Observing Microscopes through a microscope Flashcards
Which metric units are used to measure microorganisms ? compare the size of bacterial cells, vs viruses and mammalian cells What is the similarity when looking at metric unit of meter to millimeter?
MICROMETERS (um) are used to measure microorganism
Viruses are smaller than bacterium, bacterium is smaller than mammalian cells Largest)
from meter to millimeter; units are are divisible by 10 each time
(decimeter =1/10; centimeter= 1/100; millimeter= 1/1000)
anything below millimeter will divide by 1000 (ex; nano, pico)
Discuss the units of measuremnt of how to convert micrometers to meters and millimeters; nanometers to millimeters, converting nanometers to micrometers and also micrometers to nanometers
Unit of Measurement
1um= 10^-6 m= 10^-3 mm (divide 10^-6/10^-3)
1 nm= 10^-9 m= 10^-6 mm
1000 nm = 1 um (micrometer)
0.001 um= 1 nm
What is an Angstrom (A) ? What units are used?
Angstrom- another unit used predominantly for short distances between atoms
A= 10^-10 M or 0.1 nm.
can be used to measure visible light or protein crystals
What is the Limit in size of organism that humans can see with a naked eye?
200 um (limit that can be seen)
What kind of microscope can viruses be seen with? what microscope cannot see viruses? what kind of microscope can view Biological samples ?
Viruses can be seen with Transmission Electron microscope (TEM)
-viruses CANNOT be seen with light microscope
TEM see biological samples
Discuss the different types of microscopy and what kind of structures that are able to be seen
TEM (transmission electron microscope) - can view viruses,(TEM limit 10 pm-100 um)
-Light microscope - can view RBC’s (limit 200 nm-10 mm)
-Scanning electron microscope (SEM) (10 nm-1mm) ;can view bacterium (1 um)
unaided eye- limit: 200 um; can view ticks
Compare and contrast the simple microscope with compound microscope
A simple microscope- has only ONE lens
Compound microscope- has TWO lens
What are the components of the Compound Light Microscope and their functions?
Compound Light Microscope:
1. Ocular lens (eyepiece) - remagnifies the image formed by the objective lens
2. Body Tube - transmits the image from the objective lens to the ocular lens
3. Arm -carries microscope
4. Objective lenses: primary lenses that magnify the specimen
5. Stage- holds the microscopic slide in position
6. Condenser- focuses light through the specimen
7. Diaphragm- controls the amount of light entering the condenser
8. Illuminator (light source)
9 Coarse focusing knob- make more movement of stage
10. Base
11. Fine focusing knobs - make smaller adjustments of stage
What part of the microscope moves when the course focusing knob is turned?
When the coarse focusing knob is turned, you move the *condenser, the stage, diaphragm, illuminator(?)
Explain how a specimen will be viewed through microscope using the different tools
process;
1. Light source will shine light up through microscope
2. light go in through condenser, that will focus light on specimen
3 This specimen will appear on the stage and enter objective lens to remagnify image
How is the image magnified in a compound microscope? what is the total magnification?
In a COMPOUND microscope, the image from the Objective lens is magnified again by the Ocular lens
-Total magnification = Objective lens x Ocular lens
What is the purpose of the prism in microscopy? What are the typical magnification ranges for objectives and ocular lens on
Prism- splits and direct the image to both oculars and bend the light towards ocular lens )
Magnification ranges for objective lenses= 5x-100x (highest)
Magnification range for ocular lens: 10x-20x (highest)
highest Magnification (of ocular and objective) will be 100m x 20 = 2000
Describe the working principle of a compound microscope
Working principle of Compound Microscope:
Light hits object, which will generate inside the tube of microscope, a magnified real image. When you look into microscope with your eye, your ocular will magnify the magnified real image, to form a Large virtual image at distance of MOST DISTINCT Vision (25 cm) from the eye
ex; if you have 100x magnification for objective, the magnification for magnified real image will be 100 times more of objective
likewise if 20 x magnification for ocular will be 20 times more of that for real image
Discuss the differences seen with inexpensive educational microscopes, compared to regular microscopes
Inexpensive Educational microscopes:
cost: $50
-Have fewer components
-No focusing knobs
-1 objective lens (you only move ocular to bend light)
1 ocular lens
-10x ocular
-40x objective
no splitting of light
-Prism at bottom of stage (to catch light from ceiling and bend it into specimen, since no real light source)
What is Resolution and how do microscopes use this for images?
Resolution: the ability of the lenses to distinguish two points a specific distance apart
A microscope with a resolving power of 0.2 nm can distinguish two points > or = 0.2 nm.
-if objects are closer to each other (like 0.05), they would form a blurry image (mesh of objects tougher, not clear)
What does Resolution depend on? What is the formula for resolution and its components? Does the value in resolution formula have to be bigger or smaller for better resolution?
Resolution depends on the source of light.
RESOLUTION= 0.61 X lambda/ n sin (theta)
lambda (wavelength of light) for white light = 0.5 um
n= refractive index of the medium (usually air) that separates specimen from the objective and condenser lenses
if air, n = 1
theta = half the angle for the cone of light rays collected by objective lens that hits the
specimen
sign 90 degrees = 1
as this objective gets closer to specimen, the angle gets bigger (Max angle= 180)
for white light, R= 0.61 x 0.53;/1 = 0.32 um
-SMALLER the resolution value, In equation, the BETTER the resolution
How do we increase resolution of a microscope with a wavelength of light?
We use an Electrons.
Electrons have a shorter wavelength, that will change
faster you can move electrons (accelerating voltage) , the shorter wavelength gets (up to .004 nm of wavelength of light)
**shorter the wavelength the better the resolution
How is the light miscroscope’s resolution limited? What is the relationship between wavelength and resolution?
The Limit to resolution of Light Microscope: is set by the type of Radiation utilized
-SHORTER wavelengths of light provide Greater RESOLUTION
What is Numerical arpeture? How does it relate to resolution. What is the cost go higher numerical arpeture and its value for dry lenses?
The Numerical Aperture (n sin (theta)) of a microscope objective is a measure of its ability to collect light and resolve fine detail
-The higher the numerical aperture, the greater the resolution and Brighter the image
-The cost of a higher numerical arpeture is very short working distances (distance between objective and specimen), and small depth of field
depth of field- how and far and below the objective and sample can be and still have everything in focus
-For dry lenses, this value can’t be more than 1.
(due to n=1 sign theta not more than 1)
Define refractive index and discuss what occurs when light rays change direction? What happens when light bends in the air and how can this be prevented?
Refractive index: the ratio of the speed of light in a vacuum to the speed of light in a particular transparent medium (ex: light travels 1.33x slower in water)
-Light rays change direction when they cross the interface from glass to the air (and vice versa). The refractive index determines how much the path of light is bent when entering a material
-The light may bend in air so much that it misses the small high-magnification. Lens- immersion oil prevents this.
higher the magnification of objective lens, the smaller the lens
What is the refractive index for immersion oil and why is this important?
Most immersion oils have a refractive index of 1.51.
This value of refractive index is beneficial because immersion oil has the SAME refractive index as glass, thus light does not bend (and there will be no loss of light at objective)
What is Light Microscopy? List the 7 types of light microscopy. How do the last three types differ from others?
Light Microscopy- uses of any kind of microscope that uses visible light to observe specimens
7 types:
Brightfield microscopy
Dark field microscopy
Phase-contrast microscopy
Differential interference contrast microscopy (DIC/Nomarksi)
*Fluorescent microscopy
*Confocal microscopy
*2-Photon microscopy
**last three require FLOURESCENT light
What does brightfield and Darkfield Microscopy have in common?
They both take advantage of the LIGHT SCATTERING properties of various cellular components
-(light will hit the specimen and some of specimens will be absorbed or diffract light)
Discuss what occurs in bright field microcopy and how they are able to view things? what is this microscopy best used for?
Brightfield illumination
-Dark objects are visible against a bright background
-Light reflected/absorbed by the specimen does NOT enter the objective lens
-Thus areas of the cell that scatter more light are darker
-generally useful for STAINED biological specimens
-Unstained cells are virtually invisible
ex: E. coli cells
What is Darkfield microscopy and does it work? What is it commonly used for?
Darkfield microscopy:
Light objects are visible against a dark background
-Light reflected off the specimen ENTERS the objective lens
-Useful for LIVE organism Not visible with a light microscope, cannot be stained or distorted by staining
-Commonly used to visualize Thin Spirochetes from syphilitic chancres (sores; used to diagnose syphyllis)
ex: treponema pallidum
what is the light path for a dark field microscope? Compare what will be seen with this kind of microscope with specimen on stage vs no specimen
Light path for Dark microscope
unique opaque disc between condenser lens and light source. The disc will block any light that will directly traverse to and objective lens
no specimens on stage- field dark
If there is a specimen on stage, and light hits it at an angle, the specimen will deflect some of light into objective lens and you will be able to see organism.
when is brightfieqld microscopy inadequate ?
Brightfield microscopy is Inadequate for Visualizing TRANSPARENT and COLORLESS specimens
(most bacteria are colorless; unless photosynthetic)
What are two ways to increase contrast in light microscopy?
Two ways to increase contrast in light microscopy;
1) Use a stain; you will have chromofore that will absorb particular wavelengths of light from white light and you will see that color to get good contrast
(amplitude of light decreased when light pass though stained cell)
2)observing Phase shift in unstained cell; that occurs when light travels through specimens and parts of specimen have different refractive index than other parts.
Light passes through sample, amplitude does not decrease, but phase of light shifted.
(initially before light passes bacterial specimens, trough and peaks of light are the same, but after light passes specimens, trough light has dropped, phase shift)
compare and contrast what happens to size and brightness of wave when two waves of light are in phase vs out of phase.
Two waves in phase: BRIGHT
Two waves OUT of phase; DIM
when two light waves combine in phase, the resultant wave is larger/brighter. When they combine out of phase, the wave is smaller/dimmer.
What do Phase contrast-microcopy and Differential interference contrast microscopy (DIC or Nomarski optics) have in common?
These 2 types of microscopy exploit light wave Interference effects to obtain greater contrast in light microscope
What are the differences between brightfield and phase contrast microscopy? which region of the specimen in the phase-contrast image produce the largest phase shift?
Phase contrast Microscopy produces much better CONTRAST than brightfield.
The EDGES- have largest phase shift in phase contrast image.
Compare and contrast images used with Phase contrast and DIC microscopy.What advantage does each method have over the other?
Phase contrast; has HALOS in the image, and you can see the internal structure of the cell
-you also cannot use phase contrast on bacteriophage (too small in terms of resolution)
DIC (aka Normarski, differential interference contrast); gives you more 3D image, and is a light gray color.
Advantages:
- DIC has INCREASED Depth of Field
-Phase contrast better visualize INTERNAL structures
How do image usually look with DIC (differential interference contrast microscopy) ?
DIC (aka Differential interference contrast, or Normarksi):
-Objects typically appear black to white on a grey background and appears nearly 3-dimensional.
(side note: the colored image from textbook requires insertion of birefringent compensator plates, Intel optical pathway of microscope; uncommon)
What things do both Phase contrast and DIC microscopy have in common?
Both phase contrast and DIC Microscopy;
can be used with LIVING cells
-DON’T require cell fixation/attachment slides
-DON’T require cell staining
what is Flurousecnt microscopy and how does it work? Describe the pathway of Fluorescence Microscope
Flourescent Microscopy: takes advantage of fluorescent dyes (fluorochromes) that absorbs short wavelengths of light (UV or near UV) and emit at another, longer wavelength(visible)
pathway for Flourescent Microscope:
the specimen will have green fluorescent protein: it absorbs blue light.
1. set first barrier filter: to let through ONLY blue light (with wavelength between 450 and 490 nm)
2. it hits Beam-splitting mirror: reflects light below 510 nm but transmits light above 510 nm (specimen absorbs blue light, and emits green light, which will go through second beam-splitter barrier, which only allows green light through ; up to eyepiece )
3. Second barrier filter: cuts out unwanted fluorescent signals, passing the specific green fluorescing emission between 520 and 560 nm
what happens when you need to use a different color flourophore for a filter ? What must you have to do?
When can you use Fluorescent microscope
If you absorb red light, or any other color, you must change barrier filter to allow red light to be deflected and another color of light go back up. There are different filters and mirrors you have to change for each fluorophore used.
**Fluorescent Microscope ONLY used If specimen itself Fluoresces or if specimen is bound to something that fluoresces.
since it requires fluorochromes.
