Fluorescent microscope users have common concerns regarding the uses, methodologies, and technologies that they have tried on their samples over time. Common concerns include slide sample preparations, prevention of cross-talk or bleed-through, auto-fluorescence, and maximizing the full potential of the fluorescent microscope in acquiring optimum, high-resolution images.
In order to obtain high-resolution images, preparation of the slide for optimum fluorescence would require immersion objectives, water or oil, as well as a glass cover slip between the specimen and the microscope objective. The use of cover slip #1 yields better results. Although cover slips #0 are acceptable, they are more fragile and have a tendency to break because of thermal stresses when flaming after dipping the slide into 70% ethanol. Use glass-cover slip-mounted dishes if the cells are being cultured in 35mm dishes. Use the appropriate mounting media or a homemade mounting medium if the cells being examined are fixed. A clear nail polish can be used to seal the cover slip. Each slide are gathered and stored in the dark at 4° C since this is the optimum storage condition for most slides. Microtubule, a cold-sensitive structure, might not last in abovementioned condition if its fixation were not done completely.
The prime concern in a fluorescent microscope is viewing only the emitted fluorescence photons while removing any background light particularly from the source of the illumination. The key for high-sensitivity fluorescence imaging is barrier filter. It does not significantly impact white-light imaging. Bleed-through, another concern for multiple-labeled samples produces artefactual co-localization. What happens is that one fluorescent dye “bleeds” through the optical filters that are to be used for another dye. It is all dependent on both the optical filters of the microscope and the quantity of dye used. A quick evaluation can be done by exciting only one fluorophore at a time at the same time watching the emission settings of all the other fluorophores. To be absolutely thorough, only observe samples or controls that are labeled with only a single fluorophore. The fraction of “bleed-through” through the “wrong” optical filters can be directly quantified compared to “correct” signal.
In theory, obtainable sensitivity can never be reached when using the fluorescent microscope because of autofluorescence from solvents, solutes, cells, tissues, the fixatives employed, and optical components of the microscope system. Whether in living cells, fixed tissues, or clinical samples, this is a common concern that affects all studies that uses fluorescent microscopes. Autofluorescence in effect lessens the signal-to-noise ratio of detection. It has a defined lifetime. Delayed luminescence microscopy (DLM) allows the separation of true fluorescence that is released from the fluorescence probe of interest from autofluorescence from solvents, solutes, cells, tissues, the fixatives employed, and optical components of the microscope system. The goal in DLM is to tag the structure with a probe and monitor the emitted fluorescence of the structure after the autofluorescence has perished. The requirement for this process is to label the molecular markers to be used with phosphorescent or fluorescence compounds with long lifetimes (as probes), excite the sample with a short pulse of light, and to detect the emitted fluorescence from the long lived probes then delay it to the micro- or millisecond range.
