Spectroscopy of single molecules
المؤلف:
Peter Atkins، Julio de Paula
المصدر:
ATKINS PHYSICAL CHEMISTRY
الجزء والصفحة:
ص504-505
2025-12-09
23
Spectroscopy of single molecules
There is great interest in the development of new experimental probes of very small specimens. On the one hand, our understanding of biochemical processes, such as enzymatic catalysis, protein folding, and the insertion of DNA into the cell’s nucleus, will be enhanced if it is possible to visualize individual biopolymers at work. On the other hand, techniques that can probe the structure, dynamics, and reactivity of single molecules will be needed to advance research on nanometre-sized materials (Impact I20.2).
We saw in Impact I13.3 that it is possible to obtain the vibrational spectrum of samples with areas of more than 10 µm2. Fluorescence microscopy (Impact I14.2) has also been used for many years to image biological cells, but the diffraction limit pre vents the visualization of samples that are smaller than the wavelength of light used as a probe (Impact I13.3). Most molecules—including biological polymers—have dimensions that are much smaller than visible wavelengths, so special techniques had to be developed to make single-molecule spectroscopy possible.
f luorescence microscopy with laser excitation. The laser is the radiation source of choice because it provides the high excitance required to increase the rate of arrival of photons on to the detector from small illuminated areas. Two techniques are com monly used to circumvent the diffraction limit. First, the concentration of the sample is kept so low that, on average, only one fluorescent molecule is in the illuminated area. Second, special strategies are used to illuminate very small volumes. In near f ield optical microscopy (NSOM), a very thin metal-coated optical fibre is used to deliver light to a small area. It is possible to construct fibres with tip diameters in the range of 50 to 100 nm, which are indeed smaller than visible wavelengths. The fibre tip is placed very close to the sample, in a region known as the near field, where, accord ing to classical physics, photons do not diffract. Figure 14.40 shows the image of a 4.5 µm × 4.5 µm sample of oxazine 720 dye molecules embedded in a polymer film and obtained with NSOM by measuring the fluorescence intensity as the tip is scanned over the film surface. Each peak corresponds to a single dye molecule. In far-field confocal microscopy, laser light focused by an objective lens is used to illuminate about 1 µm3 of a very dilute sample placed beyond the near field. This illumination scheme is limited by diffraction and, as a result, data from far-field microscopy have less structural detail than data from NSOM. However, far-field microscopes are very easy to construct and the technique can be used to probe single molecules as long as there is one molecule, on average, in the illuminated area. In the wide-field epifluorescence method, a two-dimensional array detector (Fur ther information 13.1) detects fluorescence excited by a laser and scattered scattered back from the sample (Fig. 14.41a). If the fluorescing molecules are well separated in the specimen, then it is possible to obtain a map of the distribution of fluorescent molecules in the illuminated area. For example, Fig. 14.41b shows how epifluorescence microscopy can be used to observe single molecules of the major histocompatibility (MHC) protein on the surface of a cell. Though still a relatively new technique, single-molecule spectroscopy has already been used to address important problems in chemistry and biology. Nearly all the techniques discussed in this text measure the average value of a property in a large ensemble of molecules. Single-molecule methods allow a chemist to study the nature of distributions of physical and chemical properties in an ensemble of molecules. For example, it is possible to measure the fluorescence lifetime of a molecule by moving the laser focus to a location on the sample that contains a molecule and then measuring the decay of fluorescence intensity after excitation with a pulsed laser. Such studies have shown that not every molecule in a sample has the same fluorescence lifetime, probably because each molecule interacts with its immediate environment in a slightly different way. These details are not apparent from conventional measurements of fluorescence lifetimes, in which many molecules are excited electronically and only an average lifetime for the ensemble can be measured.
Fig. 14.40 Image of a 4.5 µm × 4.5 µm sample of oxazine-720 dye molecules embedded in a polymer film and obtained with NSOM. Each peak corresponds to a single dye molecule. Reproduced with permission from X.S. Xie. Acc. Chem. Res. 1996, 29, 598.
Fig. 14.41 (a) Layout of an epifluorescence microscope. Laser radiation is diverted to a sample by a special optical filter that reflects radiation with a specified wavelength (in this case the laser excitation wavelength) but transmits radiation with other wavelengths (in this case, wavelengths at which the fluorescent label emits). A CCD detector (see Further information 13.1) analyses the spatial distribution of the fluorescence signal from the illuminated area. (b) Observation of fluorescence from single MHC proteins that have been labelled with a fluorescent marker and are bound to the surface of a cell (the area shown has dimensions of 12 µm×12µm). Image provided by Professor W.E. Moerner, Stanford University, USA.
الاكثر قراءة في مواضيع عامة في الكيمياء الفيزيائية
اخر الاخبار
اخبار العتبة العباسية المقدسة
