Atomic force microscopy sees a bright future with simultaneous fluorescence imaging

Researchers modified a commercially available atomic force microscope (AFM) to enable simultaneous fluorescence and AFM imaging. Atomic force microscopy utilizes an atomically sharp tip to map the surface of samples, while also allowing site-specific mechanical measurements, such as sample stiffness and adhesive forces. Being able to detect fluorescence emission alongside AFM data is useful in biological imaging, where the fluorescent labeling of samples allows locations of structures of interest to be determined. The mechanical properties of these structures can then immediately be measured by the AFM-tip to track changes over time.

In Review of Scientific Instruments, authors reported creating the instrument by first mounting an AFM stage onto an inverted optical microscope and illuminating the sample from underneath with a laser beam. By introducing a galvanometer into the beam path, they were then able to control the incident position of the laser beam at the sample and bring it into alignment with the AFM-tip above. They then developed two computer programs, one manual and one automated, which correlate features from both the AFM and fluorescence images. Information about the current offset between the two images was used to induce a response in the instrument to maintain alignment between the laser beam and the AFM-tip, thereby allowing simultaneous imaging to occur.

Their technique demonstrated its capabilities using a variety of samples, including fluorescent polymers and fluorescently labeled biological samples. While both versions of the program were able to align the AFM-tip with the laser beam from an initial offset of greater than 15 microns, and also maintain alignment for several hours, the researchers argue that the manual version gives the users more flexibility and is more reliable across a wider range of samples.

Their modification will enable future investigators to track forces and mechanical properties of specific structures in real time. Moreover, aspects of their correlation program can reduce the amount of manual labor required for experiments in correlative microscopy, by providing an automated process for overlaying images from both techniques.

Source: “Simultaneous AFM and fluorescence imaging: A method for aligning an AFM tip with an excitation beam using a 2D galvanometer,” by A. N. Moores and A. J. Cadby, Review of Scientific Instruments (2018). The article can be accessed at https://doi.org/10.1063/1.5006724 .