Кононов / 8

Synthetic Fluorescent Molecules

Metallic chelates

Aromatics & polyaromatics (dyes)

Semiconductor quantum dots

Applications

PHOTOPHYSICAL DATA

•Quenching and energy transfer information

•Molecular size and rotational relaxation

•Excited state life-time

LUMINESCENT ANALYSIS

•Fluorescence is linear with concentration

•Multicomponent analysis

•Excitation spectrum is similar to absorption spectrum

•Emission spectrum can provide unique fingerprint

•Three-dimensional matrices for complex mixtures

ENVIRONMENTAL PROBE

• pH, temperature, viscosity, polarity

CONFORMATION ANALYSIS

CHROMOTOGRAPHY

ELECTROPHORESIS

Single molecule spectroscopy

There are some major advantages of looking at the dynamics and spectroscopy of single biological molecules.

Single molecules exist at any given time in particular conformational states with a particular solvent environment. Observing only population averages can hide dynamic or mechanistic features of biological molecules that are important to function. Working with single molecules allows one to measure optical spectra in the absence of inhomogeneous broadening, learning both a great deal about the number and identity of overlapping transitions as well as the underlying conformational heterogeneity that gives rise to broad spectra in populations of molecules (Fig. 1).

When combined with scanning probe microscopy (SPM), single molecule spectroscopy allows the measurement of mechanical or electrical properties of single molecules: binding forces, forces holding together secondary and tertiary structure of molecules, torque, bond strength, conductivity, etc.

Four experimental approaches to detect single molecules: a) near-field microscopy, b) confocal microscopy, c) wide-field microscopy and d) total internal reflection (dark-field) microscopy . From

ChemPhysChem, 2:647-360, 2001

Different illumination techniques used for imaging. (b) Confocal scanning microscopy. (d) TIR microscopy using a prism. When light propagating within a dense medium (e.g. the quartz prism) reaches an interface with a less dense medium (e.g. aqueous solution), Snell’s law describes how light is refracted or reflected at the interface as a function of the incident angle, depending on the refractive index differences between the glass and water phases. At a specific critical angle, however, the light is totally reflected from the glass–water interface and an evanescent field is generated that extends into the aqueous solution. The penetration depth is typically in the range of half the wavelength of the incident light, thus ensuring high surface

selectivity. Only fluorophores located within this thin layer will be imaged by the area detector (c) Epifluorescence microscopy. This is a form of conventional wide-field microscopy. Collimated laser light is directed via a dichroic mirror into the microscope objective, which acts as condenser. As the sample is illuminated by parallel light and there is no additional confinement in z, this technique only works for thin samples.

Schematic representation of a (simple) confocal microscope setup. From http://www.microscopyu.com

Confocal scanning microscopy. A parallel laser beam is deflected by a dichroic mirror and focused by a high-numerical aperture objective onto a diffraction-limited spot. The fluorescence signal then passes through the dichroic mirror, is focused onto a pinhole and finally reaches the (point) detector (i.e. a photomultiplier or an avalanche photodiode [APD]). The excitation volume is confined in the x–y plane by focusing; the z-resolution in the detection pathway is provided by the aperture, which blocks light emanating from regions not in the immediate vicinity of the focal volume. Sequential scanning in two or even three dimensions is controlled by a

computer, which also calculates the final image.