A better knowledge of biochemical and structural properties of human chromosomes is important for cytogenetic investigations and diagnostics. Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of chromosomal details. Localizing specific gene probes by FISH combined with conventional fluorescence microscopy has reached its limit. Also, microdissecting DNA from G-banded human metaphase chromosomes by either a glass tip or by laser capture needs further improvement. By both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), local information from G-bands and chromosomal probes can be obtained. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. Besides new strategies towards a better G-band and fluorescent probe detection, this study is focused on the combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA.
An atomic force microscope (AFM) head designed for nanometrology is accomplished in this study. It is the sensing component of the nano-measuring machine, a nanometrological instrument with a working range of 50 mm × 50 mm × 2 mm, as well as a part of the metrological system of the instrument. Three reference mirrors are mounted on the head and arranged without Abbe error. Relative displacement of the AFM head and the specimen is measured by interferometers and results are traceable. The optical beam deflection method is used to detect the atomic force. The laser beam is introduced through a single-mode polarization-maintaining optical fibre from an external laser diode. With a compact design, a 100 mm optical lever is realized inside the AFM head that is less than 20 mm in thickness and 200 g in weight. A force–distance curve is obtained using a gauge block in a test. Furthermore, online tests of the measurement of a step scale have been made. According to the calculation and experimental verification, the resolution of our AFM head reaches 0.05 nm.
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