Combining Confocal and Atomic Force Microscopy Using The MFP-3D-CF Combined Confocal and Atomic Force Microscopy System From Asylum Research

Topics Covered

Background
Features
Application Examples

 

Background

For years, biologists have turned to laser scanning confocal microscopy for 3D functional imaging within thick samples such as cells or tissues. AFM also uses a scanning process to form an image, but is not limited by the wavelength of light. Now you can bring these complementary technologies together with the MFP-3D-CF. This new system (Figure 1) combines the powerful features of the MFP-3D-BIO™ with your choice of a commercial confocal microscope. With the MFP-3D-CF, you can select a sample region based on its fluorescent characteristics, then zoom in for a high-resolution AFM scan; correlate topography with fluorescence; or mechanically stimulate your sample with the tip and measure an optical response. This powerful combination will bring new capabilities to your research.

Figure 1. The MFP-3D-CF integrates the MFP-3D, shown here with the transmitted light option, with the Olympus FluoView™ laser scanning confocal.

Features

  • Preferred platform is Olympus FV1000 with spectral detector.
  • Also compatible with Olympus FV300; Nikon C1; inquire regarding Zeiss, Leica.
  • Low-noise, highly linear closed loop scanner – essential for precise registry of data.
  • Infrared AFM source allows use of red fluorophores (Texas Red, Cy5). Blocking filter (included) prevents crosstalk from the AFM into confocal.
  • Laser safety interlock automatically shuts off confocal lasers when AFM head is lifted.
  • The isolation package includes acoustic hood with 30dB isolation and active vibration isolation platform.
  • Transmitted light option adds transmission channel to confocal scans; also provides illumination for optical phase contrast.
  • Extended 28µm Z-range head and BioHeater™ options recommended.
  • All the same key features found in our MFP-3D-BIO.

Application Examples

Figure 2 shows correlation of topography and fluorescence on a sample of multicolored beads. The RGB confocal image is displayed as color overlaid on the 3D- rendered topography. Beads are easily identified by their fluorescent labels. The AFM reveals features below the confocal resolution limit, such as very small spheres and a salt crystal (foreground).

Figure 2. Fluorescent microspheres on glass coverslip.

The confocal can directly image the cantilever and tip geometry (Figure 3). This shows a maximum projection along the Z axis and X axis of a volume data set containing a Si3N4 cantilever. The tip location is seen precisely.

Figure 3. Olympus TR800 cantilever.

In Figure 4, the confocal was used to engage the tip precisely on 50µm tall pollen grains. The AFM image shows fine surface detail, whereas the confocal image distinguishes the internal structure of different pollen species.

Figure 4. Mixed pollen grains.

Figure 5 illustrates AFM and confocal imaging of living cells. Because the transmitted light signal is not optically sectioned, the shadow of the tip is visible even when disengaged from the surface. Transmitted light makes it easy to align the AFM tip with the confocal for imaging of a particular cell.

Figure 5. Living cells imaged with Nikon C1 and MFP-3D-CF.

Figure 6 illustrates AFM and confocal imaging of bone-marrow-derived mast cells. This 3D image of mast cells combines two distinct yet correlated datasets. The AFM provides high-resolution surface information; the confocal shows the distribution of the fluorescent label throughout the cell.  The confocal data is overlaid on the rendered AFM topography. Note the four dark regions near the center left of the image; the upper two are beneath the cell surface, whereas the lower two are surface features visible in the topography.

Figure 6. Confocal data overlaid on rendered AFM topography of mast cells.

Source: Asylum Research
For more information on this source please visit Asylum Research

 

Date Added: Jul 26, 2006 | Updated: Jun 11, 2013
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