Abstract

ISOTHERMAL TITRATION CALORIMETRY, CONFOCAL LASER SCANNING MICROSCOPY AND ATOMIC FORCE MICROSCOPY IN LATEST SUPRAMOLECULAR LIGAND TECHNOLOGY

Hardik H. Chaudhary and Prof. Dr. Dhrubo Jyoti Sen

ABSTRACT

Isothermal titration calorimetry (ITC) is now routinely used to directly characterize the thermodynamics of biopolymer binding interactions and the kinetics of enzyme-catalyzed reactions. This is the result of improvements in ITC instrumentation and data analysis software. Modern ITC instruments make it possible to measure heat effects as small as 0.1μcal (0.4μJ), allowing the determination of binding constants, K’s, as large as 108–109M-1. Modern ITC instruments make it possible to measure heat rates as small as 0.1μcal/sec, allowing for the precise determination of reaction rates in the range of 10-12 mol/sec. Values for Km and kcat, in the ranges of 10-2-103μM and 0.05– 500sec-1, respectively, can be determined by ITC. Laser scanning confocal microscopy has become an invaluable tool for a wide range of investigations in the biological and medical sciences for imaging thin optical sections in living and fixed specimens ranging in thickness up to 100 micrometers. Modern instruments are equipped with 3-5 laser systems controlled by high-speed acoustooptic tunable filters (AOTFs), which allow very precise regulation of wavelength and excitation intensity. Coupled with photomultipliers that have high quantum efficiency in the near-ultraviolet, visible and near-infrared spectral regions, these microscopes are capable of examining fluorescence emission ranging from 400 to 750 nanometers. Instruments equipped with spectral imaging detection systems further refine the technique by enabling the examination and resolution of fluorophores with overlapping spectra as well as providing the ability to compensate for autofluorescence. Recent advances in fluorophore design have led to improved synthetic and naturally occurring molecular probes, including fluorescent proteins and quantum dots, which exhibit a high level of photostability and target specificity. Atomic force microscopy (AFM) is a technique to obtain images and other information from a wide variety of samples, at extremely high (nanometer) resolution. AFM works by scanning a very sharp (end radius ca. 10 nm) probe along the sample surface, carefully maintaining the force between the probe and surface at a set, low level. Usually, the probe is formed by a silicon or silicon nitride cantilever with a sharp integrated tip, and the vertical bending (deflection) of the cantilever due to forces acting on the tip is detected by a laser focussed on the back of the cantilever. The laser is reflected by the cantilever onto a distant photodetector. The movement of the laser spot on the photodetector gives a greatly exaggerated measurement of the movement of the probe. This set-up is known as an optical lever. The probe is moved over the sample by a scanner, typically a piezoelectric element, which can make extremely precise movements. The combination of the sharp tip, the very sensitive optical lever, and the highly precise movements by the scanner, combined with the careful control of probe-sample forces allow the extremely high resolution of AFM.

Keywords: Isothermal titration calorimetry , Confocal Laser Scanning Microscopy, Atomic Force Microscopy , DNA, Free Energy, Entropy, Ligand, Topography, Piezoelectricity, Photodetector, Laser Doppler Vibrometry, Scanning Tunneling Microscope, Force Spectroscopy