Cytokinesis is a consecutive process during cell division. For systems biological studies, it is important to precisely
monitor and quantify proteins in different cell stages and mitosis processes. However, the absolute quantities in living
cells are usually difficult to quantify. Fluorescent protein tagged protein is one of the techniques that are usually applied
to monitor biological behaviors and phenomena. In this study, an insect cell line, DPnE, which can stably express both
green fluorescent protein (EGFP) and red fluorescent protein (DsRed) was established. This dual fluorescent cell line
was chosen as a model system to monitor the protein partition during cytokinesis. A spectrum analysis system was established and integrated in an inverted microscope. The two-dimensional distribution of the full fluorescent spectra of the two fluorescent proteins was obtained in a time-lapse series. Furthermore, we also developed an algorithm to analyze the quantities of both fluorescent proteins in the daughter cells and parent cells during the process of cytokinesis, respectively. With this innovative optical system and algorithm, the proteins partition during cytokinesis can be monitored and quantified precisely.
The cloning and transcription techniques on gene cloned fluorescent proteins have been widely used in many
applications. They have been used as reporters of some conditions in a series of reactions. However, it is usually difficult
to monitor the specific target with the exactly number of proteins during the process in turbid media, especially at
micrometer scales. We successfully revealed an alternative way to monitor the cell cycle behavior and quantitatively
analyzed the target cells with green and red fluorescent proteins (GFP and RFP) during different phases of the cell cycle
by quantitatively analyzing its behavior and also monitoring its spatial distribution.
Exogenous fluorescent agents such as green fluorescent protein (GFP) have been widely used as biological indicators in
bioimaging techniques. Although GFP and its mutants have been used in many applications, their optical properties have
not been completely investigated, especially when they are under various environmental conditions. In this research, we
developed a spectrum-analyzing system to investigate the fluorescent properties of GFP in the environments of different
temperatures. We found that the fluorescent spectrum of GFP consisted of two components that might come from the
transitions between different electronic energy states where the quantum efficiencies of the two components varied with
different temperature. This effect was expected to come from the thermal effect on the electron populations in the
molecular energy states of GFP. Furthermore, GFP was used as fluorescent marker to monitor the infection process of
cells by viruses with a dynamic spectral imaging system. The recombinant baculoviruses containing the red and green
fluorescent protein gene that can simultaneously produce dual fluorescence were used as vectors in insect cells. The
system was used to monitor the spatial distribution of fluorescent spectra of cells infected by virus during the process of
infection.
We constructed a dynamic spectroscopy system that can simultaneously measure the intensity and spectral distributions
of samples with multi-fluorophores in a single scan. The system was used to monitor the fluorescence distribution of
cells infected by the virus, which is constructed by a recombinant baculoviruses, vAcD-Rhir-E, containing the red and
green fluorescent protein gene that can simultaneously produce dual fluorescence in recombinant virus-infected
Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. The system was composed of an
excitation light source, a scanning system and a spectrometer. We also developed an algorithm and fitting process to
analyze the pattern of fluorescence distribution of the dual fluorescence produced in the recombinant virus-infected cells.
All the algorithm and calculation are automatically processed in a visualized scanning program and can monitor the
specific region of sample by calculating its intensity distribution. The spectral measurement of each pixel was performed
at millisecond range and the two dimensional distribution of full spectrum was recorded within several seconds. We have
constructed a dynamic spectroscopy system to monitor the process of virus-infection of cells. The distributions of the
dual fluorescence were simultaneously measured at micrometer resolution.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.