Z-Stacking of Single Plane Digital Widefield Images Incorporation of the Cytation™ 3 Cell Imaging Multi-Mode Reader and CombineZP Software to Create Deconvoluted, Stacked Images of 3D in vitro Cell Models

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Authored by: Brad Larson, Ellaine Abueg and Peter Banks, BioTek Instruments, Inc.; Grant Cameron, TAP Biosystems

Originally published as a white paper by BioTek Instruments, Inc., October 21, 2013.  Reprinted with permission.

Three-dimensional (3D) cellular models have the potential to become a fundamental research tool in cell biology because cell culture performed in this manner re-establishes cell-cell and cell-extracellular matrix interactions that mirror what’s seen in vivo. These reorganized cell structures present complications for optical microscopy due to their thickness in the z-axis. Here we present a method to capture and deconvolute multiple single z-plane images using digital widefield fluorescence microscopy.



Z-stacking (also known as focus stacking) is a digital image processing method which combines multiple images taken at different focal distances to provide a composite image with a greater depth of field (i.e. the thickness of the plane of focus) than any of the individual source images[1] [2]. It is particularly useful for capturing in-focus images of objects under high magnification, as depth of field (DOF) decreases with magnification primarily because microscope objectives with higher magnification have typically higher numerical apertures (NA). According to the Shillaber equation, DOF relates to NA for a given wavelength of light (λ) and medium refractive index (n):

BioTek Z-stackingEq1.jpg 

Table 1 illustrates this concept for a series of commercially available microscope objectives using 500 nm light and air as the medium (n = 1.00) between microscope objective and object.

BioTek Z-stackingTable1.jpg 
Table 1. Relationship between magnification, numerical aperture and depth of field.

Note that the dimensions of a typical mammalian cell (~50 μm) is only within the depth of field (in focus axially) using a 4x microscope objective. Magnification of 4x is inadequate to provide sub-cellular resolution in either axial or longitudinal axes, thus localization of structures of interest within the cell through its width requires use of higher magnification and means of removing out of focus objects.

This can be done using confocal microscopy where the field of view is restricted both axially and longitudinally, much like in a pin hole camera, such that in-focus "slices" of the object can be acquired and z-stacked to form a composite 3D image of the object. However, because the excitation light illuminates the entire structure, photobleaching and phototoxic effects extend to all planes. While the lack of longitudinal restriction seen in widefield microscopy helps to eliminate these complications, parts of the object will appear in-focus and parts out-of-focus. In this case, z-stacking is still possible, but requires the use of deconvolution, a technique to get rid of this out-of-focus information by applying a mathematical algorithm. This provides sharper images that can be combined to yield more realistic 3D impressions of the structure of interest. In this application note, we demonstrate this technique using the freeware CombineZP to perform z-stacking of images of HCT116 tumoroids in a 3D cell culture scaffold.

Materials and Methods



Colorectal carcinoma HCT116 cells (Catalog No. CCL-247) were obtained from ATCC (Manassas, VA). The cells were propagated in McCoy's 5A Medium (Catalog No. 16600) plus Fetal Bovine Serum, 10% (Catalog No. 10437) and Pen-Strep, 1X (Catalog No. 15140) from Life Technologies (Carlsbad, CA). The cells were plated at a final density of 2.5x105 cells/mL for 72 hours prior to performing the assay.

Fluorescent Probes

DAPI (Catalog No. D1306), Alexa Fluor® 488 phalloidin (Catalog No. A12379), and CellMask™ Orange plasma membrane stain (Catalog No. C10045) were purchased from Life Technologies (Carlsbad, CA).

RAFT™ Reagents and Plates

96-well RAFT Plate (Catalog No. A-0051), and 96-well Culture Plate (Catalog No. A-9WE2) are part of the 4 x 96 RAFT Plate Kit (Catalog No. 016-0R93). Collagen Solution (Product Code A-0052), 10X Minimum Essential Medium (Product Code A-0053), and RAFT Neutralising Solution (Product Code A-0054) are part of the RAFT Reagent Kit. All RAFT components were supplied by TAP Biosystems (Hertfordshire, UK).


Cytation™ 3 Cell Imaging Multi-Mode Reader

Cytation 3 is a cell imaging multi-mode microplate reader that combines automated digital microscopy and conventional microplate detection. The patent pending design provides phenotypic cellular information and well-based quantitative data. The microscopy module provides cellular visualization and analysis and the multi-mode detection system uses BioTek's patented Hybrid Technology™, containing both high sensitivity filter-based detection and a flexible monochromator system. Cytation 3 has temperature control to 45°C, shaking and CO2/O2 gas control. Gen5™ Data Analysis Software controls the system and provides powerful analysis for all measurement modes.

Manual imaging was performed using a 20x objective to analyze the tumoroid structure.

3D Cell Culture Components

RAFT™ 3D Cell Culture System

BioTek IncorporationofaNovel3DFig1.jpg 
Figure 1. Creation of 3-Dimensional Cell/Collagen Hydrogel using RAFT System. (A) Cell/collagen mix dispensed to wells of 96-well plate. (B) 96-well RAFT plate containing individual sterile absorbers. (C) Absorber insertion into plate well. (D) Absorption of medium, concentrating collagen and cells to in vivo strength. (E) Completion of absorption process creating 120 μm thick hydrogel. (F) Removal of absorber prior to dispense of fresh cell medium.

The RAFT (Real Architecture for 3D Tissue) cell culture technique developed by TAP Biosystems allows researchers to culture cell type(s) of their choice in an in vivo like collagen environment. The technology uses the most abundant matrix protein in the body, type I collagen. The RAFT process raises the collagen concentration to physiological levels quickly and reproducibly. It takes less than 1 hour to generate cell cultures which are ~120 μm thick, biomimetic, dimensionally stable and transparent with high cell viability.


3D Tumoroid Formation Process

Day 1

HCT116 cells were added manually to the prepared collagen solution. The cell suspension was then dispensed to the 96-well plate in a volume of 240 μL per well. The final cell concentration equaled 25,000 cells/well. The cell plate was then incubated at 37°C/5% CO2 for 15 minutes, followed by manual addition of the absorbers in the RAFT plate, and an additional 15 minute incubation at 37°C/5% CO2 during which the RAFT process increases the collagen density to a physiologically relevant strength. The absorbers were then removed and 100 μL of new medium was then added to the concentrated cell/collagen hydrogel. The plate was once again incubated at 37°C/5% CO2 for three days to allow the tumoroids to form.

Day 4

Following the incubation period, the spent medium was removed and the tumoroids were stained with the DAPI, Alexa Fluor® 488 phalloidin, and CellMask™ Orange plasma membrane fluorescent probes.

Creation of Z-Stacked Images

The creation of z-stacked images follows the workflow described in Figure 2. Essentially Gen5 is used to create a series of source images for each fluorophore desired (in this case 3 fluorophores). Then these images are ported to deconvolution software to create a z-stack for each color, then ported back to Gen5 to create a composite image of all z-stacked colors. Details of the process can be found in the appendix.

BioTek Z-stackingFig2.jpg 
Figure 2. Z-Stacked Image Workflow.

Results and Discussion

Single Plane and Z-Stacked Images

Upon visualization of the images displayed in Figure 3, it was apparent that z-stacking (Figure 3D) allowed the cells to be seen with more detail and greater clarity in each tumoroid structure compared to the single plane images (Figures 3A-C). Comparisons were also made between the final deconvoluted images created by CombineZP and ImageJ (not shown), which confirmed that the algorithms used by CombineZP were superior in this instance in providing a clearly defined image for use in cellular analysis.

BioTek Z-stackingFig3.jpg 
Figure 3. Single plane and z-stacked images of HCT116 tumoroids. (A-C) Multi-color overlaid images captured at multiple z-planes within the 120 μm hydrogel, and (D) final CombineZP stacked image. 22 images captured with each fluorescent probe were combined using the "Maximum Contrast" algorithm.

Cellular Analysis of 3-Dimensional Tumoroids using Z-Stacked Images

Cell counting of DAPI stained HCT116 nuclei was then conducted using Gen5™.

BioTek Z-stackingFig4.jpg 
Figure 4. HCT116 tumoroid cellular analysis. (A) Z-stacked image of DAPI stained nuclei, and (B) cell count performed using optimized analysis settings (328 cells counted).

The z-stacked image served to increase the definition of individual cell nuclei (Figure 4A). The incorporation of a smaller "rolling ball diameter" within the "Advanced Options" analysis settings tool of Gen5 (Figure 4B) also contributed to a more accurate cell count by providing a consistent background value for the entire analysis area.


The results shown here illustrate that the z-stacking procedure previously described allows widefield fluorescence microscopy and the Cytation 3 to be incorporated for imaging and analysis of 3D cellular structures.

Alexa Fluor® is a registered trademark of Molecular Probes Inc.


Image Capture

In the manual imaging "Image Capture" mode, the Z-Focus was used to determine the upper and lower boundaries of the 3D structure of interest. The Z-height was then moved to one boundary and an image captured. The Z-Focus was then moved 4 μm into the image and a second image captured. The process was repeated until four images had been taken.

BioTek Z-stackingFig5.jpg 
Figure 5. Manual image capture of tumoroid structures using DAPI channel.

"Review/Save" was then chosen to open the "Image Review" mode. This allows for detailed inspection of the image without continual excitation of the fluorescent probe.

BioTek Z-stackingFig6.jpg 
Figure 6. Review of all individual images captured.

Each individual image was saved as an uncompressed gray-scale 16-bit TIFF file.

BioTek Z-stackingFig7.jpg 
Figure 7. Save options for individual single plane images.

The process was repeated until the opposite boundary of the structure had been reached. This procedure was also completed for each of the three different fluorescent probes.

Creation and Deconvolution of Z-Stacked Image

Once the entire image set was saved, z-stacking was performed to create a single representative image for Gen5™ analysis. Freeware, such as CombineZP or ImageJ, was used for z-stacking the individual colors within the set. Each method is described as follows:


A single color image set was loaded into CombineZP, followed by an alignment and balancing of the frames. This software offered multiple projection algorithms, such as weighted average and maximum contrast, for combining images in a z-stack. The choice of algorithm was based upon the resolution of the final image which is automatically saved as an 8-bit TIFF file. Next, Adobe Photoshop was used to convert this file into a 16-bit TIFF file for Gen5 compatibility.


A single color image sequence was imported into ImageJ, followed by selecting "Image" in the toolbar, then "Stacks", and finally "Z Project". This allowed the user to select from several projection algorithms including max intensity and standard deviation. The algorithm selected was dependent on the resolution of the final image, which was saved as a Gen5-compatible 16-bit TIFF file in ImageJ.

Importation of Z-Stacked Images into Gen5

The final z-stacked images for each fluorescent probe were then imported back into the Gen5 Data Analysis Software.

BioTek Z-stackingFig8.jpg 
Figure 8. Image importation into Gen5 software.

The appropriate color was assigned to each individual gray-scale image.

BioTek Z-stackingFig9.jpg 
Figure 9. Assignment of red coloration to gray-scale image originally captured using Texas Red imaging filter cube.

Once the images had been successfully imported, "Review/Save" was again chosen to open the final full color image, and perform any necessary analysis of the tumoroids.

BioTek Z-stackingFig10.jpg 
Figure 10. Overlaid multi-color image for analysis.


  1. Johnson, D. How to Do Everything: Digital Camera [Online]; McGraw Hill Professional: New York, 2008; 336. a2cC&q=inauthor:%22Dave+Johnson%22&dq=inaut hor:%22Dave+Johnson%22&hl=en&sa=X&ei=ihdcU sj_HM_okAeuloHQBA&ved=0CGkQ6AEwCA (accessed October 14, 2013).
  2. Ray, S. F. Applied Photographic Optics, 3rd Edition; Focal Press: Oxford, 2002, 231-232.

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