This article briefly describes the basics of both optical and atomic force microscopy, followed by a discussion of some of the technical challenges of integrating these two distinct imaging modalities. In certain cases, the benefits and disadvantages of different approaches to design and integration are discussed. Lastly, a few examples of successful application of these combined imaging modalities are presented.

Introduction

Since its invention in 1986 by Binnig, Quate, and Gerber [1] the atomic force microscope (AFM) has become an indispensable tool for investigators in the physical, materials, and biological sciences. The AFM quickly gained acceptance in these fields due to its ability to capture topographical maps of surfaces in either air or liquid with sub-angstrom (in Z) and nanometer (in XY) resolution. Further, the ability of the AFM to measure a variety of forces with pico-Newton precision quickly led to measurements of single-molecule and intra-/intermolecular forces. Force measurements were also extended to the measurement of the elasticity of samples such as living cells and polymers that are typically too soft to measure precisely with traditional instrumented nanoindentation techniques. Integral to the success of AFM-based techniques is the relatively simple and label-free preparation of most samples when compared to other ultramicroscopical techniques.
Today, extensive imaging modalities have been implemented on the AFM under the umbrella of scanning probe microscopy (SPM). In addition to topographical imaging, SPM has been used to measure magnetic fields, friction gradients, potentials, capacitance, current flow, piezo response, and temperature (to name a few) across a diverse array of samples. Wider commercial availability of user-friendly instrumentation has put the AFM into the hands of more researchers, not only pushing the boundaries of its application in particular fields, but also bringing together scientists at the interfaces between disciplines.
An exciting and promising area of growth for AFM has been in its combination with optical microscopy. Although new optical techniques developed in the past few years have begun to push traditional limits, the lateral and axial resolution of optical microscopes are typically limited by the optical elements in the microscope, as well as the diffraction limit of light.

However, its ability to image through the entire depth of certain samples with chemical specificity using a plethora of label-conjugated markers allows researchers to identify specific structures or molecules within a dynamic event. Coupled with AFM's ability to measure high-resolution topographical images, forces, and/or elasticity on a sample, a more complete understanding of structure-function relationships can be elucidated with a combined AFM/optical system. While the two imaging modalities have been used in combinational studies for over a decade, significant challenges of direct correlation of the two data sets have existed primarily due to the scaling differences between the two data sets. Recent developments in software now allow for user-friendly and intuitive routines for direct overlay and comparison between the two data sets. Further, various optical techniques are now being used to modify or stimulate samples of interest in concert with AFM measurements, and vice-versa. Indeed, AFM researchers find themselves in a diverse, multi-interfacial area of microscopy, made even more powerful by combining AFM with optical microscopy.

Microscopy Basics

While the first commercial AFMs were produced in the late 1980s, the origins of optical microscopy are much less clear, but are thought to lay with simple magnifying glasses in the mid-9th century with further developments in the 16th century. However, it wasn't until the 17th century that history records scientific observations made with simple and compound microscopes, most notably in the field of biology by Hooke and van Leeuwenhoek (Figure 1A,B). Despite this long history, the most exciting time in optical microscopy has arguably been the past 100 years or so, as diffraction-limited optics, chemically-specific stains, and fluorescent markers and indicators have become widely available. In most modern applications, optical microscopy resolution is on the order of 200-300nm in X and Y, and 500nm in Z.
The AFM (Figure 1C) uses a microfabricated cantilever made of silicon or silicon nitride with a sharp tip that physically touches the surface of interest. The cantilever raster-scans the sample while its deflection or oscillation amplitude is measured. These measurements are performed with an optical tracking system that uses a segmented photodetector to track the reflection of a laser or superluminescent diode (SLD) off the back of the cantilever (Figure 2). Detected changes in cantilever deflection or oscillation are corrected to a setpoint value by actuating the cantilever in Z via a feedback-controlled piezo. These correction voltages sent to the Z piezo are recorded and correlated to a voltage-distance calibration factor in order to determine the height at a given XY coordinate. Because piezos suffer from nonlinearities due to hysteresis, creep, drift, and aging effects, most modern AFMs incorporate sensors that can linearize and correctly measure actual piezo actuation in XYZ. While a variety of sensors are available, the highest performance typically comes from linear variable differential transformers (LVDTs) because of their high linearity and low noise, which result in accurate tip and sample positioning to 0.06-0.6 nanometers. Additionally, the tip and the sample can be mounted on flexure stages that further linearize actuation.
One of the great benefits of AFM is its ability to measure at multiple spatial scales. AFM resolution in XY is limited by the size of the tip, and is typically on the order of a few nanometers, while the upper measurement limit is on the order of 100 microns. Resolution in Z, however, is limited by electronic and thermal noise and is on the order of an Angström, with an upper measurement range that can be several tens of microns. In addition to measuring the physical topography of samples, the AFM cantilever can be used to measure forces such as adhesion, deformation, and sample elasticity by measuring the deflection of the cantilever versus tip-sample separation and applying simple spring mechanical models. With this approach, forces in the picoNewton range can be readily discriminated.
A combined AFM/optical microscope is an excellent instrument for characterizing various samples. Optical microscopy's chemical specificity and ability to image live processes within the depth of a sample is well complemented by the higher resolution capability of the AFM. For example, a popular technique for identifying internal components in cells utilizes multiple fluorescent markers that bind specifically to molecules of interest (Figure 3A). Overlaying the AFM data directly onto the optical data can allow for correlation, while the higher resolution of the AFM can resolve structures that are not composed of the target molecules for fluorescence, or structures that are too small or weakly labeled (Figure 3B,C). Integrating these two technologies is challenging, and different design criteria must be met to ensure success.