news
RESEARCH PROFILES Probing single molecules in living cells Anyone who has taken a biology course knows that cells are complicated creatures—filled with all kinds of molecules and structures, each with a name and function to memorize for the next exam. So some eyebrows might be raised by the report in the November 15 issue of Analytical Chemistry (pp 5606–5611) that Shuming Nie, Tyler Byassee, and Warren Chan at Indiana University have found and identified single molecules inside a living cell. Like the four-minute mile or the first spaceflight, this marks the breach of an important barrier. Before now, several research groups had detected individual macromolecules in solution or on the surface of a cell using fluorescence microscopes. The challenge when looking inside the cell is that the internal environment contains billions of fluorescent molecules as well as complex organelles and is known to produce intense background fluorescence. Thus, a major concern is that this intracellular background could overwhelm the relatively weak signals arising from single molecules. Further, molecules inside a living cell are either in constant motion or are attached to surfaces—perhaps even hidden. How can you chase down one of these molecules so you can identify it? Rather than pursue the molecule, the Indiana scientists wait for it to come by. They use a confocal fluorescence microscope to focus a laser beam on a selected spot inside the cell. The volume of the analytical spot is tiny—about 1 f L. Individual target molecules simply migrate through this volume. “The emissions from each passing molecule are recorded as bursts of photons,” Byassee explains. “The background fluorescence from inside the cell is noticeable, but is constant and much less intense than the bursts of photons resulting from a passing fluorescent probe molecule.” To en-
732 A
those found in pure solvent. Single molecule data were obtained for three types of fluorescent molecules inside human HeLa cells. First, the iron transport protein, transferrin—labeled with tetramethylrhodamine, which is rapidly taken up by the cell by a process called receptor-mediated endocytoLaser beam sis—was detected inside living focused inside cell cells. Second, the cationic dye rhodamine 6G (R6G) entered cultured cells by a Objective (100X) potential-driven process, and single R6G molecules were observed as intense Objective (100X) photon bursts when they (a) (b) moved in and out of the focused laser beam. Third, the researchers found that certain synthetic oligonucleoObjective Objective tides (oligos), tagged with a (100X) (100X) fluorescent dye, were taken up Focus inside cytoplasm Focus inside nucleus by living cells via a passive pathway that does not involve endocytosis. “We envision that further developments will include real-time tracking (A) Laser beam focused inside a single living and two-color fluorescence measurecell by the confocal microscope. The beam can be focused (B) in the cytoplasm, or (C) in ment at the single molecule level,” says Nie. “Such capabilities will allow the the nucleus. direct observation of key intracellular events, such as the transport of gene scope is similar to that previously retherapy vectors, hybridization of antiported by William Lyon and Nie (Anal. sense oligos to mRNA, and ligand– receptor binding and internalization.” Chem. 1997, 69, 3400–3405). The James P. Smith and excitation source is a continuous-wave Vicki Hinson-Smith argon ion laser (514-nm emission), directed into the back port of an inverted microscope. The laser light reflects from a dichroic beam splitter and is focused onto the sample by a high numerical Raman studies bacteria aperture oil-immersion objective. Fluoone by one rescence photons are collected through the same objective and detected by an As the biotechnology industry grows, avalanche photodiode. bacteria are gaining importance as bioThe recorded peak heights are typicatalysts in various processes, including cally more than 3 times that of the the treatment of waste and the recovery background. Peak width is an indication of metals. One outcome of the increased of the diffusion rate of the fluorescing interest in such “bioprocesses” is the molecule passing through the focused need to characterize the metabolism and spot. Surprisingly, the diffusion rates production of bacterial cell populations inside the cell appear to be similar to to optimize their growth and performsure that the cells are still viable after the experiments, the researchers limit the laser exposures to ~12 s/run. The confocal fluorescence micro-
A N A LY T I C A L C H E M I S T R Y / D E C E M B E R 1 , 2 0 0 0
news
The cell-by-cell instrumental analysis of a bacterial population is new and advantageous. Traditional biochemical tests to characterize bacteria are often performed on isolated colonies grown from a single cell. Because these colonies are grown outside the fermentation vat, the conditions may be different, yielding a nonrepresentative sample. In addition, some methods of on-line analysis give average composition data over the entire population. Single cell analysis by MALDI MS has been performed on much larger eukaryotic cells, but the point-and-shoot ease of confocal Raman microspectroscopy is attractive. Also attractive is the cost. Schuster is using a
K. CHRISTIAN SCHUSTER
ance. In the November 15 issue of Analytical Chemistry (pp 5529–5534), Bernhard Lendl, K. Christian Schuster, and colleagues at the Vienna University of Technology (Austria) and Jobin-Yvon/ Dilor GmbH (Germany) reveal a unique way to study a nonhomogeneous population of Clostridium beijerinckii as it grows and changes: Raman microspectroscopy of the individual cell members. Cells within a bioprocess population will, at times, differ in properties and physiological status. In the case of C. beijerinckii, which is of interest because of its ability to convert agricultural waste into basic chemicals and fuels, the early growth and division stages produce butyrate and acetate. But in a later phase, cells differentiate into spore-forming cells or into large forms for storing starchlike granulose. Solvents such as acetone and butanol are produced in this stage, too. The performance of this fermentation and the related changes in metabolic pathways of the organism are not completely understood. Confocal Raman microspectroscopy enables single cells to be chosen visually, focused upon manually with the laser beam (which is at an attenuated power level to avoid damaging the cells), and finally analyzed. When focused, the beam illuminates about the same area as the cell (~1 µm2 for C. beijerinckii). “For many organisms, the bulk composition of [a] biomass is known or can be analyzed,” perhaps by FTIR spectroscopy, says Schuster. “[And] this data can potentially be used for ‘calibrating’ single cell spectra.”
The Raman microscope with (left) bacterial cells as seen through the light microscope attachment (actual size: 65 3 43 µm).
commercially available system that costs $60,000–70,000, which is significantly less than a MALDI mass spectrometer. Lendl and his co-workers point out that Raman microspectroscopy has advantages over the other techniques for single cell analysis. IR microspectroscopy has a spatial resolution of only ~10 µm, which
is too poor to single out smaller bacterial cells. Flow cytometry and microscopy with image analysis can zero in on a single bacterium, but these techniques introduce chemical stains and see only one, or a few, components. Raman microspectroscopy, on the other hand, does not introduce interfering substances; it uses only distilled water for washing and suspending the cells. And several different compounds might be detected from Raman spectral information. Nevertheless, there were some challenges in optimizing the system. To achieve a high enough sensitivity, and thus avoid more complicated methods like UV-excited resonance Raman or surface-enhanced Raman spectroscopy, the researchers chose a HeNe laser at 632.8 nm for excitation. They also had to direct their attention to finding the optimal carrier material. Sodium glass— from which microscope slides are made— had a high fluorescent background at the desired excitation wavelength. Quartz glass produced a lower noise level. But calcium fluoride carriers were best, producing little noise and a very flat baseline with only one sharp peak at 322 cm21. Schuster says that they will soon perform experiments to identify granulose and other suitable markers in the life cycle of C. beijerinckii. When asked if he has pointed his Raman microspectroscopy apparatus at other varieties of bacteria, he notes, “Not extensively, but experiments are on the way.” He cautions, “Other applications for different bioprocesses are feasible, but have to be studied in detail from case to case.” Gerald Keller
MEETINGS Frederick Conference on CE—Britt Erickson reports from Frederick, MD. Microbes meet CE It’s not too often that words such as prokaryotes, fungi, and protists show up in Analytical Chemistry, and if you are
like most of our readers, you probably don’t remember exactly what they mean. It may be time to pull out those dusty
biology books. On the basis of a group of talks presented at the 11th Frederick Conference on CE in October, it is
D E C E M B E R 1 , 2 0 0 0 / A N A LY T I C A L C H E M I S T R Y
733 A
