Mucoadhesion analysis by fluorescence microscopy

2.6.2.1. Theory and Principles of fluorescent microscopy

The commonly known equation of energy and wavelength shows that energy is inversely proportional to wavelength (Max Plank’s equation).

E= ℎ𝑐_λ--- Equation 2 (Hellmut Fritzsche, 2007)

There are number of chemical substances that absorb the light with higher energy and low wavelength and emits the low energy and shorter wavelength light, such substances are known as florescent substances. These substances are irradiated with light of high intensity (photon), the electron in the outer shell excited to higher energy level where they dissipate the part of energy in molecular collision and after short time when they drop again to ground level they lose their energy in the form of photon with less energy and longer wavelength. The phenomenon is called Stork’s shift (Kenneth, 2003) This lead to the basis of florescence. Number of compound and dyes are used for florescence and in current study coumarin-6 was used as a florescent dye to confirm mucoadhesion of liposomes on gastric mucus. Coumarins have remarkable physicochemical properties which make them suitable for the use as florescent probes in the field of nano science, micelles and polymeric systems (Wagner, 2009) Coumarin dye has very poor florescent quantum yield therefore substitution of coumarin results in strong florescent coumarin compound emitting in blue green region (Acar et al., 2015). In florescent microscope these dyes absorb the blue light with short wavelength and release green light with longer wavelength (Valeur, 2001).

60 Working principles of florescent microscopy

Basic working of florescent microscope starts when the light with high energy usually mercury lamp put on to the material stained with florescent light through the filter. The filter separates blue light from rest of the components of white light. After filtration only blue light enters into system and reflected by dichroic mirror mounted in such angle that it bends the blue light to the specimen (figure 2.5). when this blue light falls on the specimen with florescent dye it excites the electron and when electron comes to ground state it emits green light. the portion where the dye is more confined is more prominent than rest of the area. In a current study the dye is accumulated in liposomes and appears as well defined small vesicles on stomach slices. Once the specimen emits green light it goes upward and pass through dichroic mirror that further filters blue light and only allow the green light to pass through. After this light pass through barrier filter that blocks any residual blue light and finally reaches to eye piece as shown in figure 2.5. Where the image is perceived as green specimen (Article library, 2016).

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Figure 2.5. Line ray diagram of florescent microscopy (Microscopy and imaging department of Duke University and medical Centre).

2.6.2.2. Preparation of microscopic slides

Freshly excised sheep stomach was cut into 0.5 x 0.5 cm slices. Each slice was coated with 0.5 ml of mucoadhesive liposomal suspension contained coumarin-6 as florescence dye. Slices were incubated in 5ml SGF at required pH i.e. 1.3 and 4.5. Vials were put on a shaker incubator (50 rpm) at 370_{C. Tissue specimens were} taken out at different time intervals over 6 hours. Then, tissue specimens were immediately snap freeze by using liquid nitrogen and OCT into a block. Each specimen was cut in 10µm slices by using cryostat (LEICA, Germany) and observed under fluorescence microscope.

62 2.6.2.3. Snap freezing and cryo-sectioning

Unfixed tissue of 2 cm diameter was put into tissue mold made up of aluminium foil before putting the tissue a drop of OCT (consisting of poly ethylene glycol and polyvinyl alcohol) was placed in the bottom of the mold and the tissue was fixed on top of OCT. Proper orientation was adjusted by using forceps ad tweezers. Tissues were than covered in OCT for few minutes. Special care was taken and the Aluminium molds were inverted before putting OCT to avoid air bubbles in labelled Aluminium molds. The molds were put on to the special scoop and immersed into the Dewar of liquid nitrogen at -190ºC for 2 minutes for snap freezing. OCT is viscous at room temperature, but freezes into a solid below −20°C. This embedding protects specimen from heating and also helps protect the tissue from drying during storage and supports the tissue during sectioning. Cryostat was the instrument used for the slicing of stomach embed in OCT. Cryostat is actually the microtome inside specialized freezing chamber where tempura can be kept at -20°C. The microtome is capable of cutting the tissue up to 1 micrometre thin slice but for this experiment 10µm was selected and the sections were picked up on a glass slide and viewed under florescent microscope.

2.6.2.4. Microscopic examination of slides

The slides and cover slips were washed with PBS before fixing. After washing prepared slides from cryostat were fixed with the mount and coverslip were mounted on top. A piece of filter paper was placed around the edge of the coverslip to absorb excess mounting medium. The slides with specimen and mount were kept overnight before viewing to fix the specimen onto the slides. For fixing regular nail polish was used on the sides of cover slip and kept for three minutes. Most common problem associated with fluorescent microscopic specimens is the fluorophores readily loses fluorescence upon excitation during viewing. This could be overcome by fixing the

63 slides with mount. Fixed slides were viewed under microscope. The microscope was turned on and the slide was place in inverted position with the specimen side facing towards the source of light. Ordinary light was used to focus the specimen to desired area of the specimen by using the 40X objective. Once it was focused the ordinary light was changed into fluorescent light by switching the shutter. Objective was also changed suitable for oil immersion. The slides were viewed and images were taken with the camera mounted on the microscope.