cell Analyzer, IN Cell Investigator, image overlap, image stitching, whole well imaging, colony imaging, tissue imaging The ability to generate composite images of a user-defined area within a sample is useful for visualizing and analyzing areas of interest that span multiple fields of view. IN Cell Analyzer 1000 v3.5 acquisition software includes image overlap features that allow the operator to define the coverage and lay out of the sample area, position, overlap, and acquire a user-defined number of fields. Acquired overlapping fields can be stitched together using an image stitching stack transformation protocol within IN Cell Investigator Software v1.5 to create accurate composite images suitable for further analysis. The image overlap features are found within the Acquisition Protocol Editor of the software and facilitate finding areas of interest within a sample, large area imaging, and whole well imaging invaluable tools when studying pathology slides, tissue microarrays, bacterial and cellular colonies, and small organisms. Here, we describe the use of IN Cell Analyzer 1000 acquisition software v3.5 to acquire multiple overlapping images of a range of sample types including cell colonies, a tissue section, and a small organism. Materials Products used IN Cell Analyzer 1000 kinetic instrument IN Cell Investigator v1.5, single seat license IN Cell Investigator v1.5, additional seat license objective (4 NIKON PLANAPO) Microscope Slide Imaging Module EGFP-2 FYVE assay Screening Research month assay evaluation month assay evaluation non-profit research well MatriPlate microplate (0.72 mm glass bottom thickness) Other materials used NIH3T3 mouse embryo fibroblasts Neuro-2a mouse neuroblasts HEP G2 human hepatocytes CellTracker Green CMFDA Hoechst Retinoic acid Monoclonal Anti-α-tubulin clone DM1A Goat anti-mouse IgG (whole molecule) - FITC Other tissue culture reagents FluoCells prepared slide #4 mouse intestine section with Alexa Fluor 350 WGA, Alexa Fluor 568 phalloidin, and SYTOX green 96-well cell culture microplate, black, µclear Nunc culture dishes ( mm) ATCC, CRL-1658 ATCC, CCL-131 ECACC, Invitrogen, C7025 Invitrogen, H3570 Sigma, R2625 Sigma, T9026 Sigma, F2012 Sigma/Invitrogen/ATCC Invitrogen, F Greiner Bio One, Costar,

Sample preparation EGFP-2 FYVE EGFP-2 FYVE cells were cultured in DMEM with GlutaMAX supplemented with 10% FBS, 1% penicillin streptomycin, and 0.5 mg/ml of G418.

2 Sample preparation EGFP-2 FYVE EGFP-2 FYVE cells were cultured in DMEM with GlutaMAX supplemented with 10% FBS, 1% penicillin streptomycin, and 0.5 mg/ml of G418. Cells were trypsinized and seeded into a 96-well µclear plate at 6000 cells/well (200 µl) and into a 96-well MatriPlate microplate at cells/well (200 µl). Cells were cultured overnight at 37 C, 5% CO 2 prior to 4% formaldehyde fixation and counterstaining with 10 µm Hoechst Spheroid colonies HEP G2 cells were cultured in MEM supplemented with 10% FBS, 1% penicillin streptomycin, 1% L-glutamine, and 1% non-essential amino acids. Cells cultured on a T75 flask were labeled with 5 µm CellTracker Green for 30 min at 37ºC, 5% CO 2 prior to washing and recovery for 3 h. Cells were trypsinized, pelleted by centrifugation, and re-suspended at a density of cells/ml. 20 µl drops of cells at cells/ml (therefore 1000 cells per drop) were dispensed onto the inverted lid of a culture dish. The lid was re-positioned onto the base of the culture dish and the hanging drops cultured in a highly humidified environment at 37 C, 5% CO 2 for 3 days to allow the spheroid colonies to form. Individual spheroid colonies were picked using a blunted 200 µl pipette tip and transferred into wells of a 96-well MatriPlate microplate containing 100 µl of media and NIH3T3 cells (8000 cells/well). Cells and spheroid colonies were fixed with 2% formaldehyde and counterstained with 5 µm Hoechst Imaging on IN Cell analyzer 1000 acquisition software v Setup IN Cell Analyzer 1000 with the required objective and suitable dichroic/polychroic. 2. Launch IN Cell Analyzer 1000 acquisition software v Select Acquisition Protocol Manager to write a new protocol or edit an existing protocol. 4. Select Filter selection and display and ensure the correct excitation and emission settings are selected for imaging the sample. Optimize exposure times and HWAF offset. 5. Select Acquisition options (Fig 1), define the number of Fields to be acquired, and select Arrange fields to change the number of rows and columns displayed in the well layout. Neurite outgrowth (Neuro-2a) Neuro-2a cells were cultured in Vitacell minimum essential medium Eagles supplemented with 10% FBS, 1% penicillin streptomycin, and 1% L-glutamine. Cells were trypsinized and seeded into a 96-well µclear plate at 5000 cells/well (100 µl). Cells were cultured at 37 C, 5% CO 2 for 6 h prior to addition of 30 µm retinoic acid and continued overnight incubation. Cells were fixed with 4% formaldehyde prior to ICC (primary antibody: 1:1000 monoclonal anti-α-tubulin clone DM1A; secondary antibody: 1:200 goat anti-mouse IgG [whole molecule] FITC) and counterstaining with 5 µm Hoechst Small organism (Zebrafish) 96-well, clear plastic Costar plate (# 3599) containing up to two Zebrafish* per well, fixed by 4% paraformaldehyde. * Zebrafish samples kindly supplied by Phylonix. Fig 1. IN Cell Analyzer 1000 image overlap features within Acquisition Protocol Editor. Acquisition options enable the user to define the number of fields to be acquired and to edit the well layout. 6. Within Acquisition options select Edit to edit the well layout. The Tile Editor window will be displayed (Fig 2A). Multiple fields can be selected using Ctrl + click and Shift + click. Selected fields can be removed or dragged and positioned within the sample area. Right click on the Tile Editor window to display the editing menu (Fig 2B) where a number of further options are available. 7. From the editing menu, select Auto-coverage to facilitate whole well sample area coverage (if required). 8. From the editing menu, select Define-coverage (Fig 2C) and input the percent of tile size that Tiles may extend beyond the sample site and the percent of tile size that Tiles may overlap by (if required). Click OK. 9. The Tile Editor window will be updated. Click OK. 10. The well layout preview in Acquisition options will also be updated. 11. Click OK to save the new/edited protocol. 12. Run the acquisition protocol to acquire overlapping images AA 02/2009

(A) (B) (C) Fig 2. IN Cell Analyzer 1000 image overlap features within Acquisition Protocol Editor. (A) Tile Editor enables the positioning and coverage of fields to be acquired.

.. of the sample area (C) by defining the percent of tile size that Tiles may extend beyond the sample site and the percent of tile size that Tiles may overlap by.

overlapping either within or beyond the sample area, if required.

In each of these examples, the 4 objective was selected and six fields were acquired, overlapping by 15% of the tile size from wells of a 96-well µclear microplate and a 96-well MatriPlate

3 (A) (B) (C) Fig 2. IN Cell Analyzer 1000 image overlap features within Acquisition Protocol Editor. (A) Tile Editor enables the positioning and coverage of fields to be acquired. Right click on the Tile Editor window to display the editing menu (B) where the user has the option to Define Coverage... of the sample area (C) by defining the percent of tile size that Tiles may extend beyond the sample site and the percent of tile size that Tiles may overlap by. Results The sample format used in the acquisition protocol is firstly defined within Plate Manager (Application menu) and the appropriate sample format selected within the Well Plate section of Acquisition Protocol Editor. The well layout preview within Acquisition options is automatically updated enabling the user to define the number of fields to be acquired, field selection, positioning, and percent of tile size overlapping either within or beyond the sample area, if required. Acquisition of images that overlap by up to 50% of the tile size facilitate image stitching within IN Cell Investigator Software v1.5. Overlapping images acquired from within the boundary of the sample area are shown (Fig 3 and Fig 5). In each of these examples, the 4 objective was selected and six fields were acquired, overlapping by 15% of the tile size from wells of a 96-well µclear microplate and a 96-well MatriPlate microplate respectively. Please note in the Tile Editor the round well layout of the µclear microplate and the square well layout of the MatriPlate microplate. Resulting overlapping images were stitched together using an image stitching stack transformation protocol within IN Cell Investigator Software v1.5. Fig 3. Sample site coverage. 4 objective. Six fields overlapping by 15% of tile size. Round well of 96- well µclear microplate seeded with EGFP-2 FYVE cells (see sample preparation). Blue channel shown (D360_40 HQ535_50M). All images acquired within sample site AA 02/2009 3

Fig 4. Sample site coverage. 4 objective. Sixteen fields overlapping by 15% of tile size.

Round well of 96-well µclear microplate seeded with EGFP-2 FYVE cells (see Sample preparation). Blue channel shown (D360_40 HQ535_50M). Fig 5.

Square well of 96-well MatriPlate microplate seeded with EGFP-2 FYVE cells (see Sample preparation). Blue channel shown (D360_40 HQ535_50M).

Acquisition of images that extend beyond the sample site by up to 50% of the tile size are permitted within the software to facilitate whole

Figure 4 shows 16 fields acquired from the well of a 96-well µclear plate that are overlapping by 15% of the tile size and extend beyond the

Figure 6 shows 30 fields acquired from the well of a 96-well MatriPlate that are overlapping by 15% of the tile size and that extend beyond

4 Fig 4. Sample site coverage. 4 objective. Sixteen fields overlapping by 15% of tile size. Tiles may extend beyond the sample site by 20% of tile size. Round well of 96-well µclear microplate seeded with EGFP-2 FYVE cells (see Sample preparation). Blue channel shown (D360_40 HQ535_50M). Fig 5. Sample site coverage. 4 objective. Six fields overlapping by 15% of tile size. Square well of 96-well MatriPlate microplate seeded with EGFP-2 FYVE cells (see Sample preparation). Blue channel shown (D360_40 HQ535_50M). All images acquired within sample site. Acquisition of images that extend beyond the sample site by up to 50% of the tile size are permitted within the software to facilitate whole well imaging (Fig 4 and Fig 6). Figure 4 shows 16 fields acquired from the well of a 96-well µclear plate that are overlapping by 15% of the tile size and extend beyond the sample site by 20% of the tile size. Figure 6 shows 30 fields acquired from the well of a 96-well MatriPlate that are overlapping by 15% of the tile size and that extend beyond the sample site by 20% of the tile size. Resulting overlapping images were stitched together using an image stitching stack transformation protocol within IN Cell Investigator Software v1.5. In both cases, the entire well area has been imaged and images successfully stitched together. The image overlap feature is compatible with all objectives used on the IN Cell Analyzer 1000 (4, 10, 20, 40, and 60 ) and the maximum number of fields required to cover the whole sample area is automatically calculated by the software. The ability to acquire overlapping images when imaging and counting colonies within the sample area ensures AA 02/2009

Fig 6. Sample site coverage. 4 objective. Thirty fields overlapping by 15% of tile size.

Square well of 96-well MatriPlate microplate seeded with EGFP-2 FYVE cells (see Sample preparation).

CellTracker Green labeled HEP G2 mono-spheroid colonies picked from hanging drop cultures and transferred into wells containing NIH

4 objective selected, Sixteen fields acquired overlapping by 15% of tile size.

(B) Green channel images (HQ480_40 HQ535_50M). Fig 8.

5 (outlined in red) and area and count measures reported (population summary). colonies are not counted more than once.

images that extend beyond the sample site is advantageous and enables whole well imaging.

hanging drops (see Sample preparation), picked, and transferred into the well of a 96-well MatriPlate microplate containing NIH 3T3

5 Fig 6. Sample site coverage. 4 objective. Thirty fields overlapping by 15% of tile size. Tiles may extend beyond the sample site by 20% of tile size. Square well of 96-well MatriPlate microplate seeded with EGFP-2 FYVE cells (see Sample preparation). Blue channel shown (D360_40 HQ535_50M). (A) (B) Fig 7. CellTracker Green labeled HEP G2 mono-spheroid colonies picked from hanging drop cultures and transferred into wells containing NIH 3T3 cells cultured as monolayer on plate bottom. 96-well MatriPlate microplate. 4 objective selected, Sixteen fields acquired overlapping by 15% of tile size. Nine fields selected for stitching within IN Cell Investigator Software v1.5. (A) Blue channel images (D360_40 HQ535_50M). (B) Green channel images (HQ480_40 HQ535_50M). Fig 8. CellTracker Green labeled HEP G2 mono-spheroid colonies segmented using IN Cell Investigator Software v1.5 (outlined in red) and area and count measures reported (population summary). colonies are not counted more than once. Colonies of cells also have the tendency to gather at the edge of the well near the well wall, therefore having the ability to acquire images that extend beyond the sample site is advantageous and enables whole well imaging. To mimic cell colonies grown on a feeder cell layer, HEP G2 spheroid colonies labeled with CellTracker Green were generated from hanging drops (see Sample preparation), picked, and transferred into the well of a 96-well MatriPlate microplate containing NIH 3T3 cells. The colonies/cells were fixed and counterstained with Hoechst The 4 objective was selected and 16 fields were acquired, overlapping by 15% of the tile size in both the blue and green channel (Fig 7A and Fig 7B). Nine of the resulting overlapping images were selected and stitched together using an image stitching stack transformation protocol within IN Cell Investigator Software v1.5. Further analysis was performed within the software to segment the spheroid colonies and report count and area measures (Fig 8). The example image shown in Figure 8 from well C12 reported 12 colonies counted with an average area of µm AA 02/2009 5

To demonstrate the usefulness of the image overlap feature when imaging tissue/histology samples that span a number of fields, the 10 objective was used to acquire thirty fields overlapping by 15% of

Ten fields spanning the tissue section were then selected and stitched together using an image stitching stack transformation protocol within IN Cell Investigator Software v1.5.

6 To demonstrate the usefulness of the image overlap feature when imaging tissue/histology samples that span a number of fields, the 10 objective was used to acquire thirty fields overlapping by 15% of tile size in the red, green, and blue channels from a 16 µm section of mouse intestine on FluoCells prepared slide #4. Ten fields spanning the tissue section were then selected and stitched together using an image stitching stack transformation protocol within IN Cell Investigator Software v1.5. This resulted in a single image of the complete tissue section being generated and available for analysis (Fig 9). The accuracy of image overlap and stitching is essential for not only generating an accurate composite image but for ensuring accurate data generation from composite image analysis. To test this, we imaged Neuro-2a cells cultured in the well of a 96-well µclear microplate (see Sample preparation). The 4 objective was selected and six fields were acquired, overlapping by 15% of tile size in the green and blue channels. The resulting images were selected and stitched together using an image stitching stack transformation protocol within IN Cell Investigator Software v1.5 (Fig 10). Neurite extensions spanning multiple fields were accurately imaged with the aid of the image overlap tools and stitched. Further analysis was performed within IN Cell Investigator Software v1.5 to segment the nuclei, cell bodies, and neurite extensions of the cells (Fig 11). Important measures for neurite analysis were reported after analysis of the composite image. For example, population summary measures such as number of cells with branch points and average branch point count per cell (Fig 12A); and cell by cell measures such as branch point count, end node count, maximum neurite length, and neurite intensity/cell (Fig 12B). Zebrafish are a common and biologically relevant model organism that can be used as a preclinical model for drug screening, developmental biology, genetic screening, and environmental toxicology. Whole well imaging is an important feature for imaging entire model organisms such as Zebrafish. Zebrafish samples* were imaged within the wells of a 96-well microplate. Brightfield imaging was used to acquire twelve fields per well, overlapping by 5% of the tile size ensuring coverage of the whole well. As the resulting stitched image shows (Fig 13), whole Zebrafish appears complete and in the correct orientation. * Zebrafish samples kindly supplied by Phylonix. Fig 9. Image acquisition and stitching of FluoCells prepared slide #4. 16 µm cryostat mouse intestine section with Alexa Fluor 350 wheat germ agglutinin (used to stain the mucus of goblet cells). Alexa Fluor 568 phalloidin (used to stain the filamentous actin prevalent in the brush border) and SYTOX Green nucleic acid stain (used to stain the nuclei). Thirty fields overlapping by 15% of tile size were acquired using a 10 objective. Ten fields were selected for stitching within IN Cell Investigator Software v1.5. Stitched overlap image (fused) shown of blue channel images (D360_40 HQ460_40M), green channel images (HQ480_40 HQ535_50M), and red channel images (HQ565_30 HQ620_60M) AA 02/2009

µclear plate. Cells stimulated with 30 µm retinoic acid.

Green channel images (HQ480_40 HQ535_50M) shown.

analyzed using IN Cell Investigator Software v1.5.

7 Fig 10. Mouse neuroblastoma Neuro-2a cells cultured in well of a 96-well µclear plate. Cells stimulated with 30 µm retinoic acid. Hoechst nuclei stain, and FITC Ab labeled. 4 objective. Green channel images (HQ480_40 HQ535_50M) shown. Six fields selected for stitching within IN Cell Investigator Software v1.5. Fig 11. Mouse neuroblastoma Neuro-2a cells cultured in well of a 96-well µclear plate. Cells stimulated with 30 µm retinoic acid, Hoechst nuclei stain, and FITC Ab labeled. 4 objective. Image segmented and analyzed using IN Cell Investigator Software v1.5. Nuclei (blue bitmap), cell bodies (red bitmap), and neurites (green bitmap). (A) (B) Fig 12. (A) Population well summary data and (B) cell by cell data reported from the neurite analysis of stitched image shown in Fig AA 02/2009 7

Conclusions The ability to acquire overlapped images using IN Cell Analyzer 1000 acquisition software v3.5 enables the user to edit the well layout and define the coverage of the sample area.

The positioning of the overlapping fields is user-defined and accurate imaging facilitates image stitching within IN Cell Investigator Software v1.5 to generate composite images.

Excellent quality composite images formed by image stitching stack transformation in IN Cell Investigator Software v1.5 can be further analyzed to generate application-relevant data.

4 objective. Whole well brightfield image acquisition of twelve fields, overlapping by 5% of tile size. For local office contact information, visit www.gelifesciences.

8 Conclusions The ability to acquire overlapped images using IN Cell Analyzer 1000 acquisition software v3.5 enables the user to edit the well layout and define the coverage of the sample area. The image overlap feature is compatible with both microplate and slide sample formats, all objectives compatible with the IN Cell Analyzer 1000, and with multiple color images. The positioning of the overlapping fields is user-defined and accurate imaging facilitates image stitching within IN Cell Investigator Software v1.5 to generate composite images. Image overlap and stitching facilitates feature finding within the sample area, whole well imaging and analysis, colony imaging and analysis, and imaging of whole tissue sections. Excellent quality composite images formed by image stitching stack transformation in IN Cell Investigator Software v1.5 can be further analyzed to generate application-relevant data. Image flat field correction can be used with the image overlap feature, although this is not a requirement. Fig 13. Zebrafish samples imaged in 96-well, clear plastic Costar plate (# 3599). 4 objective. Whole well brightfield image acquisition of twelve fields, overlapping by 5% of tile size. For local office contact information, visit GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway, NJ USA GE, imagination at work, and GE monogram are trademarks of General Electric Company. MatriPlate is a trademark of GE Healthcare companies. The IN Cell Analyzer 1000 is the subject of US patent application number 10/514925, together with other granted and pending family members, in the name of GE Healthcare Niagara, Inc. The IN Cell Analyzer 1000 and associated analysis modules are sold under use licenses from Cellomics Inc. under US patent numbers US , , , , , , , , , , , , , , , ; Canadian patent numbers CA , , , ; Australian patent number AU ; European patent numbers EP , , , , , ; Japanese patent numbers JP , , and equivalent patents and patent applications in other countries. EGFP-2 FYVE domain cells/assay. This product is sold under license from Fisher BioImage ApS (formerly BioImage A/S) under patent numbers US , US , US , CA , EP , EP , JP and equivalent patents and patent applications in other countries. This product is sold under license from Invitrogen IP Holdings Inc (formerly Vertex Pharmaceuticals (San Diego) LLC, formerly Aurora Biosciences Corporation) under patent numbers US , US , US , US , US , US , US , US , US , US , US , US , EP , JP and equivalent patents and patent applications in other countries. This product is sold under license from Columbia University under patent numbers US and This product is sold under license from Fisher BioImage ApS (formerly BioImage A/S) under patent number US and equivalent patents and patent applications in other countries. This product is sold under license from University of Florida under patent numbers US , US , US , US and equivalent patents and patent applications in other countries. This product is the subject of patent numbers US , US and EP and equivalent patents and patent applications in other countries in the name of GE Healthcare Limited. The CMV promoter is covered under patent numbers US and and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA52242, USA. This product was developed in collaboration with Fisher BioImage ApS (formerly BioImage A/S). This product is sold under license from Fisher BioImage ApS (formerly BioImage A/S) under patent numbers US and EP and equivalent patents and patent applications in other countries. This product is sold under license from Cedars Sinai Medical Centre under patent number US and equivalent patents and patent applications in other countries. All third party trademarks are the property of their respective owners General Electric Company All rights reserved. First published Feb All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Bio-Sciences AB, Björkgatan 30, Uppsala, Sweden GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare Europe GmbH, Munzinger Strasse 5,, D Freiburg, Germany GE Healthcare Bio-Sciences KK, Sanken Bldg., , Hyakunincho, Shinjuku-ku, Tokyo , Japan AA 02/2009

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