Techniques for Microscopy

G.D.Bagchi
Central Institute of Medicinal & Aromatic Plants, Lucknow

Microscopy has a major role in the study of cells. In recent years, developments in microscopes, dyes, staining protocols and preparation techniques have helped in understanding of structure and function of the cell. Below 250mm human eye cannot depict objects clearly. At 250mm the resolution of the human eye is about 0.15 - 0.2 mm. For seeing object below this magnifying glass is required.

If you want to magnify an object even more, two lenses in tandem have to be used (objective and eyepiece or ocular). The resulting construct is a simple microscope.

Microscopes that are used for histological studies are Light microscope Where the source of illumination is light. Light beam is controlled by sets of lenses to get the clear view of the object. Electron microscope- Where the source of illumination is electron. The electron beam is controlled by sets of electromagnetic lenses to get the clear view of the object

Resolving powers of eye, light and electron microscopes:. Sizes of cells and their components drawn on a logarithmic scale, indicating the range of objects that can be readily resolved by the naked eye and in the light and electron microscopes. The following units of length are commonly employed in microscopy: m (micrometer) = 10-6 m nm (nanometer) = 10-9 m (ngstrm unit) = 10-10 m

Resolution: The measure for the ability to tell two points apart is called resolution. 0.2m is the highest resolution that can be reached with a light microscope. Types of light microscope: Diascopic illumination: In a typical compound microscope the light passes through the specimen and is collected by the image forming optics is called diascopic illumination. Episcopic illumination: The light is reflected on to the specimen and then into the objective lens. Stereomicroscopes use episcopic illumination for use with opaque specimen.

Typical compound microscope with diascopic illumination

Ruta Chalepensis -Oil glands

Stereomicroscope with episcopic illumination

Plumbago zeylanica - Glandular trichomes

Compound microscopes It creates a magnified, detailed image of seemingly invisible objects or specimens, based on the principles of transmission, absorption, diffraction and refraction of light. Light is controlled by the condenser. It evenly illuminates the specimen and help in increasing the contrast of the object. In microscopes there are usually two magnifying systems in tandem, One defined as the objectives and the other defined by the eye piece. Objective: It is the most critical part of the microscope. It determines the depth of focus, resolution and contrast of the specimen. Eyepiece: It simply magnify the resolved details of the image formed by the objective.

Bright field microscopy: It is the most commonly used technique, ideal for fixed, stained specimens or objects with high natural absorption. In BF, the light source and condenser are arranged to fill the objects aperture fully or partially with bundles of rays symmetrical to the optical axis. In this case, the background is bright and absorbing structures within the specimen appear darker

Dark field microscopy:

It renders high resolution and excellent detection of features far below the resolution limits of the microscope. It is helpful in the study of the microbes and cytoplasmic structures. In DF, no direct light enters the objective. Only where specimen features difract, refract, reflect or disperse the incoming, highly oblique wavefronts do they become visible.Only higher orders of diffracted light participate in image formation, resulting in contrast reversal. Specimen features appear bright on a dark background.

Phase contrast
It is widely used method in studies of live cells or unstained fixed material. In PC microscopes an annulus in the condenser aperture plane generates a hollow cone of 0-order illumination that is projected into the back focal plane of the objective. A matching phase ring in the objective performs two functions (1) it attenuates or absorbs the nondiffracted 0-order light by 7080% and (2) it shifts its phase position by one quarter of the wavelength (O/4) of the light. This arrangement alters the amplitude and phase relationships of diffracted versus nondiffracted wavefronts to achieve better interference, thereby enhancing contrast.

Two ways to obtain contrast in light microscopy. The stained portions of the cell in (A) reduce the amplitude of light waves of particular wavelengths passing through them. A colored image of the cell is thereby obtained that is visible in the ordinary way. Light passing through the unstained, living cell (B) undergoes very little change in amplitude, and the structural details cannot be seen even if the image is highly magnified. The phase of the light, however, is altered by its passage through the cell, and small phase differences can be made visible by exploiting interference effects using a phase-contrast or a differential-interference-contrast microscope

Polarized light microscopy Ergastic components of the cells are contrasted effectively in polarized light. Polarizing filters or prisms are used to convert unpolarized light to polarized light that oscillates in only one plane.

Fluorescence microscopy A wide range of specimens absorb light radiation, become excited and then re-radiate or emit light. When the emission continues for some time after excitation, the process is called phosphorescence. Emission that ceases almost instantly after excitation is called fluorescence. Many organic and inorganic substances display auto fluorescence or primary fluorescence. Labeling tissues and cellular components with proteins (eg anti bodies) and nucleic acids derivatized directly or indirectly with specific fluorochromes has become one of the most important methods for research and clinical diagnostic studies.

Fluorescent dyes. The structures of fluorescein and tetramethylrhodamine, two dyes that are commonly used for fluorescence microscopy. Fluorescein emits green light when activated by light of the appropriate wavelength, whereas the rhodamine dye emits red light. The portion of each molecule shown in orange denotes the position of a chemically reactive group; at this position a covalent bond is commonly formed between the dye and a protein (or other molecule). Commercially available versions of these dyes with different types of reactive groups allow the dye to be coupled either to an -SH group or to an -NH2 group on a protein

Four types of light microscopy. (A) The image of a fibroblast in culture obtained by the simple transmission of light through the cell, a technique known as bright-field microscopy. The other images were obtained by techniques discussed in the text: (B) phase-contrast microscopy, (C) Nomarski differentialinterference-contrast microscopy, and (D) dark-field microscopy. All four types of image can be obtained with most modern microscopes simply by interchanging optical

components.

Video microscopy and image enhancement

Video microscopy is the combination of videotechnology and microscopy. This combination opens doors to two new fields of microscopy: 1.Video enhanced contrast microscopy (VEC): It involves the production of an image from a specimen that is invisible to the eye either due to a lack of contrast or due to its spectral characteristics (UV or IR). 2.Video intensified microscopy (VIM): It involves imaging a specimen when the light level are too low for standard cameras or in some cases even for the eye. Images are produced by VIM using image analysis computers.

Specimen preparation for light microscopy

1.Fixation: FAA (EtOH-50cc, Gl acetic acid-5cc, Formaldehyde 3740%-10cc, Distilled water-35cc)-12-24 hrs. 2.Washing: In distilled water-3x5min. 3. Dehydration In EtOH or TBA series 30%, 50%, 70%, 90%- 100% for 15min each 4. Infiltration In xylol 30%, 50%, 70%, 100% (in EtOH), wax chips added at 40C and 50% volume decanted , process repeated at 60 C till material infiltrate in 100% wax 5. Block preparation fresh wax is liquefied, infiltrated material is dropped in it. Cooled and cut into square blocks. 6. Microtomy prepared block fixed to microtome and cut into ribbons of wax containing fine sections.

7. Slide preparation and staining wax ribbons placed on the slide pre coated with Mayers albumen (50% egg albumen+ 50% glycerin + a crystal of thymol). Quickly move the slide over low flame to fix the ribbon on slide. For staining- Slide is dipped in 100% xylol (1/2 hr), then in decreasing grades of alcohol. At the step of 50% alcohol safranin staining is given while at 90% alcohol step light green stain could be used. Finally the slide is kept in 100% xylol and then again in 100% xylol containing a drop of clove oil for hr each. 8. Mounting done in canada balsam to make permanent slide.

Water proof paper tray for paraffin block preparation

Paraffin blocks with different types of specimens

Trimming of paraffin blocks for microtomy

Microtome

Microtomy

Paraffin ribbons containing sections

Making tissue sections. How an embedded tissue is sectioned with a microtome in preparation for examination in the light microscope.

Slides containing serial sections

Staining

Some common stains

Hematoxylin- Hematoxylin 5g + Aluminium ammonium sulphate 3g dissolve in 50% EtOH-1000ml (keep in dark bottles (for nucleus, chloroplasts). Acid Fucsin- 1% in 70% EtOH or 0.5-1% in DW (for cellulose walls, mitochondria, cytoplasm. Anilin blue- 1% in 90% EtOH (slightly acidified with dil HCl (cellulose walls, fungi, algae). Crystal violet- 1% in DW- freshly prepared (for nucleus, mitochondria, chloroplast). Fast green- 1% in DW, 0.1% in EtOH (for cellulose walls, cytoplasm). Light green- 0.5% in 95% EtOH (algae, cytoplasm, cellulose walls). Methylene blue- saturated EtOH and DW solution (bacteria). Safranine- 2.25g in 225ml of 95% EtOH- diluted if required (cutinized, suberized, lignified tissues and nucleus). Picric acid- saturated solution in EtOH or DW (cytoplasm)

Some specific stains Fat- Sudan III & IV o.5g in 70% EtOH- 100ml Starch- 0.3g iodine+1.5g potassium iodide in 100ml DW. Tannin- 10% aq ferric chloride + little sodium carbonate Protein- Saturated solution of picric acid and eosin in absolute EtOH.

Electron microscopy It was realized that because of diffraction and wavelength of light, there was a limit to the resolution achievable with optical microscopes. Resolution (d)= 0.612O / n sin E 0.612 Abbes constant O- wavelength of image forming radiation n-index of refraction E-half angle of cone of light From this equation it is obvious that to get get the best possible resolution, one should have the smallest possible Oand largest possible n sin E. Using light with a wavelength of 500nm, best resolution is 200nm. Electron microscopes use electron beam for viewing having wave length of 0.0038nm, one can get resolution up to 0.24nm.However, due to limitations in specimen preparation one can get resolution up to 2nm in biological materials

Electron microscope

Electron microscopes possess following components: Electron gun Electromagnetic lenses Deflection coil Vacuum system Electronic control system

Electron gun: It consists of filament of tungsten or lanthanum hexaboride. It is surrounded by cathode cap. High votage (60-100 keV) to both. Below the cathode, there is a metal disc with a hole called anode. The high voltage potential between the cathode and anode determines the energy of electron beam and its penetration power. Electro-magnetic lenses: These control the electron beam. (1) Condenser lens- It regulates the convergence of the beam and controls brightness. (2) Objective lens- It focuses the image of the specimen.specimen is actually positioned inside this lens. (3) Intermediate lens- It is actually magnifying lens. (4) Projector lens- It projects the image on the fluorescent screen. Because electrons cannot be seen with the eye.

Vacuum system
To propagate the electron beam down the column, a high vacuum (104 torr) is required to prevent air molecules from interacting with beam. Two different types of pumps are employed. Mechanical Rotary pump- It brings down the vacuum up to 10 torr. Oil diffusion pump- It brings down the vacuum from 10 torr to 104 torr.

There are two types of electron microscopes: 1.Transmission Electron Microscope (TEM): This is used for seeing the internal structures / organelles of the cell. For this the sections of the specimens must be extremely thin (50250nm) because electrons at 60-100 keV range can not penetrate the specimen thicker than this.

Electron micrograph of a root-tip cell stained with osmium and other heavy metal ions. The cell wall, nucleus, vacuoles, mitochondria, endoplasmic reticulum, Golgi apparatus, and ribosomes are easily seen.

2.Scanning electron microscope: In this the electron beam scans over the specimen surface and helps in observing the magnified surface characteristics of the specimen. In contrast to TEM, which derives its information by collecting electrons after they have passed through the sample, the SEM produces information from scattered electrons (secondary or backscattered).

Scanning electron microscope

SEM microphotographs

TEM specimen processing 1. Fixation In 2% gluteraldehyde in 0.15M sodium cacodylate buffer- 1-4 hrs. 2. Wash- In 0.15M sodium cacodylate buffer-3x10 min. 3. Post fixation-1% osmium tetraoxide in cacodylate buffer-1hr. 4. Rinse-In distilled water-3x5 min. 5. Stain with 1% aq Uranyl acetate- 30min-overnight. 6. Dehydrate in 20%, 50%, 70%, 90%acetone-10min each: 100% acetone-2 changes- 15min each 7. Infiltrate with Epon / Araldite- 1:1 acetone : resin-overnight 100%resin-2changes over 3hrs. 8. Embed tissue and polymerize- 24-48 hrs at 60C. 9 Ultramicrotomy sectioning. 10. Post stain with UA(DNA, membrane) and lead citrate (ribosomes, glycogen, cytoplasm) 11. View under TEM.

Silicon rubber flat embedding molds

Trimming of resin blocks

Glass knife

Glass knife with water boat

Ultra microtome

SEM specimen preparation 1. Fixation In 2% gluteraldehyde in 0.15M sodium cacodylate buffer- 1-4 hrs. 2. Wash- In 0.15M sodium cacodylate buffer-3x10 min. 3. Post fixation-1% osmium tetraoxide in cacodylate buffer1hr. 4. Rinse-In distilled water-3x5 min. 5. Dehydrate in 20%, 50%, 70%, 90%acetone-10min each: 100% acetone-2 changes- 15min each 6. Critical point drying- here acetone is replaced by liquid carbon dioxide (at critical point 31.3C&1072 psi pressure) then it is removed by heating and exhaust phase. 7. Mount specimen on metal stub with silver paint 8. Sputter coat the sample with Au/Pd. 9. Observe under SEM

Critical point drier

Sputter coater