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First published online March 4, 2009
doi: 10.1242/10.1242/jcs.033837

Journal of Cell Science 122, 753-767 (2009)
Published by The Company of Biologists 2009
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Live-cell microscopy – tips and tools

Melanie M. Frigault1, Judith Lacoste2,3, Jody L. Swift4 and Claire M. Brown2,*

1 Molecular Oncology Group, McGill University Cancer Centre, Montreal, Canada
2 McGill University Life Sciences Complex Imaging Facility, Department of Biochemistry, Montreal, Canada
3 Cell Imaging and Analysis Network, Department of Biology, McGill University, Montreal, Canada
4 Department of Chemistry, McGill University, Montreal, Canada


Figure 1
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  		Fig. 1. Effects of the type of medium and the presence or absence of CO2 on cell proliferation. CHO-K1 paxillin-EGFP-expressing cells were plated on 2 µg/ml fibronectin-coated Lab-Tek II (Thermo Fisher Scientific, NY) eight-well chamber cover-glass slides 24 hours before imaging. Cells were maintained at 37°C in a Chamlide TC system (Live Cell Instruments, Seoul, Korea) with 5% humidified CO2. (A) Complete Dulbecco's modified Eagle's medium (DMEM) (10% FBS). (B) Serum-free DMEM. (C) Complete DMEM supplemented with 25 mM HEPES. (D) Leibowitz medium (CO2-independent) with 10% FBS (Gibco). (A-D) Images were collected on a WaveFX SD-CM (Quorum Technologies, Guelph, ON) mounted on a Leica (Wetzlar, Germany) DMI6000B motorized microscope with a 20x (0.7 NA) DIC oil-immersion lens, a custom-modified Yokogawa CSU10 head and a Synapse Diode Laser merge module. EGFP was excited with ~30% of a 25 mW, 491-nm line, using a custom 440/491/561/638 dichroic mirror and a 520/35-nm band-pass (BP) filter. Images were collected with 400-ms exposure times with a Hamamastu (Shizuoka, JP) C9100-12 EM-CCD camera. Maximum projections of seven images of z-planes 0.3 µm apart are shown for each time point. Brightness, gamma and contrast were adjusted to the same settings for all images within a given panel. (E) Plot of cell growth relative to the initial time point for the four different types of culture media; error bars represent s.d. for three image fields. Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. Cell proliferation and tracking can be measured from bright-field images such as DIC. CHO-K1 cells expressing paxillin-EGFP were plated on 35-mm glass-bottom dishes (World Precision Instruments, Sarasota, FL) coated with 2 µg/ml fibronectin (Sigma-Aldrich) 24 hours prior to imaging. Cells were maintained at 37°C with 5% humidified CO2 in a Solent Scientific (Segensworth, UK) incubation chamber. DIC images were collected on an Olympus (Tokyo, Japan) IX81 microscope with a 20x (0.4 NA) objective lens, using a Photometrics (Tuscon, AZ) CoolSNAP EZ camera with no binning. Raw images (A,A') were corrected for shading using the MetaMorph (MDS Analytical Technologies, Sunnyvale, CA) shading-correction algorithm and a shading image collected above the focal plane (B) to give corrected images (C,C'). Areas enclosed by red boxes in A and C are shown at higher magnification in A' and C', respectively. Cells were imaged every 5 minutes for 15 hours with a 20x (0.4 NA) objective (D) with 1-second exposure with the halogen lamp at low intensity. Cell numbers were counted manually for cell-proliferation measurements (E), and the manual cell-tracking feature in MetaMorph was used to generate cell tracks for the Rose plot (F). Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Loss of light across DIC optics and WFM plus deconvolution increases S/N and resolution. Images of CHO-K1 cells prepared and imaged as in Fig. 2. Fluorescence images of paxillin-EGFP-expressing CHO cells without (A) and with (B) DIC optics (prism and analyzer) in place. Using DIC optics decreases the cellular intensity (i) by 72%, from 298 to 83. (C) The same cells labeled with the nuclear dye Draq5 (0.5 µM) were imaged using 6% lamp power. Using a 0.2-µm step size, 241 z-planes were collected with 2x2 pixel binning and a 1-second exposure time using a custom triple cube for EGFP-mCherry-Cy5 (Chroma Technology). EGFP was imaged with a 480/20 excitation filter and a 515/30 emission filter, whereas Draq5 was imaged with a 630/30 excitation filter and 685/70 emission filter. Maximum-intensity projections of the raw wide-field images are shown for the central 21 image planes, and contrast, gamma and brightness were adjusted to see the intensity of labeling. (C,D) For intensity comparisons, the same projected images of the raw data are shown (C') on the same display scale as the deconvolved images (D), but are a bit difficult to see. (D) The native.stk MetaMorph-format files were transferred into the Autoquant X2 deconvolution software (http://www.mediacy.com/index.aspx?page=Home). Each file was subjected to a 20-iteration deconvolution using the `adaptive blind' deconvolution algorithm starting with the theoretical point spread function. Following this, the deconvolved files were imported directly into the Imaris 6.1.5, 3D/4D Image Analysis software (www.bitplane.com). Rendered 3D iso-surface plots are shown (C,D). Scale bars: 10 µm.

 

Figure 4
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  		Fig. 4. Lamp photobleaching, image segmentation by absence of fluorescence and CLSM of living cells. Images of CHO-K1 cells prepared and imaged as in Fig. 2. Excitation was from a 100 W mercury lamp using a custom EGFP filter cube for both WFM and total internal reflection fluorescence microscopy (TIRFM) (Chroma Technology, Rockingham, VT, hq480/20x, z488rdc, hq525/50m). Cells were found by using DIC optics. Images using an Olympus 60x, 1.45 NA oil-immersion lens from a time series with 500-ms exposure time using either 100% (A) or 6% (C) power from the lamp. (B) Decrease in cellular intensity for three separate image series measured using a custom journal written for MetaMorph, which measures the intensity of all of the cells in the field of view over time. Error bars are s.d. for the three experiments. (D) Image of paxillin-EGFP expression, which is absent from the nucleus, smoothed using a low-pass filter to remove noise. (E) Image in D inverted to show high intensities and absence of nuclear labeling. (F) A morphology filter was applied using MetaMorph followed by an integrated morphometry analysis (IMA) to select and fill only the large holes. A red mask of the regions of interest detected by the IMA analysis was overlaid on the raw image that is shown in D. Bright focal adhesions below the nucleus can cause some underestimates of the nuclear area (cell on the far left near the bottom of the image). Note that all display properties (brightness, gamma, contrast) have the same settings for images within a panel. (G,H) Confocal images were collected on a Zeiss (Jena, Germany) LSM510 confocal microscope using a 63x Plan-Apochromat 1.4 NA oil-immersion lens. Cells were kept on the stage at 37°C with a Zeiss incubation chamber equipped with a gas mixer and 5% humidified CO2. Resolution was at zoom one with 0.14 µm in x and y, and the pinhole was set to 194 µm or ~2.7 Airy units. Six z-axis image plans were collected at 0.5-µm separation every 2.5 minutes using 2% of the 488-nm laser (30 mW Ar at 6.0 A current) and a 505 LP emission filter. Two representative cells migrated well under this level of laser-light exposure. A median filter was applied to the images to reduce noise. Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. SD-CM and total internal reflection fluorescence microscopy (TIRFM). MDCK cells expressing EGFP-Gab1 were seeded 1 day prior to imaging on a 35-mm glass-bottom dish (MatTek Corp.) in complete DMEM. Cells were imaged live by SD-CM as in Fig. 1 with a 63x oil-immersion objective. Images were acquired in z-stacks of 28 planes at 0.3-µm intervals with 400-ms exposure times every 20 seconds over a period of 30 minutes. (A) Images at the indicated time points of one z-plane are shown. (B) Percentage intensity of the two cells shown in A over time. (C,D) Cells were stained with the nuclear dye Draq5 (0.5 µM) and a z-stack of 61 planes at 0.2 µm was acquired 15 minutes after treatment with hepatocyte growth factor (HGF; 100 ng/ml). Exposure times were 400 ms and camera gain was set to 203-243. Draq5 was imaged using 69% of the 30 mW, 638-nm laser line, whereas EGFP-Gab1 was imaged using 63% of the 25 mW, 491-nm laser line. The raw data (C) and deconvolved data (D) are shown as maximum projections and 3D rendered iso-surfaces (Bitplane). Deconvolution was performed as in Fig. 3. (E) Selected 3D rendered iso-surface views are shown and can be viewed in supplementary material Movie 11. (F) CHO-K1 cells expressing paxillin-EGFP were imaged as in Fig. 2. An Olympus TIRF illumination system was used and lasers were attenuated with an AOTF (Prairie Technologies, Middleton, WI). EGFP was excited with ~3% of the 488-nm laser line of a 200-mW Ar laser and Draq5 was excited with ~50% of a 5-mW 633-nm laser. A custom triple dichroic mirror and triple BP emission filter were used (Chroma Technology). Images were collected with a 1-second exposure with no camera binning. The laser was tuned to go straight through the sample for the wide-field images (F) and at the crucial angle for TIRF images (G). Color overlays of WFM and TIRFM images were done using MetaMorph. Images were filtered with a median filter to reduce noise. (H) Intensity profile of paxillin-EGFP signal for five parallel lines averaged from the wide-field image (red line) and the TIRF image (blue line) showing the increase in signal versus background for the TIRF image. (I) Time-lapse images of paxillin-EGFP expressed in CHO-K1 cells, showing a cell rounding up and disappearing from the TIRF field during cell division (panel 4). All display properties (brightness, gamma, contrast) have the same settings for images within a panel. Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. Suggestions for selection of imaging platform on the basis of sample type, requirement for 3D imaging, and the speed of the dynamics under study. TLM, transmission light microscopy.

 

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© The Company of Biologists Ltd 2009