Combining Atomic Force Microscopy (AFM) and fluorescence microscopy (FM) has long been of interest for biologists. These two types of microscopes individually provide unique information about samples.
Combining Atomic Force Microscopy and Fluorescence Microscopy
Fluorescence microscopy is typically used to detect specific molecules. However, this microscopy technique frequently has a number of drawbacks including limited spatial resolution and complex sample preparation. Another limitation of this microscopy is that the molecule must be bound to the fluorochrome, which in turn, can alter the state of the molecule.
On the other hand, AFM is a measurement made with a cantilever that is scanned cross the surface.
Figure 1. The MFP-3D -BIO allows simultaneous optical and AFM measurements on an inverted optical microsope, ideal for bioscience applications.
It provides three dimensional molecular resolution of biological molecules with no additional contrasting agent (such as fluorochrome, dyes, etc.) or compromise to the sample. In addition, AFM is also a higher resolution measurement that is non-fleeting.
Using these microscopy techniques simultaneously provides the user with high resolution imaging on specific molecules and also opens the door to a wide variety of research experiments. In this article, we will describe the instrumentation and setup, sample preparation, applications, and give experimental examples of the technique.
Successful simultaneous AFM and FM measurements require specific configurations of both instruments. First and foremost, the AFM must not interfere with FM measurement. Because most commercial AFMs use red laser diodes to illuminate and detect the deflection of the cantilever, the fluorescent wavelengths cannot be observed. However, the Asylum Research MFP-3D uses an infrared superluminescent diode with reduced emission at most fluorescent wavelengths. This allows viewing of both the fluorescence and AFM image (see Figure 2).
Figure 2. Fluorochrome Excitation (blue)/Emission (red) and MFP-3D SLD light wavelength.
In addition, both measurements have potentially conflicting spatial constraints. The FM microscope requires that the sample be close to the inverted microscope objectives. For the AFM, the cantilever must also reside close to the sample for imaging. The MFP-3D is designed specifically so that the sample can be accessed both by the inverted optical microscope and the scanning stage of the AFM.
The MFP-3D scanner rests on the top of an inverted microscope and provides almost unlimited optical access to the sample from below, making it easy to combine with fluorescence microscopes, including epifluorescence, confocal, TIRF and FRET.
Description and Setup
The MFP-3D-BIO AFM is the ideal instrument to perform the simultaneous measurements. This model has a base plate and an X-Y scanner that rest on top of an inverted optical microscope (manufactured by Olympus, Zeiss or Nikon). This stage and scanner arrangement allows both simple alignment of the AFM tip with the optical axis and submicron positioning of the sample in two dimensions. It is also compatible with any inverted optical objectives including high numerical aperture 100X and TIRF objectives. Figure 1 shows a setup of the MFP-3D-BIO mounted on a Nikon TE2000-E equipped for fluorescence.
During operation, the MFP-3D AFM infrared SLD emits between 840 and 880nm. Because most fluorophores have excitation and emission wavelengths below these values, there is no interference (see Figure 2).
The sample preparation for simultaneous measurements is similar to that used for AFM imaging. However, in fluorescence microscopy, light signals can be faint. Some common methods of increasing the efficiency of fluorescent emissions cannot be used in the MFP-3D because it would prevent the AFM probe from coming into contact with the sample ¨C for example, using a hydrophobic medium and replacing water with glycerol, or adding antioxidants, to increase the efficiency of the fluorescence.
These methods typically require that a coverslip be placed on top of the sample to protect and preserve it. Since such a coverslip prevents the AFM probe from interacting with the sample, this step in sample preparation must be omitted. Instead, preparation must be tailored to each sample and the amount of fluorescent dye has to be adapted to the new imaging conditions. For example, it might be necessary to increase the amount of fluorochrome that is going to bind to the specimen to obtain a larger signal for the FM measurement. Note, however, that since this may result in a loss of binding specificity of the dye, and a compromise may need to be found.
Figure 3 shows localization of actin filaments relative to the surface topography of single cells using a combined TIRFM/AFM. HeLa cells were grown on glass microscope coverslips, formaline-fixed, permeabilized with Triton X-100, labeled with TRITC-phalloidin, washed with acetone and dried. Imaging was carried out using a custom built combination of the MFP-3D AFM and a TIRF microscope constructed on an Olympus IX81 microscope. Topographic and fluorescence data were collected simultaneously. Topographic data were acquired in non-contact mode using an SiN cantilever (Olympus AC160). The image shows the TIRF data channel overlaid on the rendered AFM topography.
Figure 3. TIRF data overlaid on rendered AFM topography of HeLa Cells. Image courtesy Miklós S.Z. Kellermayer, Nanobiotechnology and Single-molecule Biophysics Group, University of Pécs, Hungary.
Figure 4 shows an example of an epithelial cell. These images were taken with an oil immersion 100X Nikon S fluor objective. The gray image is a brightfield image with some additional white light illuminating the sample region. This allows the cantilever (Olympus AC 240) to be visualized as well. The second image in the sequence is the fluorescence image. The third image is the AFM image of the same cell taken in AC mode. You can also see the zoom of the specific area within the cell region.
Figure 4. (A) optical phase contrast, (B) fluorescence, (C) AFM, and (D) combined AFM/fluorescence of fixed human lung fibroblasts, 60µm scan.
Figure 5 is an optical image of bacteria (Pseudomonas aeruginosa) showing a Green Fluorescent Protein (GFP) and corresponding force curves. The image was taken with a 10X objective and used a Nikon FITCHYQ cube fluorescent filter. White light from a fiber source was also used to illuminate the cantilever (visible on the left side of the image). The bacteria was first grown in a medium suspension. The suspension was then flushed over a glass slide for a few hours in order for the bacteria to grow a biofilm adherent to glass. The glass slide was then rinsed with the cell culture medium and imaged in the same medium. The cantilever used in this measurement is a 60¦Ìm long Olympus Bio-Lever (visible as a dark shadow in the brightfield image).
There are many application examples of AFM and epifluorescence ¨C Near Field Scanning Optical Microscope (NSOM), confocal, Total Internal Reflectance Fluorescence (TIRF), and Fluorescence Resonance Energy Transfer (FRET) methods. Researchers may be concerned with the risk of modifying the conformation of the specimen with the fluorochrome, thus slightly compromising the AFM as well. However, there are labeling techniques that minimize this problem. One technique is to insert a modified and fluorescent amino acid in a protein (such as inserting methylglyoxal in collagen). Many experiments do not present this issue at all, particularly when the AFM is used to mechanically trigger a specimen and not to image it.
Epifluorescence and AFM
- Labeling proteins or other components on a cell membrane. Epifluorescence is used to locate the membrane optically and AFM is used to obtain a high molecular resolution image (as shown in Figure 4) or measure mechanical properties of the samples (see Figure 5).
- Labeling certain lipids (like DPPC) to study the fluidity of cell membrane by correlating FM and AFM images.
- Studying the composition and distribution of lipids in vesicles through epifluorescence and using simultaneous AFM to obtain data about the mechanical and viscoelastic properties of the same vesicles.
- Studying the structure of chromophores, like carotenoids and chlorophyll, in relation to their optical behavior.
- Mechanically excite a cell or even some receptors at the surface of a cell membrane with the AFM and using the FM to optically follow a triggered cascade of biochemical reactions, like Ca2+ flow inside the cell.
Figure 5. (Top) The optical image was used to locate the bacteria and position the cantilever. Force measurements were then made on the bacteria to assess the hardness of the cell (bottom image). The sample is Pseudomonas aeruginosa showing GFP. In the force curves, the blue curve corresponds to the deflection of the cantilever when it is being withdrawn from the surface, and the red curve corresponds to the deflection as it is approaching the surface. Compare the force curve taken on the bacteria to that on a glass substrate. The most striking feature is the large adhesion (visible as a negative deflection on the blue retraction curve). Sample courtesy of Terry Beveridge, University of Guelph, Canada.
FRET and AFM
FRET is used for measuring proximity relations in proteins, nucleic acids, and membranes. Using a FRET method, it is possible to study the dynamic of changes in conformation like DNA, or the molecular interaction between two entities. AFM can be used simultaneously to visualize these events.
NSOM and AFM
Simultaneous AFM and NSOM imaging of cells permit topographic imaging and the determination of the elastic properties of the membrane.
TIRF and AFM
This technique can be used to study the response of cells to localized forces applied via the cantilever on the top of the cells. More generally, it is used to manipulate cells on the nanometer level with the AFM probe used to follow the cell reaction with fluorescence.
The MFP-3D-BIO from Asylum Research is the instrument of choice for simultaneous AFM and fluorescence measurements. Its unique design allows these measurements to be made easily and precisely. By simply altering the normal AFM sample preparation, researchers can perform these measurements and broaden their experiments to a limitless number of bioscience applications.
This information has been sourced, reviewed and adapted from materials provided by Asylum Research - An Oxford Instruments Company.
For more information on this source, please visit Asylum Research - An Oxford Instruments Company.