Microscopy slide preparation

Though the previous methods allow for long term storage, fresh smear-slide preparation arguably retains the most fidelity to in vivo systems as the tissue is not processed in any way prior to observation. In smear-slide preparation, samples are harvest from the embryos at different stages. Each sample is then immediately smeared on a slide wetted with PBS (PH 7.4) buffer then mounted with a cover slip. This method is best observed by polarization microscopy, which can be conducted immediately following the sample smear preparations. 

Mounting media help the coverslip to adhere to the slide bearing the tissue section or cytological preparation, protect the specimen and the immunohis-tochemical staining from physical damage and improve the clarity and contrast of the image during microscopy. 

Optical microscopy and scanning electron microscopy (SEM) were used to evaluate the drug incorporation and surface shape of the microspheres prepared under the various conditions. Particle size was determined using a Tiyoda microscope. Samples of microspheres (180-200) were dispersed on a slide and their diameter was then sized using suitable objectives. 

We studied samples of La lecithin with 16-29% water at room temperature. The samples were prepared between two microscope glass slides that were first treated with a sulfochromic solution, then carefully rinsed with distilled water and dried. They were observed by transmission polarizing microscopy. Just after deposition between the slides, the samples present a highly disordered texture which strongly diffuses light. After compression (squeezing) and/or shearing parallel to the plates, one obtains an overall homeotropic orientation that is perturbed by defects. The thickness of the samples is in the order of 20/. ... 

Once the sample was prepared, meristems were scored with light microscopy using the x40 objective, and the mitotic index was estimated by counting up a total number of 1000 cells in three slides of the same sample. 

For optical microscopy, hairy root specimens were fixed with 3 % formaldehyde in 50 mol m phosphate buffer (pH 6.8) for 1 h at ambient temperature. Cross-sections of the specimen were prepared using a sliding microtome equipped with a specimen-freezing stage. The sections were examined with microscopes under conventional or fluorescent light (excitation wavelengths of 520-550 nm and emission wavelength of >580 nm). 

Microscopy slides are ideally analyzed within a few days of preparation. The slides can, however, be stored in the dark at 4°C for weeks or months with acceptable staining quality. 

Two types of samples were prepared for detailed morphological examination. Sections of the as-polymerized material were examined directly by WAXS and DSC. Samples also were prepared by casting films from a solution of the polymer in dimethyl formamide (DMF) at 52°C. Solvent was allowed to slowly evaporate, and the films were dried by annealing for 670 hr at 52°C, followed by slow cooling to room temperature. Thin films suitable for electron microscopy were cast from 0.5 wt % polymer in DMF onto clean glass slides, and after 20 hr of annealing at 52°C, floated off on distilled water and mounted on 300-mesh copper grids. 

Sample preparation must be done carefully because biases in sample preparation will lead to inaccurate results. The statistical diameters that are popular for characterizing particles in microscopy are based on a random orientation thus, biases in orientation due to improper sample preparation will affect the values. Any factors that cause the particles to preferentially orient on the microscope slide will affect the results. For example, spreading the particles out with a spatula may causes a preferential orientation. Another example is particles dispersed in a liquid when the liquid is sprayed or poured onto the microscope slide the particles could orient themselves with the flow lines of the liquid and this could lead to their non random orientation. 

Samples used in microscopes can be placed on the slide dry or wet. Typical liquids used for wet-immersion are non-aqueous based like Nujol (International Crystal Laboratories, Garfield, New Jersey, U.S.A.), which is liquid paraffin, or Sirax, which is cedar wood oil. Again, there are many practical aspects of preparing samples for microscopy and the requirement of proper microscope settings can be found in Refs. 7, 9, 40. 

The green hairy roots cultured in the light for 3 weeks were used for the fluorescent microscopy and transmission electron microscopy (TEM). For the fluorescent microscopy, frozen sections (15 pm in thickness) were prepared using microtome cryostat (HM 500 OM, Microme, Heidelberg, Germany) and sealed with 50% glycerol on glass slides. Localization of chloroplasts was observed by fluorescent microscopy (VANOX AH-3, Olympus). 

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