SPM Applications in Biology

     It is hardly imagine more fundamental as well as complicated biological object than a living cell. In a short period of time applications of AFM has been extended to such complex field of biology as science of the cell [357, 959, 960, 961].

     In this field Atomic Force Microscopy features not only high-resolution imaging of cellular structures below the optical limit, that is quite "natural" for this method, but also evaluation of micromechanical properties of the cell and ability to monitor cell dynamics and processes running in it even in real time. At present, no other microscopic techniques are able to provide directly both structural information of a biological sample and related functional information at such high spatial resolution [357]. Using AFM cells can be imaged directly requiring little or no sample pre-treatment and, that is quite noticeable, in most native physiological media such as aqueous solutions. Offering several advantages over conventional microscopic techniques AFM is successfully employed in combination with another methods such as electron microscopy, SNOM, PCT and others [981, 982, 983, 997, 998].

     Direct imaging of fixed or living cells and subcellular structures provides important information on the architecture of the membrane, organelles and cytoskeleton of cells. AFM offers unique opportunity to image, localize and identify integral membrane proteins at the surface of living cells [962, 963, 964, 965, 966, 967]. Although one couldn't avoid staining or fluorescent labeling to mark the proteins of interest due to indiscriminative nature of AFM probing relative to chemical composition and nature of the objects, further improvements and extensions promise this problem to be solved. For instance, using method proposed in [999], the functionality of membrane proteins such as ion channels can be identified using Patch Clamp Technique (PCT) [980] and the density of the protein distribution over the membrane patch can also be estimated. This combination of AFM and PCT allows for lateral resolution of cytoskeletal elements from the patches as low as 10 nm [981]. To observe membrane structural features such as ruffles, lamellipodia, microspikes and microvilli, cell fixation is used [968, 969]. Because plasma membrane prevents from observing the intracellular structure the means of its careful removal were developed [970].

     Contact mode, commonly used in AFM, is not a perfect mode for cell imaging since it affects the membrane in a destructive way. So, tapping mode or intermittent contact AFM is preferable in such studies for high-resolution imaging of subcellular structures. The main problem arisen in this case is to remove damping involved by liquid environment and develop appropriate contrast mechanism to improve quality [971, 972, 973, 974].

     Another major AFM application in cell studies is real-time monitoring of living cells dynamics, intercellular interactions and response to internal and external perturbing factors [357, 975]. One can mention exploration of exocytosis of virus from an infected cell in real time [976], platelets shape transformation upon the activation [977], cultured pancreas cells secreting the starch-digesting enzyme amylase [978].

     The main problem in monitoring dynamic behavior of the cell is diminishing of perturbation involved by cantilever during scanning process as well as maintaining stable environmental conditions for both temperature and pH value [357, 979]. Another technical challenge is to make higher temporal resolution since time to acquire full scan of a living cell often exceeds characteristic alterations being happened in it. Diminishing of undesirable cellular stimulation can be achieved by implementation of modified tapping mode technique in liquid or development of a new technique in which much lower cantilever loading forces are required, and/or the design of novel cantilever probes which are biochemically and mechanically compatible with biological samples [357].

     The simplest remedy to make temporal resolution higher is, obviously, to speed up scan rate, often though at the expense of spatial resolution [984, 985, 986, 987]. So, existing AFM apparatus and techniques allow to monitor certain dynamic cellular processes, such as cell growth, exocytotic and endocytotic events, which are not so quick in time and requires less power in spatial resolution, and to study the cell morphology in real time in the presence of growth factors, hormones, and other biological reagents. With the development of high scan rate AFM it may be possible to use AFM to monitor the processes that occur at the cell membrane during an antibody binding, vesicle transfer, channel blocking or gating, etc., and to obtain information on the delivery of a specific drug with molecular resolution [357].

     Information about micromechanical properties is quite important for cellular systems because it helps to understand cell architecture and it's functions [975, 988, 989, 990, 991, 992, 993, 994]. Local elastic properties of a cell can be quantitatively derived from the force versus distance (F-S) curves obtained at fixed surface points using AFM. Cytosceleton is the main characteristic feature observed in AFM images and responsible for it's mechanical properties of cell. Cytosceleton generally defines the shape, activity and mobility of the cell. Data acquired from AFM measurements contains information about both topography and elasticity so it must be distinguished from each other. This can be done using elasticity mapping technique. The accuracy of elasticity measurements depends upon a number of factors considered in [357]. So care should be taken in the quantitative study of cell micromechanical properties [593, 988, 995].