Advantages of Simultaneous Imaging Using an Atomic Force Microscope Integrated with an Inverted Light Microscope
W. Travis Johnson Agilent Technologies, Nanotechnology Measurements Division 4330 West Chandler Blvd, Chandler, AZ 85226
w-travis_johnson@agilent.com
Introduction Atomic Force Microscopy (AFM) permits measurements
on biological samples below the limits of light microscopy resolution under physiological environments and other controlled conditions [1]. Consequently, AFM has become an increasingly valuable technique in cell biology [2]. One of the most exciting advances in AFM instrumentation has been its integration with the light microscope [3]. Tis permits investigators to take advantage of the power and utility of light microscopy and scanning probe microscopy simultaneously. In combining a light microscope with an AFM, scanner components must be specifically designed so that they do not adversely impact the light microscope’s optical imaging capabilities. For example, an AFM-ILM (inverted light microscope) hybrid system should be fully compatible with the highest quality, off-the-shelf 0.50–0.55 NA numerical aperture (NA) OEM objectives and condensers. An example of an ILM-AFM system that does not
compromise AFM performance, nor interfere with the ILM’s imaging capabilities,
is Agilent’s 6000ILM AFM, which is
pictured in Figure 1. Tis instrument seamlessly integrates the high-resolution AFM with the researcher’s choice of light microscope: Zeiss Axio Observer (A1, D1, or Z1), Nikon Ti, or Olympus IX series 71/81.
Methods and Materials Instrumentation. With the AFM in place, the ILM-AFM
is capable of operating in the most popular and effective light microscopy modes, including bright field (Kohler) illumination, differential interference contrast (DIC), phase contrast, fluorescence, and even confocal imaging. Tus, translucent cells and other semi-transparent samples can be easily located with the ILM and further investigated at higher resolutions with the AFM. Te ILM-AFM also supports the most popular and useful AFM imaging modes, including contact mode, acoustic AC (AAC) mode [4], magnetic AC (MAC) mode [5], and topography and recognition imaging (TREC) [6]. TREC, which is not directly addressed further in this article, is a useful technique that maps molecular interactions, much like force–volume spectroscopy [7], albeit with much greater speed and better resolution. Separate X-Y and Z AFM scanners eliminate horizontal–
vertical crosstalk, which can occur when X-Y and Z piezos are combined in the same scanner. Closed-loop sample scanning stages that provide at least 100 × 100 µm of range in the horizontal plane are necessary for cell imaging because they minimize the impact of hysteresis and permit extremely accurate positioning and repositioning of samples under the AFM probe. An unobstructed optical path through the Z
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scanner avoids any need for a complicated system of lenses and mirrors, which can be detrimental to high-quality light microscopy applications. A closed-loop Z scanner with a large Z range (for example 30 µm) is also an important feature because it permits the ILM-AFM to be compatible with the relatively large sample sizes that are oſten encountered with cultured cells and tissue samples. Sample Formats. Hybrid ILM-AFMs should accommodate
sample formats that are commonly used in cell and tissue imaging applications in both air and in-liquid environments. Tese include sample plates for 1 × 3 inch microscope slides, 35 and 50 mm Pitre dishes, and 22–25 mm glass cover slips. Other very useful features for prolonging cell viability are sample plates that are able to maintain cells at physiological temperature (37oC) and maintain optimal cell culture atmospheres (for example, 5% CO2) [8]. Specimens and Data Acquisition. Data and images for
the T24 and MyEND cells were obtained in professor Peter Hinterdorfer’s group at the Institute for Biophysics University of Linz, Linz, Austria. Te other cells used in this study were
Figure 1: An example of an AFM that was designed specifically for combined ILM-AFM studies; the Agilent 6000ILM AFM mounted on a Zeiss D1 ILM.
doi:10.1017/S1551929511001222
www.microscopy-today.com • 2011 November
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