Microscope
DEFINITION
- A microscope (Greek: micron = small and scopos = to look)
- MICROSCOPE: Is an instrument for viewing objects that
are too small to be seen by the naked eye
- Microscope must accomplish three tasks:
* Magnification
* Resolution
* Contrast
3
MAGNIFICATION
Degree of enlargement
No of times the length, breadth or diameter, of an
object is multiplied.
Which of these images
would be viewed at a
higher power of
magnification?
4
RESOLUTION
Ability to reveal closely adjacent structural
details as separate and distinct
LIMIT OF RESOLUTION (LR): The min distance between two visible bodies at
which they can be seen as separate and not in contact with each other
Types of microscope
Resolving power
Compound Microscope
200 nanometers
Scanning Electron Microscope
10 nanometers
Transmission Electron Microscope
0.2 nanometers
5
Contrast
- Giving the details visible to the eye, camera, or other imaging
device
HISTORICAL
BACKGROUND
nd
Century BC: Ptolemy describes a stick that appears to bend in water
st
Century: Romans experiment with glass and observe objects larger when
viewed through glass
12thCentury: Salvino DArmante made first eye glass
1590: Hans Janssen and his son Zacharias Janssen, mount two lenses in a
tube to create first compound microscope
1609: Galileo Galilee develops compound microscope with concave and convex
lens
1665: Robert Hookes book Micrographia officially documents observations
through the microscope
1674: Anton van Leeuwenhoek achieves greater magnification allowing him to
observe bacteria. Generally credited with bringing the microscope to the
attention of biologists.
10
Safety
IS FIRST
Biological contamination: Gloves
Grounded electrical connection (ideal)
Immersion oil in eyes
Location & Storage
Do not use under vibration
Do not store in high humidity
Avoid intense temperatures
Keep clean
Wear Personal Protective Equipment
Always check PPE for defects or tears before using
If PPE becomes torn or defective remove it and replace with new
equipment
Do not reuse disposable equipment
Dispose of contaminated PPE properly
Wash hands immediately after removing PPE
Treat all blood and bodily fluids as if they are contaminated
Wash thoroughly during cleanup and decontamination
Properly dispose of all contaminated material
The following symbols are found on the microscope. Study the meaning of the symbols
and always use the equipment in the safest possible manner
TYPES OF MICROSCOPE
- Simple microscope
- Compound microscope
- Electron Microscope
- Phase Contrast
Microscope
- Dark Ground Microscope
- Fluorescent Microscope
- Others
SIMPLE MICROSCOPE
More than five hundred years ago, simple glass magnifiers were
developed. These were convex lenses (thicker in the center than the
periphery). The specimen or object could be focused by use of the magnifier
placed between the object and the eye. These simple microscopes, along
with the cornea and eye lens, could spread the image on the retina by
magnification through increasing the visual angle on the retina.
Convex Lenses are curved glass used to make microscopes (and glasses etc.)
Convex Lenses bend light and focus it in
one spot
The image produced by such a magnifier, held close to the observers eye,
appears as if it were on the same side of the lens as the object itself. Such an
image, seen as if it were ten inches from the eye, is known as a virtual image.
These magnifiers had severe limitations in specimen positioning, illumination,
lens aberrations, and construction.
Simple magnifier. A simple magnifier uses a single lens system to enlarge the object in
one step
COMPOUND
MICROSCOPE
18
Around the beginning of the 1600s, through work
attributed to the Janssen brothers in the Netherlands and
Galileo in Italy, the compound microscope was developed. In
its basic form, it consisted of two convex lenses aligned in
series: an object glass (objective) closer to the object or
specimen, and an eyepiece (ocular) closer to the observers
eyewith means of adjusting the position of the specimen
and the microscope lenses. The compound microscope
achieves a two-stage magnification. The objective projects a
magnified image into the body tube of the microscope and
the eyepiece further magnifies the image projected by the
objective
For example
The total visual magnification using a 10X objective and a 15X eyepiece
is 150X
Compound magnifier. In the compound microscope, the intermediate image formed by
the objective and tube lens is enlarged by the eyepiece.
Parts of Microscope
i. Optical component
ii.Mechanical component
iii.Electrical component
Optical component
OBJECTIVE LENS
1. Its basic function is to gather the light passing through the specimen
and then to project an accurate, real, inverted IMAGE of the specimen
up into the body of the microscope.
2. Other related functions of the objective are simply to magnified the
specimen with a given magnification rate i.e 4x,10x ,40x and 100x
magnification power
Scan - 4X
Low Power - 10X
TYPES
High Power - 40X
Oil immersion - 100X
OIL IMMERSION OBJECTIVE
- Highest magnification
- Oil prevents refraction of light outwards and
allows it to pass straight in to objective
G
FBEG - OIL
ABCD - AIR
OIL
C GLASS
B
F
25
Using the 100x immersion objective
1.
2.
3.
Focus on the specimen using all objectives,
starting from the lowest-power to the
highest-power objective
Before engaging immersion objective, place
a drop of oil onto the specimen.
Engage the immersion objective
4.
5.
Make sure oil is free of bubbles
Use the fine adjustment knob to bring the
specimen into focus
After use, remove oil from objective front
lens by wiping with gauze moistened with
70% alcohol
Working distance
Objective
Magnification
Working
Distance
4X
18.5
10X
10.6
40X
0.6
100X
0.13
EYE PIECE
1. Its basic function is to look at the
focused, magnified real image
projected by the objective.
2. They are usually 10x,15x,40x and
45x
- TYPES
(a) Monocular(Huygenian)
(b) Binocular (Ramsden)
(c) Trinocular (Compensating)
31
Whats the power of this lens?
To calculate the power of magnification, multiply the power of the
ocular lens by the power of the objective, e..g.: 10x40=400 times
What are the powers of
magnification for each
of
the objectives we have
on our microscopes?
Fill in the table on
your worksheet.
Comparing Powers of Magnification
We can see better details with higher the powers of
magnification, but we cannot see as much of the image.
10x
Which of these images
would be viewed at a
higher power of
magnification?
40x
SUBSTAGE CONDENSER
1. Its basic function is to gather the light coming from the light source and to
concentrate(focus) that light in a collection of parallel beams onto the
specimen.
COLLECTOR LENS.
This lens is placed in front of the light source. Its function is to project an
image of the light source onto the plane of the condensers aperture
diaphragm. In some instruments a diffusion or frosted filter is placed just
after the collector lens (side closer to the specimen) in order to provide
more even illumination.
FIRST SURFACE MIRROR
Its function is to reflect the light coming from the lamp up into the
substage condenser. Just before that mirror (closer to the lamp side)
is another variable diaphragm known as the field diaphragm
TUBE LENS
Its function is to gather the parallel rays of light projected by the objective
and bring those rays to focus at the plane of the fixed diaphragm of the
eyepiece.
FILTERS - Blue, Green,
Heat absorbing filters,
Barrier filters.
ii. Mechanical component
Body Tube
The body tube holds the objective lenses
and the ocular lens at the proper distance
Diagram
Nose Piece
The Nose Piece holds the objective lenses
and can be turned to increase the
magnification
Diagram
Stage Clips
These 2 clips hold the slide/specimen in
place on the stage.
Diagram
CONDENSER HOLDER.
This holder receives any one of several types of condenser
Diaphragm
The Diaphragm controls the amount of light
on the slide/specimen
Turn to let more light in or to
make dimmer.
Diagram
Arm
Used to support the microscope when
carried. Holds the body tube, nose piece
and objective lenses
Diagram
Stage
Supports the slide/specimen
Diagram
Coarse Adjustment Knob
Moves the stage up and down (quickly) for
focusing your image
Diagram
Fine Adjustment Knob
This knob moves the stage SLIGHTLY to
sharpen the image
Diagram
Base
Supports the microscope
Diagram
PARTS OF COMPOUND MICROSCOPE
48
iii. Electrical component
ILLUMINATION - It is light source
Lamp, sunlight, battery
operated lamp, 60 W bulb,
Quartz halogen light.
* step-down transformer
* switch
* Rheostat
* lamp (halogen or incandescent)
* fuse
* Electronic sub system
Light Source
Projects light upwards through the
diaphragm, the specimen and the lenses
Some have lights, others have mirrors
where you must move the mirror to reflect
light
Diagram
Operational Principle
Light
Knowledge of the behavior of light and the effects resulting when light passes from
air through a glass convex lens and out into air again is fundamental to the
understanding of image formation. When light passes from air into a convex lens,
the speed of light is slowed. The various colors, differing in wave length, are slowed
at different rates (dispersion). This bending (refraction) effect differs for different
colors. Those rays which strike the central area of the lens at a perpendicular
emerge unrefracted. Light passing through the other parts of the convex lens are
refracted or bent. The blue rays are bent more than thegreen rays, more than
the red rays.
When white light passes through convex lenses of objectives, eyepieces, or
condensers, two main kinds of aberrations may occur; chromatic aberration and/or
spherical aberration. These aberrations can be corrected in the design of the
lenses.0
53
Installation and commission
DETAILED OBSERVATION PROCEDURE
General Maintenance
Preventive Maintenance
Daily Preventative Maintenance
Yearly Preventative Maintenance
Objective (after using oil immersion lens)
Body
Microscope frame
Observation Tube optical axis
Observation Tube left/right axis
Observation Tube revolving axis
Observation Tube parfocality
As needed
Grease replacement parts
General Maintenance
General Maintenance
General Maintenance
Cleaning the eyepieces
Never rub the lens surface strongly
Do not use the same lens tissue to clean more than one lens
Do not moisten lens tissue with excessive amounts of cleaning solution
When cleaning with tweezers, be careful not to protrude its tip from the lens tissue
General Maintenance Cleaning the Objective Lens
*When cleaning oil immersion objective, first wipe objective with a dry lens tissue
General Maintenance
General Maintenance
General Maintenance
Preparing for Inspection
General Maintenance
Inspection Sheet
Use the CX21 inspection sheet
and check the electrical unit,
mechanical and optical
performance
General Maintenance
Inspection: Checking for Dust
Image influence caused by dirt on each component
Close aperture iris diaphragm
When aperture iris diaphragm is closed
the depth of focus increases and the dirt
position is brought into focus
Observe a specimen through the
eyepiece
General Maintenance
Inspection: Checking for Dust
Note: If dirty portion
can not be
identified, it is
assume that the
internal lens and
prism are
contaminated.
General Maintenance
Inspection: Checking Lenses
General Maintenance
Inspection: Checking Lenses
General Maintenance:
Checking Left/Right Alignment
Place the cross eyepiece into the right
sleeve
Place a slide with an image with a
recognizable reference point (e.g. center
of a spiral), secure it on stage
Align the reference point in the center of
the cross of the eyepiece (looking
through right eyepiece) by moving the
stage with the control knob
General Maintenance:
Checking Left/Right Alignment
Cont.
General Maintenance:
Checking Left/Right Alignment
Cont.
General Maintenance:
Checking Left/Right Alignment
Switch the cross eyepiece to the left
sieve
If the crosshair is aligned with the same
reference point, the left/right alignment is
correct
If the crosshair is not aligned, do NOT move
the stage. The left eyepiece sleeve needs
adjusted.
General Maintenance: Fixing
Left/Right Alignment
General Maintenance: Fixing
Left/Right Alignment
General Maintenance: Fixing
Left/Right Alignment2`--------------
Common Failures
Electrical/Power Failure
Light does not turn on when power switch is turned on
Light bulb burnt out
Objective not placed in alignment
Dirty components
Eyepiece
Objectives
Filters
General User Error
Not in focus
Brightness not adjusted properly
Objective not rotated fully into secured position
Troubleshooting
What is troubleshooting?
A logical, systematic process that is able to determine "what" happened, "why" it
happened and a method to develop effective fixes for the "why" it happened.
Why do we Troubleshoot?
To prevent the equipment and machinery from repeat incidents and failures
5 Steps of Troubleshooting
o Preparation
o Step 1 Observation
o Step 2 Define Problem Area
o Step 3 Identify Possible Causes
o Step 4 Determine Most Probable Cause
o Step 5 Test and Repair
o Follow-up
Preparation
o Before you begin to troubleshoot any piece of
equipment, you must be familiar with safety rules and
procedures for working on electrical equipment.
Be Safe!
Turn off power, ground yourself, and use gloves when
appropriate
o Next, gather information regarding the equipment and
the problem.
Be sure you understand how the equipment is designed to
operate it.
Operation or equipment manuals and drawings are great
sources of information and are helpful to have available.
If there are equipment history records, you should review
them to see if there are any reoccurring problems
Step 1: Observation
o What Happened?
o Most faults provide obvious clues as to their cause.
o Through careful observation and a little bit of reasoning,
most faults can be identified as to the actual component
with very little testing.
o When observing malfunctioning equipment, look for
visual signs of mechanical damage such as indications
of impact, chafed wires, loose components or parts lying
in the bottom of the cabinet.
o Look for signs of overheating, especially on wiring, relay
coils, and printed circuit boards
o Do not forget to use your other senses
when inspecting equipment.
The smell of burnt insulation is something you would not
miss.
Listening to the sound of the equipment operating may
give you a clue to where the problem is located.
Checking the temperature of components can also help
find problems, but be careful while doing this, some
components may be alive or hot enough to burn you.
o Pay particular attention to areas that
were
identified either by past history or by the person that
reported the problem.
Here is a note of caution! Do not let these
mislead you, past
problems are just that past problems, they are not
necessarily the problem you are looking for now.
Also, do not take reported problems as a
fact, always check
for yourself if possible. The person reporting the problem
may not have described it properly or may have made their
own incorrect assumptions.
Step 2: Define the Problem Area
o At this stage you apply logic and reasoning to your
observations to determine the problem area of the malfunctioning
equipment.
Often times when equipment malfunctions, certain parts of
the equipment will work properly while others not.
o The key is to use your observations (from step 1) to rule out
parts of the equipment or circuitry that are operating properly and
not contributing to the cause of the malfunction.
You should continue to do this until you are left with
only the
part(s). If faulty, it could cause the symptoms that the
equipment is experiencing.
o To help you define the problem area you should have a
schematic diagram of the circuit, in addition to your noted
observations.
o Starting with the whole circuit as the problem area, take each
noted observation and ask yourself, "what does this tell me about
the circuit operation?".
If an observation indicates that a section of the circuit
appears to be operating properly, you can then eliminate it
from the problem area.
As you eliminate each part of the circuit from the problem
area, make sure to identify them on your schematic. This will
help you keep track of all your information.
Step 3: Identify Possible Causes
o Once the problem area(s) have been defined, it is necessary
to identify all the possible causes of the malfunction.
This typically involves every component in the problem
area(s).
o It is necessary to list (write down) every fault which could
cause the problem no matter how remote the possibility of it
occurring.
Use your initial observations to help you do this.
During the next step you will eliminate those which are
not likely to happen.
Step 4 Determine the Most Probably
Cause
o Once the list of possible causes has been made, it is then
necessary to prioritize each item as to the probability of it being the
cause of the malfunction.
The following are some rules of thumb when prioritizing
possible causes.
Although, it may seem to be possible for two components to
fail at the same time, it is not very likely. Start by looking for
one faulty component as the culprit.
o The following list shows the order in which you should check
components based on the probability of them being defective:
1. Components that burn out or have a tendency to wear out: i.e.
mechanical switches, fuses , relay contacts, and light bulbs.
(Remember, fuses burn out for a reason. You should find out why
before replacing them.)
2. Coils, motors, transformers, and other devices with windings:
These usually generate heat and with time, can malfunction.
3. Connections: especially screw or bolted type. Over time these
can loosen and cause a high resistance. In some cases this
resistance will cause overheating and eventually will burn open.
Connections on equipment that is subject to vibration, are
especially prone to coming loose.
4. Defective wiring: Pay particular attention to areas where the
wire insulation could be damaged causing short circuits. Do not
rule out incorrect wiring, especially on a new piece of equipment.
Step 5 Test and Repair
o Once you have determined the most
probable cause, you must either prove it to be
the problem or rule it out by
inspection/observation or by using test
instruments
o Test Instruments: used to help
narrow the problem area and
identify the problem component.
specialized instruments designed
to measure various behaviors of
specific equipment
General instruments (i.e. multimeters)
o Important Rule: when taking meter readings, predict what the
meter will read before taking the reading.
Use the circuit schematic to determine what the meter will
read if the circuit is operating normally.
If the reading is anything other than your predicted value, you
know that this part of the circuit is being affected by the fault.
o Depending on the circuit and type of fault, the problem area
as defined by your observations, can include a large area of the
circuit. It creates a very large list of possible and probable
causes.
Use a divide and eliminate approach to eliminate parts of the
circuit from the problem area.
The results of each test provides information to help you
reduce the size of the problem area, until the defective
component is identified.
o Once you have determined the cause of the faulty operation
of the circuit, replace the defective component.
o After replacing the component, you must test operate all
features of the circuit, to be sure you have replaced the
proper component and that there are no other faults in
the circuit.
It can be very embarrassing to tell the customer that you
have repaired the problem only to have him find another
problem with the equipment just after you leave.
Follow-Up
Not an official step of the troubleshooting process, but it
should be done once the equipment has been repaired and
put back in service.
o Try to determine the reason for the malfunction.
Did the component fail due to age?
Did the environment the equipment operates in cause
excessive corrosion?
Are there wear points that caused the wiring to short out?
Did it fail due to improper use?
Is there a design flaw that causes the same component to
fail repeatedly?
o Through this process further failures can be minimized.
o Many organizations have their own follow-up documentation
and processes
If you determine there was human performance (or user
error) involved in the equipment issue, the troubleshooter
needs to step out of the equipment
analysis role and
interview people who have interacted with the equipment or
machinery in question.
May need to properly train users on equipment usage.
Troubleshooting
Troubleshooting
Electrical Unit
If lamp does not turn on when power switch
is turned on, there is a problem with the
electrical unit, unless
Troubleshooting
Electrical Unit
After verifying the light bulb is not the issue,
check that the microscope is plugged in and
that outlet has power(use another device
you know to be working to validate this)
Verify the chord shows no obvious damage
Verify the fuse is intact
Troubleshooting
If the previous steps revealed no culprit
The problem is most likely the circuit board
itself
Replacing
individual components is not feasible,
entire board needs replaced
If an identical microscope exists that is not
usable for another reason, harvesting its circuit
board is an option. See the manual for
instructions on removing the circuit board
Troubleshooting
Coarse/Fine adjustment knobs do not
turn smoothly
New grease needs applied to knobs
If using a different microscope, turn to
manual for knob-removal instructions
Instructions for CX-21 provided next
Troubleshooting: Focus Knobs
Dissassemble fine
adjustment knob
assembly (A) and fine
adjustment knob(B) by
turning them in arrow
directions
Remove the spring
washer (C) and washer
(D)
Remove the fine shaft
mount (E) with a
spanner (wrench) while
holding the coarse
adjustment knob (F)
Troubleshooting: Focus Knobs
Remove the coarse adj.
knob (G) by turning it
counterclockwise while
holding the coarse adj.
knob (F).
Remove the tension
knob (H) by turning it
counterclockwise.
(The washer (I) is
attached to tension
knob (H) with grease.)
Troubleshooting: Focus Knobs
Pull out the tension
ring (J) while holding
the convex part using
pliers
Place grease wherever
indicated in figure
Reassemble them in
the reverse order
Troubleshooting
Stage falls down spontaneously or an infocus image quickly moves out of focus
Cause: tension on coarse adjustment knob
is too loose, needs to be adjusted
Troubleshooting: Coarse Knob Tension
On the CX-21, the tension of the
coarse adjustment knob can be
adjusted by inserting a flat head
screwdriver into (2). Rotating
clockwise increases tension,
rotating counterclockwise
reduces tension
If the stage is falling on its own,
increase the tension.
If the knob is hard to turn and
grease has already been
applied, reduce the tension
slightly
Troubleshooting: Stage Movement
If the stage does not move smoothly in
the X or Y directions, the tension of the
X/Y wires must be adjusted
Instructions for CX-21 are provided in
following slides, for other models see
manual
Troubleshooting: Stage Movement
Troubleshooting: Stage Movement
X-tension adjustment
Troubleshooting: Stage Movement
Y-tension adjustment
Troubleshooting: Backlash
CARE OF THE MICROSCOPE
- Handling
- Proper storage
- Care of Lenses
- Care of mechanical part
- Care of electrical part and lamp
129
Care and maintenance of your microscope
The microscope has 3 major enemies:
1.
Dust
Protect the microscope from dust- make sure you use a dust
cover.
NEVER CLEAN THE OPTICS WITH A DRY TISSUE /
SWAB/ COTTON BUD OR YOUR SHIRT SLEEVE! This will
scratch the lens and cause irreparable damage. This is
because the lens is usually coated with an anti-glare layer
(bluish in colour). Try to remove dust from the optics by
blowing gently on them, or use an air blower.
SOLUTION: make sure you use a dust cover when the
microscope is not in use or is being stored.
Dusty microscope image
Source: www.funsci.com/fun3_en/dust/dust.htm
2.
Humidity
Humidity (warm and wet) offers the perfect environment for
fungus to grow.
Fungal growth will occur primarily on the optics of the
microscope. Once you have a fungal infection on your
microscope it is difficult to remove and may permanently
damage your optics.
Using a plastic dust jacket (like the one supplied with the
scope) is a bad idea.
SOLUTION: Buy some thick calico and use the plastic cover as a
template to make dust covers that are washable and can
breathe. This prevents condensation inside the cover and the
development of ideal conditions for fungal growth (warm and
wet)
3.
Water
Microscopes are electrical items. Water
and electricity are a dangerous mix!
Storing your microscope when it is wet
may lead to corrosion of parts and hygiene
problems. See also enemy number 2!
If wet mounts are used, the stage is to be
cleaned and dried before returning the
scope to storage. The optics must be kept
dry to prevent fungal attack.
Summary
Safety is the first priority: microscopes are used to
examine biological specimens, gloves are imperative
Microscopes have many moving parts which must be
properly positioned for clear viewing
Always check that the microscope is being used
properly before assuming there is another problem
Dirt and dust can reduce image quality, when trying to
deduce debris location
