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You Are Visitor
The Preparation
The following video clip was filmed outside the hood because there was no space to film inside. Note that conditions should be as sterile as those for tissue culture preparation.
The following video clip shows a nucleus being formed by aggregation. I apologize for the dirt in the lenses. We had a flood.
The following sequential clips show the same single nucleus using a filament to draw material to its interior. Again, I apologize for the dirt in the lenses.
Clip 1 , Clip 2 , Clip 3 , Clip 4 , Clip 5 and Clip 6
The following clip shows the procedure to form a tigh-seal/gigaseal. Note that suction should not be applied to form the gigaseal. Suction may be applied to attract the nucleus toward the pipette. Once the seal is formed, the pipette is lifted to prevent mechanical vibrations from dislodging the seal. Note also that the focus was adjusted as the nucleus was lifted. The same nucleus may be useful for a few days if sterile conditions and syncytial fluid is provided. Coconut water is an extremely good food for bacteria and the cause of many diseases for those who consume improperly handled coconuts. Again, sorry for the dirt in the lenses.
Contrary to the established dogma that nuclear pores are freely permeable to small particles (e.g. <40 kDa), our results demonstrate that living nuclear pores engaged in macromolecular traffic (e.g. mRNAs) are plugged by these macromolecules and, therefore, that they do not allow the translocation of small particles (including monoatomic ions such as K +, Na +, Ca 2+, etc.). In the following figures, FITC-labeled 4 kD dextran was added to the bath (see paper text). The control, transmission light microscopy images are given in the a panels. The fluorescent light microscopy images are given in the b panels. As shown by these figures, most nuclei excluded the FITC-labeled dextran (the black holes in the fluorescent images of the b panels). Note that the microscope images are not confocal. Note also that bleaching was avoided and tested for. Thus, the dark nuclei indicate genuine exclusion of dextran while the bright nuclei indicate inclusion. Included also are some cutoff images deblurred with AutoDeblur (AutoQuant Imaging). The AutoDeblur software uses Blind Deconvolution. The AutoDeblur algorithm does not require symmetric images (cf. Dr. Timothy Holmes, AutoQuant). Blind Deconvolution does not require knowledge of the Point Spread Function (PSF) usually required for other deconvolution algorithms. Deconvolution software is used to produce confocal images.
The settings I used for the AutoDeblur program were the following.
Launch => 2D Deconvolution => Standard
Total Iterations=1
;
PSF Correction Factor=2
;
Super Res Factor=1
Noise Smoothing=Low
;
Background=Black
After this single iteration processing of the original fluorescence image, the initial AutoDeblur inset window is replaced by a new window entitled 2D Deconvolution Results. I the further processed the result from the first iteration, one iteration at a time. I did not require more than 5 additional iterations. The settings in the new window were as follow.
Further Processing=1 More Iterations ; Current PSF=Checked
For more information on the deblurring procedure, please use the key phrase Blind Deconvolution with your internet search engine (e.g. Yahoo).
Deblurred Nucleus =>
Deblurred Nucleus =>
Deblurred Nucleus =>
Deblurred Nucleus =>
