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On the surface Objective lens
Antigens that adhere to the surfaces of living cells are difficult to detect, particularly at the single cell level. Traditional detection methods require fixation treatment or cell lysing procedures, which are detrimental to living cells. Now, Tsuguo Sawada and co-workers at the University of Tokyo and Juntendo University School of Medicine (both in Japan) have found a way to detect surface antigens in single, unfixed living cells. The key to their success is thermal lens microscopy, a technique based on the measurement of a laser-induced photothermal effect. Thermal lens microscopy is unique in its ability to measure localized molecules on the surface of a single cell without having to extract the cell. Rather than using a fluorescently labeled antibody, the researchers use colloidal gold as the labeling material. A laser beam irradiates a microscopic area of the gold-coated sample, and the light-into-heat conversion at that area is measured. This is accomplished by measuring the reduction in intensity of the laser beam as it passes through
Excitation beam
Thermal lens
Colloidal gold CCD camera
Sample
Spreading of probe beam~ absorbance of gold
Dichroic mirror
Light chopper (Cycle, 1.5kHz)
Ar+ laser (514.5 nm, 20 mW)
He-Ne laser (633 nm, 1mW)
Sample
Pre-amplifier Lens
Filter
Microscope
Photodiode
Lock-in amplifier
Apparatus used in thermal lens microscopy. (Adapted with permission. Copyright 2000 Academic Press.)
the so-called thermal lens, which forms as a result of a change in refractive index that occurs when the gold absorbs the laser beam. The laser beam is capable of targeting cells with a spot size as small as 1.5–2 µm. The researchers used the technique
to detect several surface antigens on lymphocytes (~5 µm in diameter) and mononuclear leukocytes (8–15 µm in diameter). Signals were shown to vary depending on the quantity of the antigen and its location. (Anal. Biochem. 2000, 283, 27–32)
MEETING NEWS News from the 52nd Annual Meeting of the American Association for Clinical Chemistry—Sandra Katzman reports from San Francisco, CA. Screening for STDs The first FDA-approved system for simultaneous amplification and detection of sexually transmitted diseases (STDs) is currently being used in limited situations, California county health officials told the annual meeting of clinical chemists in July. The system, called the BDProbeTec ET, is commercially available from Becton, Dickinson and Company, and it incorporates a urinebased amplified DNA probe assay. Because it is noninvasive, it eliminates the pain, expense, and lengthy analysis
time characteristic of traditional probes, which have been used since the 1980s. In addition, it uses amplified molecular technology, which is 20–50% more sensitive than nonamplified methods. The BDProbeTec ET uses a closed reagent system and a proprietary amplification technology, Strand Displacement Amplification (SDA)—a technique that uses a restriction enzyme and a polymerase. One advantage of SDA is that it allows real-time amplification and detection without thermocycling. Jeffrey D. Klausner of the San Francisco Department of Public Health
commended the use of the test in nightclubs, and Dennis V. Ferrero of San Joaquin County Pub-lic Health Services described an 18-month success story, in which the probe was used to screen youths for STDs. “Screening prevents costly health implications with its high-sensitivity, noninvasive amplified DNA tests. The solely urine-based test is 20 to 40 times faster than traditional nonamplified systems,” Klausner says. Also, the technology has led to better acceptance of testing from the overwhelmingly nonsymptomatic affected population. Last year is the
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