medical microscopy

1. MEDICAL MICROSCOPY
Dr. ANNA KURIAN

2. Microscopy
• defined as the use of a microscope to magnify objects too
small to be visualized with the naked eye so that their
characteristics are readily observable.

3. HISTORY
• 1676 –ANTONIE VAN LEEUWENHOEK
• Constructed simple microscope composed
of double convex glass lens held between
two silver plates
• Magnification: 50 -300 times

4. GENERAL PROPERTIES
1. GOOD MAGNIFICATION
2. GOOD RESOLUTION
3. GOOD CONTRAST

5. MAGNIFICATION
❖Refraction-phenomenon of bending of light
rays when it enter from one medium to another
❖Focal length
➢Magnification is inversely proportional to focal length(f)
➢ Short focal length object is magnified more

6. RESOLUTION
❖- Described by Ernst Abbe , Ability of lens to separate or distinguish between small
objects that are close together.
.
Abbé equation : Minimal distance (d) between two objects that reveals them as separate
entities depends on
d = 0.5 λ
n sin ϴ
( λ) : the wavelength of light used to illuminate the specimen
(n sin ϴ ) : the numerical aperture of the lens

7. NUMERICALAPERTURE (NSIN ϴ)
defined by two components:
• RI of the medium(n)
• Angular aperture(ϴ) : 1/2 the
angle of the cone of light entering
an objective
• Maximum NA of lens obtained in
air =1 as,
RI (air) =1
sinϴ =1 ( sin90)

8. GOOD RESOLUTION
1. increasing the numerical aperture
 increase the refractive index with
immersion oil
(light rays that did not enter the objective
due to reflection and refraction at the surfaces
will now do so.)
2. decreasing the wavelength
light at the blue end of the visible spectrum in the range of 450 to 500
nm

9. Working distance
➢Characteristic of an objective lens.
➢Distance between front surface of the lens and surface of the
cover glass or the specimen when in focus.
➢Objectives with large NA and great resolving power  short
working distance

10. GOOD CONTRAST
•Measure of the differences in image luminance.
•Under optimum conditions, the human eye can detect 2%
contrast .

11. LIGHT MICROSCOPES
TYPES
▪Bright-field microscpe
▪Dark-field microscope
▪Phase-contrast microscope
▪Fluorescence microscopes

12. BRIGHT FIELD MICROSCOPE
• Routinely used in microbiology labs.
• Employed to examine both stained and unstained specimens.
• forms dark image against brighter background.
• 2 types:
1. Simple Microscope
✓ contains single magnifying glass
2. Compound Microscope
✓ uses series of lenses for magnification.
✓ Total magnification =mag of objective lens * mag of ocular lens

13. STRUCTURE
1. base: Light source
2. arm.
• Two focusing knobs  on the arm.
• Stage at halfway up arm with slide
clip or stage clip.
• Sub stage condenser
3. metal body or stand
• Connects the nosepiece and ocular
lens or eyepiece
• Nose piece - 3 to 5 objective lenses of
different magnifying power.

14. PRINCIPLE
• Parfocal - Image should remain in focus when
objective are changed.
• Light from illuminated specimen is focused by
the objective lens enlarged image within
microscope ocular lens further modifies the
primary image.

15. APPLICATION
•Observation of morphology of microorganisms.
• Detection of cell structures.
•Observation of intracellular structures.
•Observation of motility.
• Measurement of size.
•Observation of blood smears

16. DARK FIELD MICROSCOPE
• Specimen : bright ; background : dark
central opaque area
peripheral annular hollow

17. PRINCIPLE
• Hollow cone of light focused on
specimen
• unreflected and unrefracted rays not
enter the objective.
• Only light reflected or refracted by
the specimen forms image.
• Field appears dark & object
brightly illuminated.

18. APPLICATION
➢Allows to observe living unstained cell
and organisms
➢Reveal internal structure in larger
eukaryotic microorganism.
➢Identify thin and distinctively shaped
Treponema Pallidum.
DISADVANTAGE :
• Not only the specimen, but dust and
other particles scatter the light

19. PHASE CONTRAST MICROSCOPE
• Invented by Frits Zernite 1930s, received
the Nobel prize in physics in 1953
• Excellent way to observe unstained living
cells.
• Contrast enhanced
• Object: dark ; background :bright
• Converts slight differences in refractive
index and cell density into easily detected
variations in light intensity.

20. COMPONENTS • Condenser annulus
• Phase plate

21. PRINCIPLE
• Light waves through a cell structure is slowed more than those in surrounding
medium
• Deviated light rays – cell structures
• Undeviated light rays –through and
around the cell
• Deviated light rays slowed more
than undeviated
• Crests and troughs do not align
• Deviated rays retarded by ¼ th the wavelength of undeviated

22. • Undeviated light rays strike phase ring in
phase plate
advanced by 1/4 wavelength
• Deviated rays miss the ring and pass through the
rest of the plate.
• Deviated and undeviated waves will be 1/2
wavelength out of phase.
• Cancel each other together form an
image.

23. • Background by undeviated light appears bright
• Unstained object appears dark and well-defined.
• This type of microscopy is called dark-phase-contrast microscopy.
• Color filters often are used to improve the image.

24. APPLICATION
• Used studying microbial motility,
• Determine the shape of living cells
• Detecting bacterial components - endospores and inclusion bodies.
• Used to study eukaryotic cells.
• Disadvantages : 1. Halo artifacts
2. low contrast specimens needs staining
.

25. DIFFERENTIAL INTERFERENCE
CONTRAST MICROSCOPE(DIC)
➢Similar to phase-contrast microscope
➢Image created by detecting differences in
refractive indices and thickness.
➢Two beams(plane-polarized light) at right
angles to each other are generated by prisms.
1. Object beam : passes through specimen
2. Reference beam : passes through clear area
of the slide.
➢ After passing through specimen, two beams are
combined and interfere with each other to form an
image.

26. APPLICATION
• Live unstained specimen : appear as a brightly colored 3 dimensional image
• Cell wall
• endospore
• granules
• vacuoles
• eukaryotic nuclei

27. FLUORESCENCE MICROSCOPE
• Principle: fluorescent dyes on exposure to uv rays get excited
• Most common- Epifluorescence microscope
• Source of light : mercury lamp
• Excitation filter
• Dichromatic mirror
• Barrier filter
• dye used: fluorochromes

28. FLUROCHROME:
FLUOROCHROMES USES
Acridine orange Stains DNA
Diamidino-2-phenyl indole(DAPI) Stains DNA
Fluorescein isothiocyanate(FITC) Attached to antibodies that bind specific
cellular components or to DNA probes.
Tetra methyl rhodamine
isothiocyanate(TRITC or rhodamine)
Attached to antibodies that bind specific
cellular components.

29. USES
• Visualize photosynthetic microbes.
• Distinguish live bacteria from dead bacteria
• Used in localization of specific proteins within
cell.
Eg:
Jelly fish (Aequorea genus)  GFP(green
fluorescent protein).

30. CONFOCAL MICROSCOPY
• Uses optical sectioning for better resolution
• When 3D objects are viewed  light enters from all areas of the
object not just the plane of focus blurred image.
• confocal scanning laser microscope (CSLM) or confocal
microscope.

31. • Uses a laser beam to illuminate a
fluorescently stained specimen
• Major component is aperture placed
above objective lens
• Eliminates stray light from parts of the
specimen that lie above and below the
plane of focus.
• Light used to create the image is from
the plane of focus clearer sharper
image formed.
• A computer attached to confocal
microscope receives digitized
information from each plane in the
specimen.
• Used to create clear composite image
• 3D reconstruction of the specimen.

32. BENEFITIS OF CONFOCAL MICROSCOPY
• Reduce blurring of image from light scattering.
• Increased & effective resolution.
• Clear examination of thick specimen.
• Z- axis scanning.
• Depth perception in Z- sectioned images.
• Magnification can be adjusted electronically.

33. USES
✓Study of biofilm
✓DNA hybridization
✓Epitope tagging
✓Stem cell research

34. THANK YOU