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MICROSCOPES AND MICROSCOPY TECHNIQUES

1. Define microscopy

Microscopy is a technique used to view objects invisible to the naked eye, or too small to be seen clearly. The size of the object being viewed can vary from nanometres – bacteria, single celled organisms, to millimetres –  small insects, & plant cuttings for example. There are 3 main microscopic techniques that are used: Optical microscopy, Scanning probe microscopy and Electron microscopy.

1. Explain the importance of microscopy in biology

In biology, microscopy is the most important way to gain insight into biological structures and processes. The majority of living organisms are far too small to be seen with the human eye, and cells and their organelles can only be seen with the aid of a microscope.

2. The diagram below represents a light microscope.  Identify the labeled parts

3. Describe
   1. Optical microscope

Also known as light microscopy, involves using glass or plastic lenses arranged in a tube, much like a telescope, to produce an enlarged image of any small object placed in the focal plane of the lens. There are many applications to Optical microscopy, but one of the main uses of an optical microscope is in medical diagnostics of tissues or free cells in a smear test. An optical microscope has limitations regarding the amount of useful magnification which can be obtained. This is due to a property of light itself, and the wave length of the light used to resolve detail in the subject. A light microscope (optical microscope), has a magnification limit of approximately 1600x.

1. Electron Microscopy / Electron Scanning Microscopy

This is a form of microscopy which uses an electron beam to scan an object instead of using light waves. The beam excites various surface structures of the probed object, which then emit secondary electrons that are detected and used to create an image on a monitor. An electron microscope has  a much higher magnification than a light microscope, so a much higher resolution and magnification is obtained. Resolution can be increased because as the electrons travel faster their wavelength becomes shorter so there is a direct correlation between reducing wavelength and increasing resolution.

There are two types of electron microscopes used: Transmission (TEM) and Scanning electron microscopes (SEM). A TEM is a microscope which fires a high voltage beam through a thin layer of a specimen to gather details of the structure. A SEM creates  images by detecting secondary electrons that have been emitted off the surface of the target object due to excitation by a primary electron beam. Although  highly detailed images are obtained using TEM and SEM microscopes, there are disadvantages as well. Unfortunately TEMs and SEMs are very expensive to build and maintain. They are very sensitive to magnetic fields and require cool water running through the lens constantly.  Using either a TEM or a SEM requires a high degree of specialised training and lies outside the scope of non-professionals.

1. Distinguish between a stereo microscope and a compound microscope?

A compound microscope has a single path of light that travels in the manner outlined above, which is split before the eyepieces into the number of ocular tubes the microscope has. This means that samples must be translucent enough to pass light through, and that each eyepiece contains the exact same image as the other.

Simple path of light in a compound microscope. Ignore the darkfield stop, as the path is the same. Each lens “flips” the image in the path of light, hence why it looks far more complicated than it is.

A stereomicroscope, or stereoscopic microscope, is one that commonly has two objective lenses for each magnification setting, which are focused slightly offset from each other. Each lens captures the image individually, meaning that each objective has its own path of light per eyepiece. 

2. Explain the function of each of the following parts of a light microscope
   1. Iris diaphragm

Controls the amount of light reaching the specimen. It is located above the condenser and below the stage. Most high quality microscopes include an Abbe condenser with an iris diaphragm. Combined, they control both the focus and quantity of light applied to the specimen.

1. Condenser

It is located under the stage often in conjunction with an iris diaphragm. Used to collect and focus the light from the illuminator on to the specimen.

1. Stage

It is where the specimen to be viewed is placed. A mechanical stage is used when working at higher magnifications where delicate movements of the specimen slide are required.

1. Objective

 Are the primary optical lenses on a microscope. They range from 4x-100x and typically, include, three, four or five on lens on most microscopes. Objectives can be forward or rear-facing.

3. Differentiate between  low power objective lens and high power objective lense in terms of

(a) magnification

(b)  Focal length

(c )Working distance

(d) resolving power

1. Field of View

The field of view is the amount of the specimen you see when you look through the objectives. The field of view decreases at higher magnifications.

 

4. (a) Define the term”:resolution of a microscope”

In microscopy, the term “resolution” is used to describe the ability of a microscope to distinguish details of a specimen or sample. In other words, the minimum distance between 2 distinct points of a specimen where they can still be seen by the observer or microscope camera as separate entities.

(b) explain the factors  which must be considered  to get maximum resolution  from a lens using a compound microscope

 The primary factor in determining resolution is the objective numerical aperture, but resolution is also dependent upon the type of specimen, coherence of illumination, degree of aberration correction, and other factors such as contrast enhancing methodology either in the optical system of the microscope or in the specimen itself.

 

5. outline the precaution  taken into consideration  before returning a microscope  into the store after use in the laboratory

When returning the microscope to the laboratory cabinet:

• Lower the stage.
• Rotate the scan objective into position over the stage.
• Remove your slide from the stage.
• Clean the slide and objective using the special lens-cleaning fluid and paper provided.
• Center the stage so that it does not project too far to either side.
• Secure the cord by wrapping it under the stage of the microscope..
• Replace the dust cover.
• Carry the microscope with TWO hands.
• Return the microscope to the same cubby from which you obtained it making sure to put it in arm out.

7. When returning the microscope to the laboratory cabinet:
   1. Lower the stage.
   2. Rotate the scan objective into position over the stage.
   3. Remove your slide from the stage.
   4. Clean the slide and objective using the special lens-cleaning fluid and paper provided.
   5. Center the stage so that it does not project too far to either side.
   6. Secure the cord by wrapping it under the stage of the microscope..
   7. Replace the dust cover.
   8. Carry the microscope with TWO hands.
   9. Return the microscope to the same cubby from which you obtained it making sure to put it in arm out.

 

6. Describe care and maintainance  of microscope lenses and slides

Never clean lenses with alcohol, ordinary tissues, cleaning paper, toilet paper, cotton wool or hand towels, which will scratch the lens surface. Cover the microscope with a dust cover when not in use. Turn off the power at the end of the day, and unplug the microscope to protect it from a power surge.

7. Outline the procedure for using oil emersion  objective techniques

When a beam of light passes from different medium i.e.  from  Air  to   Glass    and  back  to  Air ,   It’s normally bent due to refraction property of light  . Such bedding can be avoided by replacing the air between the specimen and the lens with an oil Immersion which have the same optical property as that of glass . These makes light to pass in a straight line as though it was passing through the same media i.e. glass all the way. These therefore provides better resolving power.

8. Define the following terms as used in microscopy

• Working distance of an objective

This is the distance from the front lens element of the objective to the closest surface of the coverslip when the specimen is in sharp focus.

• Resolving power of a lens

The resolving power of an objective lens is measured by its ability to differentiate two lines or points in an object. The greater the resolving power, the smaller the minimum distance between two lines or points that can still be distinguished.

9. Name four types of light microscope

• Bright-field Light Microscope.
• Phase Contrast Light Microscope
• Dark Field Light Microscope
• Fluorescent Light Microscope

10. State the principle of darkfield microscopy

Dark-field microscopy is ideally used to illuminate unstained samples causing them to appear brightly lit against a dark background. This type of microscope contains a special condenser that scatters light and causes it to reflect off the specimen at an angle. Rather than illuminating the sample with a filled cone of light, the condenser is designed to form a hollow cone of light. The light at the apex of the cone is focused at the plane of the specimen; as this light moves past the specimen plane it spreads again into a hollow cone. The objective lens sits in the dark hollow of this cone; although the light travels around and past the objective lens, no rays enter it.

The entire field appears dark when there is no sample on the microscope stage; thus the name dark-field microscopy. When a sample is on the stage, the light at the apex of the cone strikes it. The rays scattered by the sample and captured in the objective lens thus make the image.

Samples observed under dark-field microscopy should be carefully prepared since dust and other particles also scatter the light and are easily detected. Glass slides need to be thoroughly cleaned of extraneous dust and dirt. It may be necessary to filter sample media (agar, water, saline) to exclude confusing contaminants. Sample materials need to be spread thinly; too much material on the slide creates many overlapping layers and edges, making it difficult to interpret structures.

Dark-field microscopy has many applications in microbiology. It allows the visualization of live bacteria, and distinguishes some structure (rods, curved rods, spirals, or cocci) and movement.

11. State  the difference in preparation  of materials for light and electron microscopy with respect to the following

• Fixation
• Staining
• Sectioning

12. Explain why higher magnification is obtained when oil emmersion  is used in microscopy

In light microscopy, oil immersion is a technique used to increase the resolving power of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens.

Without oil, light waves reflect off the slide specimen through the glass cover slip, through the air, and into the microscope lens (see the colored figure to the right). Unless a wave comes out at a 90-degree angle, it bends when it hits a new substance, the amount of bend depending on the angle. This distorts the image. Air has a very different index of refraction from glass, making for a larger bend compared to oil, which has an index more similar to glass. Immersion oils are transparent oils that have specific optical and viscosity characteristics necessary for use in microscopy.

Specially manufactured oil can have nearly exactly the same refractive index as glass, making an oil immersed lens nearly as effective as having entirely glass to the sample (which would be impractical). Oil immersion objectives are used only at very large magnifications that require high resolving power of X100. Objectives with high power magnification have short focal lengths, facilitating the use of oil. The oil is applied to the specimen (conventional microscope), and the stage is raised, immersing the objective in oil. (In inverted microscopes the oil is applied to the objective).

13. Describe the path of light in  a compound microscope

  The path of light through a microscope.

1Light, originating in the light source

(2), is focused by the condensor

(3) onto the specimin

(4). The light then enters the objective lens

(5) and the image is magnified. Light then passes through a series of glass prisms and mirrors, eventually entering the eyepiece

(6) where is it further magnified, finally reacing the eye.

7.Outline the working principle of transmission light microscope Transmission electron microscope; the electrons pass through the specimens . solid specimens are bombarded with a beam of electrons which causes secondary electrons  to be emitted  from the surface  of specimen . These electrons are recorded  on a photoelectric plate  as in transmission  electron microscope . images seen on these  microscope are three  dimensional

8. Compare the functioning of a light and electron microscope

• The light microscopes uses light for illumination and sets of  glass lenses for magnification while the electron microscope use electron beams instead of light and electromagnet instead of glass lens.
• In the electron microscope, the electrons are recorded in efflorescent screen which then forms a viewable image on the screen  (photomicrograph ). This is missing in the light microscope.
• In electron microscope, electrons travel in cathode ray tube which is a vacuum chamber. The specimens are mounted in total vacuum   in order to enable the electrons to travel with high velocity without colliding with air or any atoms in space within. These electrons pass through an electromagnet which acts as lenses where they produce electric fields.
• Also the specimens are prepared by sectioning using an extremely delicate microtome which produce extremely thin slices then fixed using osmic acid or glutaraldehyde for cytoplasm components
• Electron microscope have very high resolving power that is 1000 times  more than a light microscope  , thus specimens can be magnified  much more without loss of clarity .
• With the electron microscope, materials which were initially described as structure less have been shown to have elaborate internal organization  and the so called homogeneous fluids have now shown to contain  a variety of complex structures

9. Explain  staining as used in microscopy

Staining is a method used to better to reveal details of cells and cell components under a microscope. Using different stains, one can stain certain cell components, such as a nucleus or a cell wall, or the entire cell with different colours. These details are often transparent or semi transparent, but staining makes them more visible and different colours (organic dyes) enable various processes in the cell to be differentiated. Stains, such as weak water soluble safranin, and crystal violet are often added to a sample. The Gram negative cells are stained red. The Gram positive cells are stained blue.

10.Explain it is important to start observation with 4x magnification.

A typical compound optical microscope has three or four objective lenses with 4x, 10x, 40x and 100x (oil immersion) magnification. 4x objective provides the smallest increase, so you will be able to observe a large area of the sample. This allows you to easily find the desired area for further microscopic observation. When you have found the area, place it in the center of the field of view and switch to a higher magnification objective. It is much easier to focus the view using a low-power objective lens, rather than a powerful one.

11. Outline to calculate the magnification of a microscope

To calculate the magnification of a microscope, simply multiply the magnification of the microscope eyepiece by the magnification of the objective lens. The total magnification of a typical compound microscope with 10x eyepiece and 4x, 10x, 40x, 100x objectives will be 40x, 100x, 400x and 1000x depending on the lenses used.

12.  Explain  the relationship between magnification of a microscope  and the focal length

In microscopy, the focal length is understood as a distance between the objective lens and the upper part of the observed object. The focal length of the optical system shows how efficiently the system collects and focuses light rays. Commonly, the greater the magnification of the microscope, the shorter its focal length.

13. Define field of view in a microscope

The field of view is the diameter of the illuminated circle seen through the eyepiece. The higher the magnification, the smaller the field of view. To obtain the exact value of your microscope’s field of view, place a transparent ruler under the microscope objective and count how many millimeter-lines fit inside the visible illuminated circle.

14.Outline the routine care and maintenance of a microscope

Carry microscopes using both hands by the limb and the base and should be in upright position to avoid dropping loose parts e.g. mirror and eye- pieces.

Do not touch the microscopes lens using   fingers

Clean microscope  lens using lens cleansing tissues

Microscopes should be kept clean and covered to avoid dust

Keep microscopes away from direct sunlight and dust i.e. always cover microscopes and keep then in locked cupboards

The optical parts should be cleaned using a little xylene on a lens cleaning tissue or soft cloth. Do not use sleeves or lab coats

Never place microscopes at the edge of benches

Avoid using a high power objective when a low power objective is satisfactory

It is dangerous to rock down the objective while you are looking into the microscope. You risk breaking the slide, the objective lens or the condenser

Repair should be left only to a trained expert

Do not place wet preparation on the stage without wiping the undersurface of the slide

14.Outline the isolation techniques of a check cell for observation with light microscope

Materials

• Glass microscope slides
• Plastic cover slips
• Paper towels or tissue
• Methylene Blue solution (0.5% to 1% (mix approximately 1 part stock solutionwith 4 parts of water))
• Plastic pipette or dropper
• Sterile, individually packed cotton swabs

Methods

1. Take a clean cotton swab and gently scrape the inside of your mouth.
2. Smear the cotton swab on the centre of the microscope slide for 2 to 3 seconds.
3. Add a drop of methylene blue solution and place a coverslip on top. Concentrated methylene blue is toxic if ingested. Wear gloves and do NOT allow children to handle methylene blue solution or have access to the bottle of solution.
4. Remove any excess solution by allowing a paper towel to touch one side of the coverslip.
5. Place the slide on the microscope, with 4 x or 10 x objective in position and find a cell. Then view at higher magnification.

Methylene blue stains negatively charged molecules in the cell, including DNA and RNA. This dye is toxic when ingested and it causes irritation when in contact with the skin and eyes. The cells seen are squamous epithelial cells from the outer epithelial layer of the mouth. The small blue dots are bacteria from our teeth and mouth

16. Describe the technique of irrigation in a microscope

Irrigating the specimen is another simple technique. It is an important microscopic technique, as the process is used to introduce a dye or reactant to a section, which is already mounted under a coverslip. For example; place a small drop of iodine at one edge of the coverslip of the slide containing starch grains. Use a small piece of filter paper and draw the iodine solution through the mount from the opposite side. Replace any ‘lost’ liquid with more iodine.

17. Describe how hanging drop is prepared

The hanging drop technique is a well-established method for examining living, unstained, very small organisms. The traditional procedure employs a glass slide with a circular concavity in the centre into which a drop of fluid, containing the ‘microorganisms’, hangs from a coverslip.

The following is the procedure for preparing a hanging drop

• Hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a toothpick.  If too much Vaseline is used, it will be squeezed toward the center and mix with the drop or squeeze out the edges and get on the objective lens of the microscope.
• Place a loopful of the culture to be tested in the center of the prepared coverslip.
• Turn the clean concavity slide upside down (concavity down) over the drop on the coverslip so that the Vaseline seals the coverslip to the slide around the concavity.
• Turn the slide over so the coverslip is on top and the drop can be observed banging from the coverslip over the concavity.
• Place the preparation in the microscope slide holder and align it using the naked eye so an edge of the drop is under the low power objectives.
• Turn the objective to its lowest position using the coarse adjustment and close the diaphragm.
• 
• Look through the eyepiece and raise the objective slowly using the coarse adjustment knob until the edge of the drop is observed as an irregular line crossing the field.
• Move the slide to make that line (the edge of the drop) pass through the center of the field.
• Without raising or lowering the tube, swing the high dry objective into position (Be sure the high dry objective is clean).
• Observe the slide through the eyepiece and adjust the fine adjustment until the edge of the drop can be seen as a thick, usually dark line.
• Focus the edge of the drop carefully and look at each side of that line for very small objects that are the bacteria.  The cells will look either like dark or slightly greenish, very small rods or spheres.  Remember the high dry objective magnifies a little less than half as much as the oil immersion objective.
• Adjust the light using the diaphragm lever to maximize the visibility of the cells.
• Observe the cells noting their morphology and grouping and determine whether true motility can be observed.
• Brownian movement should be visible on slides of all the organisms, but two should also show true motility.
• Wash the depression slide and after soaking in lysol buckets.

 

NOTE:  The bacteria are still alive in a hanging drop slide.  Slides made from possible pathogens should be soaked in lysol for 5-10 minutes with the coverslip pulled aside to expose the drop before they are washed.

18. Outline the procedure for estimating  the size of a cell in microscopy
    Objects observed with microscopes are often too small to be measured conveniently in millimeters.  Because you are using a scale in millimeters, it is necessary to convert your measurement to micrometers. Remember that 1 μm = 0.001 mm.
    To estimate the size of an object seen with a microscope, first  estimate what fraction of the diameter of the field of vision that the object occupies. Then multiply the diameter you calculated in micrometers by that fraction. For example, if the field of vision’s diameter is 400 μm and the object’s estimated length is about one-tenth of that diameter, multiply the diameter by one-tenth to find the object’s length.

 

19. Outline the preparation of temporary slide  of a stained onion tissue

Material required:

Onion, forceps, watch glass, water, dropper, iodine solution, cover slip, filter paper and microscope.

Procedure or steps involved:

Remove a fleshy leaf from the onion bulb.

Strip a thin layer with forceps.

Place a piece of it in a drop of water on a clean side.

With a dropper, add a drop of iodine onto it.

Gently cover with a cover slip.

Use a piece of filter paper to remove excess liquid from the slide. This type of slide is known as wet mount.

Place the slide on the microscope and observe.