Introduction and Question
Visualization of the DsRed1 protein is enabled because of its relatively longer wavelength of its red-emitting fluorescent protein that presents fewer challenges during in vivo labeling because of the reduced light scattering effect (Rodrigues, Hemert, Steensema and Leao, 2001). The importance of application of the DsRed1 protein tool in Trichoderma reesei cellulose analysis is its ability to describe the mechanism of cellulose synthesize. The process involved in this mechanism requires the DsRed gene to be imbedded within the chromosomal DNA’s of the Trichoderma reesei that is under investigation (Rodrigues et al, 2001).
The resulting recombinant of Trichoderma reesei strain that exhibits the desired DsRed protein of the specific promoter that was applied is then isolated and tagged as T. reesei (TR2) (Xue Bao, 2007). It is this TR2 strain that has been isolated that is subjected to DsRed1 protein under various cultures and analyzed using the Confocal microscopy which generates the results of the specific gene expression mechanism that is necessary for interpretation of the T.reesei (Xue Bao, 2007).
The principle that Confocal Microscopy applies in its functioning is the fluorescence; this is the phenomenon that is observed when light energy is shone on molecules leading to dispersion of light rays of different colors (Weeks, 2007). The fluorescence process that takes place in Confocal Microscopy is absorption of light energy by the molecules which gets excited; some light energy is lost in the process and the rest is emitted (Weeks, 2007). This emition is what identifies the specific element in the molecule since it only gives out color specific photon; the functioning of the Confocal Microscopy applies the same concept described by substituting light factor with fluorescein dye.
The Laser Scanning Confocal Microscopy technique involves the use of laser beam that is emitted at very high intensity and directed towards a sample target routed through a series of several mirrors (Weeks, 2007). Upon absorption of the light beam in the sample, the fluorescein dye is dispersed and photons of specific color are captured and routed through the same mirrors towards a pinhole that measures it intensity inside the photomultiplier tube.
DsRed1 is a variant of DsRed Antibodies which belongs in the group of Red Fluorescent Protein & Fruit Fluorescent Protein Antibodies (Clontech,com, 2008). The importance of the DsRed1 red fluorescent protein is its ability to combine with other protein factors for purposes of identifying their sub-cellular localization, in this way the DsRed1 protein ability to determine the localization of sub-cellular is very similar to how Green Fluorescent Proteins (GFP) functions in monitoring gene expression and pinpointing sub-cellular protein localization (Clontech,com, 2008).
DiOC6 (3,) is an important fluorescent dye in the field of biotechnology due to its effectiveness in staining the endoplasmic reticulum, mitochondria’s and vesicle membranes (Sabnis and Deligeorgiev, Jachak and Dalvi, 1997). The dye has several hydrophilic groups that attracts these cells and enable the binding to occur which can be determined by observing the green fluorescence that is emitted. However, this dye is limited in its application due to its characteristic of Photodynamic Toxicity (Sabnis et al, 1997).
Confocal Microscopy was invented as an improvement to the Fluorescence Microscope; as such it has several advantages not only against the epifluorescence microscope but also against wide ranging types of the existing fluorescence microscopes equipments. Its major advantage is its confocal pinhole that enables it to focus on the desired fluorescence and eliminate the disbursed fluorescent light thereby preventing external fluorescence interferences that would affect the accuracy of the results (Weeks, 2007).
The result is a very high resolution 3D image that cannot be achieved by the other types of microscopies. In addition, the pinhole screen of the microscopy ensures that emitted light that emanates from the focal plane is blocked allowing only the light transmitted via the focal point; this also serves to increase the resolution of the microscopy (Weeks, 2007).
References
Clontech. (2008). Fluorescent Protein Antibodies. Web.
Rodrigues, F., Hemert, M., Steensema, Y. & Leao, C. (2001). Red Fluorescent Protein (DsRed) as a Reporter in Saccharomyces Cerevisiae. Journal of Biotechnology, 16: 3791-3794.
Sabnis, R., Deligeorgiev, T., Jachak, J. & Dalvi, T. (1997). DiOC6(3): A Useful Dye for Staining the Endoplasmic Reticulum. Web.
Weeks, Eric. (2007). How does a Confocal Microscopy Work? Web.
Xue Bao, W. (2007). Analysis of Cellulose Synthesis Mechanism in Trichoderma Reesei Using Red Fluorescent Protein. Web.