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Atomic force and optical microscopy align themselves A new type of instrument simultaneously carries out total internal reflection fluorescence and atomic force microscopy.
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cantilever tip and the evanescent field when tugged on. “The common idea is roving once again that necessity is was so small that if the cantilever did that the domains unfold in a stochastic the mother of invention, Miklós enter the field, the resulting fluoresmanner. The more mechanically unstaKellermayer and colleagues at the Unicence was seen only as a very small dot ble domains unfold first, followed by versity of Pécs (Hungary), the Universion the screen. The investigators demonthe rest. But it’s not known if there is a ty of Heidelberg (Germany), Supertech strated that they could carry out widespatial pattern in the distribution of the (Hungary), and Asylum Research have field, as well as focused, developed an instrument that TIRF imaging in combicombines total internal reflec(a) (b) nation with atomic force tion fluorescence with atomic microscopy. force microscopy so that they The most critical ascan advance their research on pect of the instrument is large biological molecules. correct alignment. “The The instrument, called a spawhole experiment begins tially and temporally synchroby coaligning the optical nized total internal reflection and the mechanical axes, fluorescence with atomic force done under video conmicroscope (STIRF-AFM), altrol, by imaging both the lows biological samples to be cantilever and a reference simultaneously analyzed both position,” explains Kelleroptically and mechanically (a) AFM image of pancreatic carcinoma cells. (b) TIRF image of a red mayer. “That still doesn’t with high spatial and temporal dye attached to the actin filaments of the same set of cells. (Adapted ensure that the coalignresolution (Biophys. J. 2006, with permission. Copyright 2006 Biophysical Society.) ment will be maintained. 91, 2665–2677). There’s drift and so forth. Frequent redomains [within titin] according to “The combination of the techniques alignment is necessary. We’re currently their mechanical stabilities,” explains has been around, but what I think is working on establishing an electronic Kellermayer. important here is the synchronization,” feedback control for this.” The investigators were stumped besays Thomas Burghardt of the Mayo Besides obtaining topographical and cause they couldn’t simultaneously visuClinic in Rochester, Minn. The investifluorescence images, Kellermayer and alize the unfolding of titin and measure gators “are doing both experiments at colleagues demonstrated that the AFM its mechanical properties with sufficient once in the same time and space.” cantilever could be used to mechanicalresolution. So they developed the Daniel Fletcher of the University of ly perturb regions of cells or molecules STIRF-AFM, in which the optical axis California, Berkeley, agrees. “It’s a real was directly lined up with the AFM can- while the process was visualized by technical accomplishment to combine TIRF microscopy. For instance, they these two forms of microscopy,” he says. tilever. At each point in a sample, they watched as they broke off parts of cells simultaneously collected both the fluoFletcher explains that previous comand actin filaments that were attached rescence and topographical or mechanibinations of fluorescence and atomic to glass coverslips. force microscopy had to deal with back- cal information of biological molecules. The investigators have gone back to TIRF with atomic force microscopy ground fluorescence that added to the the original question about the spatial wasn’t an instant pick for the investiganoise and confused the detector signal. distribution of titin’s domains that had He says that Kellermayer and colleagues tors. They tried to join a confocal mitriggered the development of STIRFcroscope to an AFM but ran into probbypassed the problem by using TIRF to AFM. They are also tackling amyloid lems. The laser beam exerted an optical confine the fluorescence to a narrow, fibrils, the protein precipitates that are force on the cantilever tip and caused well-defined slice of the sample in z. the hallmark of Alzheimer’s disease. the cantilever to fluoresce with a rich Kellermayer says the idea for the in“We are zipping them apart and trying red color. “The most important thing strument was sparked by a specific exto measure the interaction forces bewas to reduce the depth of the excitaperiment. In 2000, the investigators tween the protofilaments and -sheets,” were trying to understand how domains tion field. This could be accomplished says Kellermayer. a with TIRF,” says Kellermayer. He exin titin, a huge protein involved in con—Rajendrani Mukhopadhyay plains that the interaction between the traction of striated muscles, unfolded © 2006 AMERICAN CHEMICAL SOCIETY
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