Collecting Z-Stack Image Sequences With the EVOS FL Auto Imaging System

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The Invitrogen™ EVOS™ FL Auto Imaging System is a fully automated, digital, inverted multi-channel fluorescence and transmitted-light imaging system with outstanding workflow efficiency. Designed to meet demanding requirements over a broad range of applications, it supports high-resolution mosaic tiling, multiple-position well scanning, cell counting with thresholding, and time-lapse studies.

Capturing images at different Z-planes can be a powerful tool in fluorescence microscopy. Depending on the sample type and prep used in the experiment performed, this capability can reveal conditions not seen with standard widefield imaging.

In this application note, a series of images were captured on the EVOS FL Auto Imaging System. Creating a Z-stack from these images allowed the observation of cellular cytoskeletal changes, which can be indicative of the loss of cell health.

Materials

• CellLight™ Tubulin-GFP, BacMam 2.0 (Cat. No. C10613)
• CellLight™ Mitochondria-RFP, BacMam 2.0 (Cat. No. C10601)
• NucBlue™ Live ReadyProbes™ Reagent (Cat. No. R37605)
• EVOS™ Light Cube, DAPI (Cat. No. AMEP4650)
• EVOS™ Light Cube, GFP (Cat. No. AMEP4651)
• EVOS™ Light Cube, RFP (Cat. No. AMEP4652)
• EVOS™ FL Auto Imaging System (Cat. No. AMAFD1000)

Methods

HeLa cells grown in MatTek 6-well glass bottom culture plates were transduced with CellLight Tubulin-GFP and CellLight Mitochondria-RFP overnight at 37°C. The following day, NucBlue Live reagent (2 drops/mL) was added to the cultures. Cells were then imaged on an EVOS FL Auto Imaging System with 100x oil immersion objective using the Z-stack function. The step size was set using the Nyquist formula and performed at 0.366 μm. All images were then processed using ImageJ software.

Results and discussion

Following transduction and staining of HeLa cells, images were taken using the Z-stack function on the EVOS FL Auto Imaging System software. Once the cell of interest was identified, a series of 41 images were automatically captured every 0.366 μm of the focal plane. Following capture, the 41 images were combined into a movie to allow visualization of the entire cell (Figure 1). Analysis of the composite overlay of step 16 of the Z-stack is indicative of a typical 3-color overlay captured by widefield fluorescent microscopy (Figure 2). In this focal plane, the HeLa cell appears healthy and normal. However, analyzing the movie of the Z-stack series from top to bottom shows the blebbing of tubulin-GFP, which can be an indication of the loss of cell health (Figure 3). These tubulin-GFP blebs are readily apparent in step 26 of the Z-stack image series and not readily apparent in previous Z-stack sections. Using the Z-stack capabilities of the EVOS FL Auto Imaging System uncovers changes in cellular morphology and possibly cell health not seen at a fixed Z-plane in standard widefield microscopy.

Figure 1. A series of 41 images taken every 0.366 μm of focal plane in HeLa cells using the Z-stack function of the EVOS FL Auto Imaging System. These images were combined to create a movie of the Z-stack sections through the cell.

Figure 2. Composite image of step 16 of the Z-stack, indicative of the typical 3-color overlay captured by widefield fluorescence microscopy. Note the intact structure of tubulin molecules (green).

Figure 3. Composite image of step 26 of the Z-stack. Note the blebbing of the tubulin seen in this image, indicative of the loss of cell health.