Microscopy And Its Details

1. MICROSCOPY
PRESENTED BY:
HASEEB AHMED (1034-FBAS/BSBT/S-18)

2. MICROSCOPY
A technique for examining tiny objects(whether living or non-living) that can’t
be seen with unaided eye by using microscopes.
OR
A science of investigating small objects and structures using a
instrument(lenses or microscope)

3. EVOLUTION OF MICROSCPES
TIME
PERIOD
HISTORY
1000C.E A glass sphere that magnified reading materials when laid
on top of them
1590C.E Zacharias Janssen invented first type of microscope
1665C.E Robert Hooke looked at silver of cork through microscope
lens
1674C.E Anton van Leeuwenhoek use self-made simple microscope
lens to examine blood, yeast and other tiny objects
1800C.E Joseph Jackson improve magnification ,developing 1st
prototype for compound microscope
1932C.E Frits Zernike invented the phase-contrast microscope for study of
colorless and transparent biological materials.
1931C.E Ernst Ruska co-invented electron microscope

4. TERMS RELATED TO MICROSCOPY
 MAGNIFICATION:
Is measure of how much larger a microscope causes an object to appear as compare to real
life.
Magnification is measured by multiples such as 2x,4x and 10x.
MAGNIFICATION
MAGNIFICATION POWER OBJECT MANIFICATION

5. CALCULATE AND ADJUSTING
MICROSCOPE’S MAGNIFICATION
 The magnification of most microscopes adjust through combining the eyepiece
and objective lens.
 The standard eyepiece magnifies 10x,objective lens magnifies up to 4x,10x and
40x.
 To calculate total magnification or magnifying power;
Total M= Me x Mo
Me=Eyepiece magnification
Mo=Objective lens magnification

6. OBJECT MAGNIFICATION
Actual Length= length of image Magnification
Magnification=Length of Image Actual length
 Magnification can be calculated using a scale bar(GRATICULE)

7. GRATICULE:
It is small transparent ruler that becomes super-imposed over the image.
Working out Magnification:
 Measure the scale bar image(beside drawing) in mm.
 Convert to micro-meter(multiply by 1000).
 Magnification=scale bar image divided by actual scale bar
length(written on scale bar).

9. RESOLVING POWER
It is the ability to distinguish distance between two dots which can be seen as
separate objects.
OR
Resolution is the measure of clarity of an image
 The smaller this value, high the resolving power of the microscope and better
the clarity and detail of image.
CONCEPT OF
RESOLUTION

10. RESOLUTION OF HUMAN EYE
The human naked eye can differentiate between two points , which are at least
0.1mm, termed as resolution of human eye.
 If we place two objects 0.05mm apart , human eye would not be able to
differentiate them as two separate objects.
 The resolution can be increased with help of lenses.

11. FACTORS AFFECTING
RESOLVING POWER
1. Objective numerical aperture
2. Type of specimen
3. Coherence of illumination
4. Degree of aberration correction
5. Wavelength of light
6. Correct alignment of microscope
7. Magnification of microscope

12. MICROMETRY
Micrometry is the science of measurement of microscopic objects especially
cells, organelles or microorganisms in terms of
 Length
 Breadth
 Diameter
 Thickness
The science involves some special types of measuring devices(micrometers), which
are well-attached and oriented in microscope , object which is to be measured is
calibrated against these scales.

13. MICROMETERS
Measurement of dimensions under microscopes involve two micro-scales called
micrometers, these are:
 Ocular Meter
 Stage Meter

14. OCULAR METER
 Ocular meter is the glass circular disc, which fits inside the circular shelf inside the
eye-piece.
 There are usually 50 or 100 divisions which are engraved on glass.
 One division of ocular = Number of stage micrometer divisions/Number of
ocular meter divisions × 10µ

15. STAGE MICROMETER
 It is for the measurement on stage of microscope, where object is to be kept.
 It is of slide’s shape and size and has mount of very finely graduated scale.
 The scale measures only 1mm(1mm=100 divisions).
L.C= 0.01mm
As 1mm=1000µ
So 1 division=10µ

16. CALIBARATION
 Calibration is very important step of micrometry, for this ocular meter is calibrated
against the standard graduations on stage micrometer.
 Calibration is required, distance bw ocular divisions varied with object used to
measure.
 For calibration both micrometers are super-imposed by rotating the eye piece.
 The number of ocular divisions coincides with stage divisions is calculated.

17. CALIBAERATION FACTOR FOR ONE
O.C DIVISION
Suppose 10 O.D with 6 S.D, then
10O.D=6S.D
BY USING FORMULA
One division of ocular = Number of S.D/Number of O.D × 10
=6/10 x 10µ
=6µ

18. PROCEDURE
1
 Remove eyepiece from the microscope, unscrew its top lid, remove it then remove
the eye lens and placed the ocular meter carefully into eyepiece, place back the
lens and fixed to its original position in microscope.

19. 2
 Clip stage micrometer into the stage and center the graduated etchings by
moving it with the help of mechanical stage
 Take lower power objective to position.

20. 3
 Rotate eyepiece till etchings on both micrometers superimposed. Search for
lines on both micrometers coinciding with each other. Count the number of O.D
equivalent to number of coinciding S.D. Calculate the calibration factor.

21. 4
 Remove the stage micrometer, place the slide containing microbes. Count the
number of O.D covered by the microbes through eyepiece. Determine the size of
microbe by multiplying number of O.D covered with that of calibration factor.

22. STAGE METER NOT USED
Stage meter is not used directly for measurement due to :-
 It is standard scale, which is costly one.
 It’s micro etchings get worn away if it used for direct measurement.
 Etchings on stage meter are more widely spaced(about 10 times) than on ocular
meter.
 Thus, the dimensions cannot be precisely measured with stage meter.

23. SPECIMEN MICROGRAPH
 MICROGRAPH
A photograph taken through a microscope is called a micrograph.
 SPECIMEN
A sample which is under observation.

24. REQUIREMENTS FOR SPECIMEN
 Cell and other elements in specimen are preserved in life-like state(process is
termed as fixation).
 Specimen is transparent one rather than opaque.
 Specimen is thin and flat one , so that single layer of cells is present.
 Some components have been differentially colored(stained),so that they can be
distinguished differentially.

25. SPECIMEN TYPES
 WHOLE-MOUNTS:
Entire organism or structure is small enough or thin to be placed directly on
microscopic slide( e.g. unicellular organism).
 SQUASH-PREPARATIONS:
Cells are intentionally squashed or crushed onto a slide to reveal their contents (e.g.
botanical specimens).
 SMEARS:
Specimen consists of cells suspended in a fluid (e.g. blood, semen, cerebra-spinal
fluid, or a culture of microorganisms).
 SECTIONS:
Specimens are supported in some way so that very thin slices can be cut from them,
mounted on slides, and stained. Sections are prepared using an instrument called
a ”microtome”.

26. MICROSCOPY CATEGORIES
LIGHT(OPTICAL)MICROSCOPES
 POLARIZING MICROSCPE
 REFLECTED LIGHT MICROSCOPE
 BRIGHT FIELD MICROSCOPE
 PHASE-CONTRAST MICROSCOPE
 FLOURESCENCE MICROSCOPE
ELECTRON MICROSCOPES
 TRANSMISSON ELECTRON MICROSCOPE
 SCANNING ELECTRON MICROSCOPE

27. LIGHT MICROSCOPE
A compound microscope is an optical instrument consisting of two convex lenses
(short focal length) used for observing highly magnified images of tiny objects.
PRINCIPLE:
when object is placed just beyond the focus of its objective lens, a virtual ,
inverted and highly magnified image of object is formed at eye piece.

28. ANATOMY OF MICROSCOPE

29. ANATOMY OF MICROSCOPE
 EYE-PIECE
The lens the viewer looks through to see the specimen. It usually comes with a
10x or 15x power lens.
 DIOPTER ADJUSTMENT
Change focus on one eyepiece so as to correct for any difference in vision
between your eyes.
 BODY TUBE(HEAD)
The body tube connects the eyepiece to objective lens.

30. ANATOMY OF MICROSCOPE
 ARM
The arm connects the body tube to the base of microscope.
 COARSE ADJUSTMENT
Brings the specimen into general focus.
 FINE ADJUSTMENT
Fine tunes the focus and increases the detail of specimen.
 NOSE PIECE
A rotating turret that spins to select different objective lens different objective
lenses.

31. ANATOMY OF MICROSCOPE
 OBJECTIVE LENSES
Major part of microscope that range from 4x to 100x.Ojective lens doesn’t touch
slide otherwise it may break the slide or damage specimen.
 APERTURE
The hole in middle of stage that allows light from illuminator to reach the
specimen.
 CONDENSER
It is used to collect and focus light on to specimen.
 IRIS DIAPHRAGM
Adjusts the amount of light that reaches the specimen.

32. RESOLVING POWER & MAGNIFICATION
 A light microscope can magnify objects up to 1500 times without causing
blurriness i.e. 1500x
 Its resolving power is 0.2µm,about the size of smallest bacterium.(1µ = 1/1000
mm).
 The image of bacterium can be magnified many times but don’t show it’s internal
structure.

33. APPLICATIONS
PATHOLOGY FORSENICS GEOPHYSICS
MICROBIAL
WORLD
ACADEMIC
EXPERIMENTS

34. ELECTRON MICROSCOPE
In EM, the object and the lens is placed in a vacuum chamber and a beam of
electrons is passed through the object. Electrons pass through or reflected from the
object and make image. Electromagnetic lenses enlarge and focus the image onto a
screen or photographic film.
 The EM has much higher resolving power than LM, which can distinguish objects
as small as 0.05 nm(1nm=1/1000,000 mm).
 The Magnification of an electron microscope may be as high as 10,000,000x times.

35. COMPARISON B/W LM AND EM
LIGHT MICROSCOPE
1. Cheap to purchase.
2. Small and portable.
3. Simple and easy sample preparation.
4. Vacuum is not required.
5. Magnifies objects up to 2000 times.
6. Stains are often needed to make the cells
visible.
ELECTRON MICROSCOPE
1. Expensive to buy.
2. Large and requires special rooms.
3. Lengthy and complex sample preparation.
4. Vacuum is required.
5. Magnifies over 500,000 times.
6. Specimens must be stained with an e-dense
chemical(like lead or gold).

36. LM VS EM
In the image above, you see clear difference, how salmonella bacteria in light
micrograph(left) vs electron microscope (right).The bacteria show up as tiny purple
dots in light micrograph, whereas in electron micrograph, you see their shape and
surface texture as well as details of human invasion by bacteria.

37. CHALLENGES WITH EM
VACUUM
• The electron beam operates within high vacuum.
• Cause problem as evaporating water destroys biological structures.
• So, samples are frozen or fixed with dehydrated solvents, stained and sectioned.
LACK OF
CONTRAST
• Biological tissue absorbs many electrons.
• Electron-dense stains are necessary to visualize samples.
TRANSPARE
NCY
•Some samples are small enough i.e. viruses are small enough can be imaged as whole.
•Most cells and tissues need to be sectioned into 50-200 nm thick slices.
CHARGING
•Biological samples are non-conductive.
•Negatively charged causes unstable, blurring the image.
•Heavy metal staining or conductive coating is applied to dissipate the charge.

38. TEM VS SEM
TRANSMISSION ELECTRON
MICROSCOPE
 TEM is based on transmitted electrons.
 Give the details of internal cell structure.
 TEM can magnify object up to 250,000
times.
 Specimen is cut into extremely thin
sections.
 In TEM,2-D image is created.
SCANNING ELECTRON
MICROSCOPE
 SEM is based on scattered electrons.
 Give the detailed architecture of cell
surfaces.
 SEM can magnify object up to 10,000 to
100,000 times.
 Require metal coating of sample, so
electrons can reflect.
 SEM is able to capture 3-D image.

39. APPLICATIONS OF EM
MATERIAL SCIENCE
FORSENIC
INVESTIGATIONS
VACCINATION
TESTING
IDENTIFYING
DISEASES &
VIRSUSES
3-D STRUCTURE OF
BIOLOGICAL
TISSUES OR CELLS