light microscope and its types, princple, uses application, advantages and limitation

1. Light
microscope
Dr. Rasika Deshmukh
(MBBS, MD)

2. • The evolution of the Microbiology field put to
perspective the need to identify, view, observe
and understand microorganisms, including
their structural morphologies and
mechanisms.

3. History
• The first simple microscope was discovered
by two Dutch scientists, Zaccharias Janssen
and his father, Hans who made spectacles,
were the first to experiment with their lenses
by combining lenses in a tube and observed
that the objects that were nearby, appeared
closer and larger.

4. History
• Antony Van Lewnehoueek an amateur
Microbiologist made the first simple
microscope, that enabled him to observe the
presence of tiny living organisms in pond
water that appeared like dots. His simple
microscope was made up of a double convex
glass lens that was held between two silver
plates.

5. History

6. What is a light microscope?
• Instrument or tool, that uses visible light to
detect and magnify very small objects and
enlarge them.
• Use lenses to focus light on the specimen,
magnifying it thus producing an image

7. Principles of Light Microscopy
• For light microscopy, visible light is passed
through the specimen and then through a
series of lenses that bend the light in a
manner that results in magnification of the
organisms present in the specimen . The total
magnification achieved is the product of the
lenses used.

9. Key component to light microscopy
Magnification
• Objective lens magnifies objects 100× (times)
• the ocular lens magnifies 10×.
• Using these two lenses in combination,
organisms in the specimen are magnified
1000× their actual size when viewed through
the ocular lens.

10. Contrast
• Needed to make objects stand out from the
background
• Contrast is most commonly achieved by
staining techniques that highlight organisms
and allow them to be differentiated from one
another and from background material and
debris.

11. Resolution
• To optimize visualization, other factors besides
magnification must be considered.
• Resolution, defined as the extent to which
detail in the magnified object is maintained, is
also essential.

12. Types of microscopes
Depending on the number of lenses, there are
two types of microscopes
• Simple light microscope (it has low
magnification because it uses a single lens)
• Compound light microscope (it has a higher
magnification compared to the simple
microscope because it uses at least two sets of
lenses, an objective lens, and an eyepiece).

13. Light microscope
The modern types of Light Microscopes include:
• Bright field light microscope
• Dark field light microscope
• Phase contrast light microscope
• Fluorescence light microscope

14. Bright field microscope
• most basic optical Microscope
• produces a dark image against a bright
background
• provide a high-resolution image

15. Parts of a bright-field microscope

16. • Objective lens: magnifies the image by 10x, 20x,
40x and 100x
• Ocular lens: magnifies the image by 10x
• Condenser: focuses a beam of light onto the
specimen
• Diaphragm: controls the diameter of the beam of
light that passes through the condenser
• Two focusing knobs: focus on the image and
sharpen the image.

18. Applications of Microscopy in
Diagnostic Microbiology
• Rapid preliminary organism identification by direct visualization in patient
specimens
• Rapid final identification of certain organisms by direct visualization in patient
specimens
• Detection of different organisms present in the same specimen
• Detection of organisms not easily cultivated in the laboratory
• Evaluation of patient specimens for the presence of cells indicative of
inflammation (i.e., phagocytes) or contamination (i.e., squamous epithelial cells)
• Determination of an organism’s clinical significance; bacterial contaminants usually
are not present in patient specimens at sufficiently high numbers (×105 cells/mL)
to be seen by light microscopy
• Provide preculture information about which organisms might be expected to grow
so that appropriate cultivation techniques are used
• Determine which tests and methods should be used for identification and
characterization of cultivated organisms
• Provide a method for investigating unusual or unexpected laboratory test results

19. DARK GROUND MICROSCOPE
• Background is dark
• Object or organism appears bright

20. PRINCIPLE OF DARK GROUND
MICROSCOPE
• To view a specimen in dark field, an opaque
disc is placed underneath the condenser lens,
so that only light that is scattered by objects
on the slide can reach the eye
• Instead of coming up through the specimen,
the light is reflected by particles on the slide.

23. Application of Dark ground microscopy
• demonstration of very thin bacteria not visible under
ordinary illumination
• method for rapid demonstration of Treponema pallidum
• demonstration of the motility of flagellated bacteria and
protozoa.
• Darkfield is used to study marine organisms such as algae,
plankton, diatoms, insects, yeast and protozoa
• Darkfield is used to study mounted cells and tissues.
• It is more useful in examining external details, such as
outlines, edges, grain boundaries and surface defects than
internal structure.
•

24. Bright field vs Dark field Microscope
Bright field microscope Darkfield microscope
Bright field microscope formed a dark
image against bright field.
Darkfield Microscope formed a bright
image against dark background
Stained, fixed, and live specimens are
observed.
We can see the living and unstained cells.
In a bright-field microscope, only the
scattered lights are able to enter the
objective lens and transmitted lights or
unscattered light rays are omitted, that’s
why the viewer sees a dark image against
the brightfield.
The only light that has been reflected or
reflected by the specimen forms an
image.
Organism appear as dark Organism appear as bright
Background appear as bright Background appear as dark.

25. Bright field vs Dark field Microscope
Bright field Microscope Dark field Microscope
It shows us the morphological as well
as the internal structure of the
specimen.
It show us External structure of
specimen in great detail.
Bright fields are inexpensive Dark fields are expensive.
Specimen preparation or staining is a
complex and lengthy process. Takes
too much time.
No need stain.
Opaque disc is Absent Opaque disc is Present
It is easy to use.
operating procedure is not easy as
compared to bright field microscope.

26. Phase contrast microscope

30. Application

31. The Fluorescent Microscope
• dye molecule added to the specimen.

36. Advantages
• helps in the study of cell behaviour.
• highlights the biomolecule of interest.
• highlight the image of particular structural
components within the microscopic organisms.
• highly sensitive technique that can detect around
50 molecules/µm3.
• analyze or track the physiochemical properties of
multiple biomolecules simultaneously.

37. • Disadvantages
• It only allows the observation of specific structures
inside a cell tagged with the fluorescent dye.
• The photobleaching due to the electron excitation
during the process of fluorescence may affect reactive
molecules of the fluorescent dyes. As a result, the
reactive dyes might lose their chemical property of
fluorescence emission intensity.
• The cells are susceptible to the phototoxic effect after
staining with fluorescent dyes, as the fluorophore
molecules absorb the high energy photons from the
short-wavelength light.

38. Thank you