You may want to use this resource for this problem. If you do, submit the output along with your solution. You have been given a confocal microscope equipped with the following lasers, excitation filters, and emission filters: Laser Emission filter 355 nm 410-470 nm 405 nm 470-500 nm 488 nm 500-550 nm 532 nm 570-610 nm 561 nm 610-650 nm 640 nm 660-700 nm 808 nm 720-780 nm Your task is to design an experiment to visualize the following:
Enzyme kinetics
In biochemistry, enzymes are proteins that act as biological catalysts. Catalysis is the addition of a catalyst to a chemical reaction to speed up the pace of the reaction. Catalysis can be categorized as either homogeneous or heterogeneous, depending on whether the catalysts are distributed in the same phase as that of the reactants. Enzymes are an essential part of the cell because, without them, many organic processes would slow down and thus will affect the processes that are important for cell survival and sustenance.
Regulation of Enzymes
A substance that acts as a catalyst to regulate the reaction rate in the living organism's metabolic pathways without itself getting altered is an enzyme. Most of the biological reactions and metabolic pathways in the living systems are carried out by enzymes. They are specific for their works and work in particular conditions. It maintains the best possible rate of reaction in the most stable state. The enzymes have distinct properties as they can proceed with the reaction in any direction, their particular binding sites, pH specificity, temperature specificity required in very few amounts.
You have been given a confocal microscope equipped with the following lasers, excitation filters, and
emission filters:
Laser Emission filter
355 nm 410-470 nm
405 nm 470-500 nm
488 nm 500-550 nm
532 nm 570-610 nm
561 nm 610-650 nm
640 nm 660-700 nm
808 nm 720-780 nm
Your task is to design an experiment to visualize the following:
1. Nuclei
2. A fluorescent protein in the cytosol
3. A cell membrane marker antibody conjugated with a fluorophore
4. Actin filaments
5. Lysosomes
You may choose from the following fluorophores for each of the five channels:
Nuclei Fluorescent protein Membrane marker Actin marker Lysosome tracker
DAPI GFP FITC AF488 Phalloidin LysoTracker Red
Hoechst 33342 YFP WGA-TRITC AF568 Phalloidin LysoTracker DeepRed
SYTO Deep Red RFP Cy7 AF594 Phalloidin LysoTracker Blue
Choose appropriate fluorophores for each of the subcellular structures to be imaged, taking into con-
sideration their excitation and emission wavelengths, photostability, and compatibility with other fluo-
rophores. If a solution is not possible with the flurophores I have provided, propose alternatives.
Specify the confocal microscope settings for acquiring images of each fluorophore, including laser wave-
lengths and the appropriate emission filters. Explain how these settings will allow for optimal imaging of
the fluorophores while minimizing crosstalk and photobleaching.
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