Techniques of Nanoscale measurement and manipulation on living cells
Yao-Xiong Huang
Institute of Biomedical Engineering, Ji Nan University, Guang Zhou, China 510632
Email:tyxhuang@jnu.edu.cn
The present paper introduces our newly developed techniques for nanoscale measurements and
manipulations on living cells, including the techniques of multi-dimensional microscopy for
measuring the structure and functions of living cells simultaneously at cellular, sub-cellular，and
intracellular molecule levels; The techniques of Confocal Raman scattering microscopy for 2D to
3D scanning measurement on the molecular composition and concentration in a living cell with
nano-scale resolution; The technique of establishing a micro-environment—or a micron scale
bioreactor for biological cells; and the techniques of manipulating chromosome/molecules and
performing micro-surgery on living cells.
The multi-dimensional microscopy system is based on an inverted fluorescent microscope, its
information acquisition and processing is organized in 8-dimensions: 3 dimensional image is
acquired by a 3 dimensional relief image construction system; the 4th dimension(wave length) is
taken by a fast micro-spectrophotometry; which，accompanied with a dynamic image analyzing
system, also gives the information in the 5th dimension(time) by giving the absorption spectra and
images in a time-lapse mode; the 6th–8th dimensions are organized by the lights from the sample: the
transmitted light, gives the information of image and absorption spectroscopy; the scattered light,
gives the information about the dynamic parameters of intracellular molecule and the cell
membrane, as well as the chemical structure and concentration of the molecules; the fluorescence,
gives the information of the labeled molecules in the cell. Therefore, the multi-dimensional
microscopy is capable of performing measurement on the parameters of single intact cells include: 1)
the chemical structure and concentration as well as the hydrodynamic radius of the intracellular
molecules; 2) the 3-D morphology and molecular distribution of the cell; 3) the functional change
of the intracellular protein; 4) the flexibility and rigidity of the cell membrane; 5) the differentiation,
proliferation and the activity of the cell.
Raman confocal scanning microscopy, including the techniques of point Raman spectra,
line-mapping and 2D mapping performed with a 514.2nm excitation laser light, was used in a
comparative study on the distribution and oxidation states of hemoglobin in young and old mature
erythrocytes. It is demonstrated that in contrast to the homogeneous distribution of the Hb in young
cells, there are more Hb distribute around the cell membrane or bound to the cell membrane in old
erythrocyte. The proteins exhibit some extent of aggregation and conformational change, present
less ability of oxidation than the Hb in young erythrocytes. Our results show the first time that
Raman confocal microscopy is a powerful tool to measure the distributions of Hb with different
molecular conformations in a living RBC, and the variation of the molecular conformation with
time. The methodologies have been also employed to the measurement on different kinds of cells,
such as lymphocyte and K562 cell.
Micro aqueous environments for biological cells with sizes of 10-20 m in diameter were
established in a drop of oil on a glass slide. Some smaller bubbles of additive solutions were also
prepared and fused into the micron scale bioreactor with a cell or microbes inside, to vary its
biochemical conditions. Since the micro-environment can help to keep the substances released from
the cell at a measurable concentration, and keep the cell stay at a right position during the
measurement of the Raman spectrum scanning, the results of the measurements can reflect more
precisely the true behavior of the cell and the situation of its molecules under different conditions.
The chromosome/molecule manipulating and living cell micro-surgery were performed by using
a super laser microscope working station which consists of a laser tweezer and a laser scissor
system. By combining both the techniques, chromosome can be cut, removed and melted with each
other to change the genetic property. The similar technique can be also used for the
micro-processing of biological cells and other biological tissues.
Resume of Yao-Xiong Huang
A Professor of Biophysics & Biomedical Engineering, and the director of the Institute of
Biomedical Engineering, Ji Nan University. He is also the Member of the Academic Degree
Committee of the State Council of China; the Vice President of the Chinese Society of Medical
Physics; the Member of the Science Committee of International Organization of Medical Physics;
the President of GuangDong Society of Biophysics; the Vice President of GuangDong Society of
Biomedical Engineering; and the Standing Member of Guang Zhou Association of Science &
Technology.
He has won eleven academic awards and honors including a First Class Award of Natural
Science, from The State Education Commission of China and 2 Second Class Awards of Scientific
and Technical Progress, from The State Education Commission of China. In the past 20 years, he
has published 5 academic books and over 200 papers.