[1] microscope Flashcards
the microscope
A magnifying glass used to magnify an object to a point where it can be seen by the human eye.
what is Microscopy?
the science of investigating small objects and structures using such an instrument.
Microscopic means?
invisible to the eye unless aided by a microscope.
history of microscope
14th century
The art of grinding lenses is developed in Italy and spectacles are made to improve eyesight.
history of microscope
1590
Dutch lens grinders Hans and Zacharias Janssen made the first microscope by placing two lenses in a tube
1667
cork
Robert Hooke studied various objects with his microscope and published his results in Micrographia. Among his work was a description of cork and its ability to float in water.
a cork has hollow areas which is why it floats
1675
Anton van Leeuwenhoek (Father of Microscopy) used a simple microscope with only one lens to look at blood, insects, and many other objects. He was first to describe cells and bacteria, seen through his very small microscope with, for his time, extremely good lenses.
18th century
Several technical innovations make microscopes better and easier to handle, which leads to microscopy becoming more and more popular among scientists. An important discovery is that lenses combining two types of glass could reduce the chromatic effect,with its disturbing halos resulting from differences in refraction of light.
1830
Joseph Jackson Lister reduced the problem with spherical aberration by showing that several weak lenses used together at certain distances gave good magnification without blurring the image.
father of antiseptic surgery
There are three structural parts of the microscope:
head, base, arms
Head
carries the optical parts in the upper part of the microscope
Base
- acts as the microscope’s support
- it also carries the microscopic illuminators
Arms
- connects the base to the head and the eyepiece tube to the base of the microscope
- it gives support to the head and used when carrying the microscope
Eyepiece or oculars
*not ppt-based
magnifies the image produced by the microscope’s objective so that it can be seen by the human eye
*not ppt-based
Eyepiece rubbers
prevents scratching of lens by the user’s eyeglass or vice versa
Numbers on the eyepiece
for differences in eye grading especially for those wearing prescription glasses
Interpupillary control
used to adjust the lateral separation of the oculars for everyone
Arm or neck
*not ppt-based
Supports the microscope head and attaches it to the base
Revolving nosepiece
used to move the objectives around
Knob/screw at the back of the microscope
what happens when u turn it clockwise or counterclockwise?
- Controls the height limit of the stage to prevent breakage of glass slides
- For example, focus the slide using 40x objective then tighten the screw. Do the same with the other objectives.
- Clockwise – to tighten the screw
- Counterclockwise – to loosen the screw
Inner knob before the coarse adjustment knob (also known as?)
what happens when u turn it clockwise or counterclockwise?
- Also known as the coarse tension adjustment knob
- Prevents the stage from going down and losing focus on the specimen
- Clockwise - to tighten
- Counterclockwise – to loose
Numbers on the objectives
- refers to the thickness of the coverslip to be used
- coverslip to be used must be compatible with the objective to be used for microscopic examination
Aperture
a hole on the microscope stage through which the transmitted light from the source reaches the stage
Objectives
*not ppt-based
lenses that allow microscopes to provide magnified, real images
*not ppt-based
Mechanical stage
*not ppt-based
holds a slide in place and allows for repositioning (lengthwise and crosswise)
*not ppt-based
Stage numbers
- determines the positioning of the cells under the microscope
- provides the coordinates or location of important cells
Stage clip
to hold the object/specimen slides in place
*not ppt-based
Condenser
*not ppt-based
gather light from the microscope’s light source and concentrate it into a cone of light that illuminates the specimen
*not ppt-based
Condenser numbers
- must be the same with the magnification of the objective for better viewing of the specimen details
- determine the angle and intensity of light focused onto the specimen
Iris Diaphragm
*not ppt-based
- below condenser
- adjust the brightness and contrast of the specimen
*not ppt-based
Coarse adjustment knob
*not ppt-based
- allows for quick focusing by moving the objective lens or stage up and down
- used in scanning and LPO
- NEVER use in HPO and OIO
*not ppt-based
Fine adjustment knob
*not ppt-based
- help maintain focus as the magnification increases
- used in all objectives but mostly HPO and OIO
*not ppt-based
Brightness adjustment knob/ Microscopic illuminator
*not ppt-based
controls the voltage supplied to the light bulb, controlling the intensity (brightness) of the light bulb
*not ppt-based
Working distance
- Working distance is the distance between the objective and the slide when objective is sharp focus.
- The higher the magnification of the objective, the shorter the working distance will be.
- Coarse adjustment should not be used when using higher magnifications.
Different types of microscopes
- Compound/Upright
- Inverted
- Stereomicroscope
Compound/Upright
used mostly in what section of the lab?
- Objective lens faces downward, and specimens is observed from above
- Suitable for observation of glass slides or prepared slides
- Used for routine microscopic examinations
clinical microscopy and microbiology
Inverted
used when?
- Objective lens faces upwards, and specimen is observed from below
- Used for observation of culture container (stem cells/unstained cells)
only if micro exam uses samples that are grown in culture bottles/plates
Stereomicroscope
used for?
- 2 separate light path, enables observation of specimen from different angles at both eyes
- Allow 3D observation of sample
- Suitable for animal works and large sample imaging
parasites
Applications of the Microscope
- Bright Field Microscopy
- Dark Field Microscopy
- Phase Contrast Microscopy
- Electron Microscopy
– Transmission Electron Microscope (TEM)
– Scanning Electron Microscope (SEM)
Brightfield Microscopy
- The simplest techniques of all the optical microscopy illumination
- Specimen’s image appears dark against a bright background
- Light source is usually halogen/LCD lamp
- Generally used with compound microscopes
Resolving Power
depends on what factors?
- Ability to distinguish between two adjacent points.
Depends on two factors: White light & Numerical aperture
White Light
Composed of a mixture of colored lights of various wavelengths
Numerical Aperture
Expression relating to the cone of light that is delivered to the specimen by the condenser and gathered by the objective
Total Magnification
give magnification for each objective
the ocular magnification multiplied by the objective magnification
magnification x ocular lens = total magnification
Scanning: 4x X 10x = 40x
Low power: 10x X 10x = 100x
High power: 40x X 10x = 400x
Oil immersion: 100x X 10x = 1000x
A factor for getting a good slide
little bonus lol
Good sample = good slide
Phase Contrast Microscopy
used for?
- Uses a special optic system which converts differences in phases into differences in intensity such that some structures appear darker than the others
- Reveals the gross details of the internal structures in a living cell
bacterial spores, kanang ga siga
Darkfield Microscopy
- Objects appear brilliantly illuminated against a black background
- Used to demonstrate spirochetes which are difficult to observe in transmitted light
Electron Microscopy
- TEM (Transmission Electron Microscope)
- SEM (Scanning Electron Microscope)
Transmission Electron Microscope (TEM)
used to observe what?
- Develops an image resulting from variable electron density of the specimen interposed in the electron beam
- Produces monotone, two-dimensional, high magnified images
- Resolving power microscopesis 0.001 um, which is 200X that of light microscopes
- Specimens must be fixed, stained, and dried
Used to observe internal ultra-structural detail of cells observations of viruses or small bacteria
Scanning Electron Microscope (SEM)
used to observe what?
- Has a practical limit of resolution of 0.005um or five-fold less than that of TEM
- Produces monotone, tri-dimensional image by detection at a 90 degree angle of secondary electrons emitted from the specimen surface as a result of bombardment by the primary electron beam
Used to observe the surface details of structures
What could (external) cause blurred image?
treatment?
Microscope is too close to mechanical appliances or machinery that can cause external vibrations
- Remove the microscope from the source of vibration
- Use a sturdy table as a support or use a vibration proof table
What external cause could prevent correct focusing?
treatment?
- microscope is near a window
- bright light from the window prevents correct focusing
- Set up the microscope near a wall
- Position the microscope in such a way that the overhead light falls slightly in front of the microscope
What external cause could reproduce room light or flares on the photo?
treatment?
microscope is in a place where the room light enters the eyepiece
- Cover the eyepiece with caps
- Shut out stray light getting into the eyepiece or the focusing telescope by changing the optical path selector
Black spots (molds) are reproduced on the specimen image?
treatment?
- microscope is in a dusty and dirty room
- microscope placed near window where the dust can enter from the outside
- Set up the microscope in another room
- Cover the whole microscope with a dust proof covering
microscope maintenance (8)
- Treat lenses with great care as they can be easily scratched. Never use anything abrasive.
- When cleaning lenses, first blow away any dust with a blower brush, then use lens tissue and lens cleaning fluid to clean the objectives and eyepieces. Do not use paper towel or regulartissues, as they will scratch the lens. Do not use other solvents.
- Do not remove eyepieces or objectives from their location but clean only their external surfaces.
- Remove immersion oil from the 100x objective immediately after use with lens tissue and lens cleaning fluid.
- Wipe dust off the body of the microscope with a damp cloth
- Never attempt to take a microscope apart. This could impairoperation, efficiency, and accuracy.
- Have the microscope serviced regularly by a professional, as most microscopes require periodic lubricating and minor adjustment of their mechanical parts.
- Follow your user’s manual for instruction in replacing the bulb.Always allow a bulb to cool before replacing it. When replacing bulbs avoid touching the glass with your hands, use a tissue.Fingerprints can reduce bulb quality and reduce its life.
No light (6)
- Power cord is not connected, power switch is off
- Wrong bulb is installed
- The bulb has burnt out
- Light intensity control is turned down too low
- Objective is not properly in position
- If using the 100x objective immersion oil has not been applied
Image is too dark (3)
- Increase light intensity
- Sub-stage iris diaphragm is not open enough
- Condenser is too low
Image is too light (2)
- Decrease light intensity
- Sub-stage iris diaphragm is open too much
Spot in the field of view that doesn’t move when the slide is moved (1)
- Lens is dirty. Clean both the objective and eyepiece.
Poor image quality, poor resolution, image not sharp (100x oil objective) (3)
- Clean objective, eyepiece and condenser
- Check if immersion oil is contaminated or cloudy or air bubbles are present
- Slide is wrong way up
Poor image quality, poor resolution, image not sharp (40x objective) (1)
- There is dirt or oil on the lens
Uneven illumination (2)
- Adjust condenser
- Make sure objective has clicked into place
Flickering light (4)
- Bulb needs replacement
- Loose connection at the outlet
- Bulb not properly inserted
- Check voltage supply
Half the viewing field is illuminated (1)
- Make sure the objective is clicked into place
Unable to focus the slide (4)
- Coverslip is too thick
- Slide is the wrong way up
- The stage is slowly dropping, adjust tension of course focus knob
- Clean the slide, objective, and eyepiece
How should you hold a microscope?
bonus ish ?
Dominant arm holds the arm, while non-dominant arm holds the base.
