Lecture 3A: Microscopy and Staining Flashcards
basic tool for viewing cells
Microscope
- specialized optical instrument designed to generate
- enlarged, visible ______________ of specimens
- key features : ______________ and _______________
- images
- Magnifying and Resolving power (which depends on the lens system)
Micrometer Symbol
µm
Unaided human eye
- 200 µm-
Compound light microscope (LM)
- 200nm-10nm
Scanning electron Microscope (SEM)
- 1nm to 1mm
Transmission Electron Microscope (TEM)
- 10nm -100 µm
Atomic force Microscope (AFM)
- 1nm-10nm
Scanning tunneling microscope (STM)
- 0.5 nm-10nm
Atom Size
- 0.1 nm
Why are cells so small?
- Cells are designed to be small for efficiency.
Why are cells so small?;
- As surface area to the volume ratio gets ______________ as the cell gets _______________.
- Smaller
- Bigger
if the cell grows beyond a certain limit, not enough material will be able to cross the _____________ fast enough to accommodate the increased cellular volume
- Membrane
(4) General Principles of Microscopy
- Wavelength of Radiation
- Magnification
- Resolution
- Contrast
Wavelength Visible for Human
- Visible light 400nm-700nm
Wavelengths from Weaker/low to strongest/biggest
- Radio waves and television, microwave, infrared, Ultraviolet light, X-rays, Gamma rays
Weakest Wavelength
- Radio waves and television
Strongest Wavelength
- Gamma rays
Simple magnifier lenses are _____
- Bi-convex
Means they are thicker at the center that the periphery.
- bi-convex
occurs when the image continues to be enlarged, but no additional detail is resolved.
- Empty Magnification
Magnification must be accompanied by _________
- Improved resolution
The _______ of a microscope is its capacity for discerning detail.
- Resolution
Microscope resolution Formula
- D= 0.61λ / n sin v
Symbol of λ in microscope resolution formula
- The wavelength of the light source
What does n in the formula?
- The refractive index of air or liquid between the objective lens and the specimen
What does ‘v’ mean in the formula?
- The aperture angle (a measure of the light-gathering ability of the lens)
Expression n sin v
Called numerical aperture.
What does the answer from formula D= 0.61λ / n sin v mean in practical terms?
- Limit of resolution, D- is the smallest dimension that we can see clearly/distinctly.
Types of microscopy
- Bright-field microscopes
- Dark-field microscopes
- Phase microscopes
- Fluorescent microscopes
- Immunoinfluorescent microscopes
- Electron Microscopes
- Probe Microscopes
differences in intensity between two objects, or between an object and background.
- Contrast
In __-_____ _______ , ______ results when cells absorb or scatter light differently from their surroundings.
- Bright-field microscopy
- Contrast
What is the theoretical limit for light microscope?
- 0.2 µm
Types of Light Microscope
- Bright-Field Microscope
- Dark-field Microscope
- Phase Microscope
- fluorescent microscope
Contain a single magnifying lens, -similar to magnifying glass, -Leuwenhoek used simple microscope to observe microorganisms.
- Bright-field microscope, simple
Contain a ____ magnifying lens, -similar to magnifying glass.
-_______ used simple microscope to observe microorganisms
- Single
- Leuwenhoek
2 lens systems
– Light rays pass through specimen and into objective lens – Specimens illuminated directly from above or below – Oil immersion lens increases ___________
– Total magnification = magnification of objective lens X magnification of ocular lens
– Most have _________________ to direct light through specimen
- Resolution (because light does not refract)
- Condense Lens
2 lens system
– Light rays pass through specimen and into objective lens – Specimens illuminated directly from above or below – Oil immersion lens increases Resolution (because light does not refract)
– Total magnification = magnification of objective lens X magnification of ocular lens
– Most have Condense Lens to direct light through specimen
- Bright-filed microscopes, compound
Advantage of Bright-filed microscopes, compound
- Convenient, relatively inexpensive, available
Disadvantage of Bright-filed microscopes, compound
- R.P 0.2 µm at best; can recognize cell but not fine details
Needs contrast of Bright-filed microscopes, compound
- Easiest way to view cells is to fix and stain.
The ____ _____ and _____ ___ combine to produce a magnified image of the specimen.
- Objective lens and eyepiece lens
Light rays from the specimen AB pass through the objective lens to give____________________________________________.
- A magnified inverted and real primary image.
The eyepiece lens magnifies this further to produce a _______ of the specimen
- Virtual Image
A typical light microscope ‘s light path consists of:
- A transillumination light source
- A condenser lens
- An objective lens
- Oculars and or camera
A typical light microscope ‘s light path consists of:
-a transillumination light source, commonly a ____________in the microscope stand;
- Halogen lamp
A typical light microscope ‘s light path consists of:
-a _______________, which focuses light from the light source onto the sample;
- A condenser lens
A typical light microscope ‘s light path consists of:
-an _____________, which collects light from the sample and magnifies the image;
- Objective lens
A typical light microscope ‘s light path consists of:
___________ and/or a _____________ to view the sample image.
- Oculars, a camera
The total magnification is obtained by multiplying this by the eyepiece value (usually __)
- 10x
Kinds of objective (lens)
- 4x Scanning
- 10x Low-dry
- 40x High-dry
- 100x oil immersion
4X scanning
- Find the object
10X low-dry
- Course focusing
40X high-dry
- Fine focusing
100X oil immersion
- Fine focusing and improved resolution.
-Best for observing pale objectives.
–Only light rays scattered by specimen enter the objective lens
– Specimen appears light against dark background
– Increases contrast and enables observation of more details
- Dark-field microscopes.
Best for observing __________________
–Only light rays scattered by specimen ____________ the objective lens
– Specimen appears __________ against _________ background
– Increases ___________ and enables observation of more details.
- Pale objectives
- Enter
- Light
- Dark
- Contrast
– occludes direct light, passes wide angle light
– angle too wide to enter objective
Special condenser diaphragm
Used to examine living organisms or specimen that would be damaged or altered by attaching them to slides or staining them –
-best for highly transparent specimen
– Treat one set of light rays differently from another set
– Light rays in phase produce brighter image, while light rays out of phase produce darker image
– Contrast is created because light waves are ½ wavelength out of phase
-– Uses two specific microscope components, the condenser annulus and the objective phase plate, to create a phase/shift light that results in an image with greater contrast perceived by the observer
- Phase microscopes
Used to examine _________________________ that would be damaged or altered by attaching them to slides or staining them –
-best for __________________
– Treat one set of light rays differently from another set
– Light rays ______________ produce brighter image, while light rays ______________ produce darker image
– Contrast is created because light waves are ______________ out of phase
-– Uses two specific microscope components, the _________________ and the _______________________ , to create a ____________________ that results in an image with greater contrast perceived by the observer
- living organisms or specimens
- highly transparent specimen
- in phase; out of phase
- ½ wavelength out of space
- condenser annulus and objective phase plate
- to create phase/shift light
Two types of phase microscopes
-Phase-contrast microscopes
-Differential interference contrast microscopes
light rays through objects of different n → change in phase, not intensity
- Phase contrast microscopy
This component is a specialized condenser meant specifically for phase contrast microscopy.
- Condenser annulus
These specialized objectives are built with a phase plate that works in conjunction with the condenser annulus to achieve the phase shift required for phase contrast microscopy.
- Phase contrast objective(s)
– Direct UV light source at specimen; causes the specimen to radiate energy back as a longer, visible wavelength
– UV light increases resolution and contrast
– Some cells and molecules are naturally fluorescent, while others must be stained
– Used in immunofluorescence to identify pathogens and to locate and make visible a variety of proteins
- Fluorescence Microscopes
– Direct _____________ source at specimen; causes the specimen to radiate energy back as a longer, visible wavelength
– UV light increases __________ and ____________
– Some cells and molecules are naturally fluorescent, while others must be stained
– Used in immunofluorescence to identify pathogens and to locate and make visible a variety of proteins UV light
- UV light
- Resolution and contrast
an assay which is used primarily on biological samples and is classically defined as a procedure to detect antigens in cellular contexts using antibodies.
- Immunofluorescence
The property of certain dyes absorbing light rays at one particular wavelength (ultraviolet light) and emitting them at a different wavelength (visible light) is known as _______
- Fluorescence
The dye which gives yellow-green fluorescence
- Fluorescein isothiocynate
Immunofluorescence tests are also termed as
- Fluorescent anti body test (FAT)
Fluorescent dyes that can be tagged with antibody molecules
- fluorescein isothiocyanate and lissamine rhodamine
– Light microscopes cannot resolve structures smaller than 200 nm
because the shortest wavelength of visible light is 400 nm
– Electrons produce wavelengths of 0.01 nm to 0.001 nm, so electron
microscopes have greater revolving power and greater magnification
– Magnifies the image (not the object) 10,000X to 100,000X
– Gives detailed views of bacteria, viruses, internal cellular structures,
molecules, and large atoms
Electron Microscopes
– Light microscopes cannot resolve structures smaller than ________ nm
because the shortest wavelength of visible light is ________ nm
200nm; 400nm
– Electrons produce wavelengths of 0.01 nm to 0.001 nm, so electron
microscopes have greater _____________ and ____________________
revolving power and greater magnification
– Magnifies the images ____________ to _______________
10,000X to 100,000X
‘Magnificies objects’ Is this statement correct??
no, because the microscopes magnifies the IMAGE, and not the object
Two Types of Electron Microscopes
- Transmission electon microscopes (TEM) and;
- Scanning Electron microscopes (SEM)
TEM stands for
Transmission electron microscopes
SEM stands for
Scanning electron microscopes
- form images using electrons that are transmitted (pass) through a specimen
- Like 2D or planar
TEM (Transmission electron microscopes)
- Utilize electrons that has bounced off the surface of the specimen
- some kind of 3D image
SEM (Scanning electron microscopes)
Points the electron beam
- Electron gun
defines the size of the electron beam
- Condenser Lens (magnet)
to focus and initially magnify the image (electron microscope)
- Objective lens (magnet)
Similarities of Electron Microscopes and Light Microscopes
- Form Larger (magnified) and more detailed images of small objects or small areas of larger objects e.g. a
leaf, part of a bone, etc. than can be formed by the human eye.
*Used in study and research in biology and medical sciences,
material sciences e.g. metallurgy and other aspects of science.
*Specimens must be carefully prepared using techniques appopriate for both the equipment and the sample
e.g. slicing, staining, mounting, etc
Differences of EM and LM Size;
Light microscopes are smaller and lighter, so are easier to move and set up.
Differences of EM and LM cost and availability;
Light microscopes are less expensive than EM
Differences of EM and LM radiation type;
LM uses UV (Visible light) approx. 400-700nm, while Electron Microscopes use electrons approx equivalength wavelength to 1 nm
Differences of EM and LM Control image;
LM via glass lenses, beams electrons can be focused using electromagnets due to negative charge on electrons
Differences of EM and LM Resolution;
EM have much higher resolution than LM
Differences of EM and LM Magnification;
EM have higher magnifications than LM
Differences of EM and LM Colour Images;
Light microscopes form images including the range of wavelengths
(colors) provided by the light source
Differences of EM and LM Preparation of specimens;
Generally involves harsher processes, e.g. using
corrosive chemicals, for viewing via electron microscope than preparation of slides for
viewing using a light microscope. Therefore more skill required
Differences of EM and LM Image Formation;
Light microscope images can be viewed directly. Electron
microscopes require use of a fluorescent screen, photographic plate or electronic
display because electrons cannot be observed directly by the human eye.
Differences of EM and LM Usage Limitations;
Living specimens cannot be viewed using electron microscopes
because electron microscopes require there to be a vacuum in the tub
Use minuscule, pointed, _________________ to magnify more
than _____________________ times
- electronic probes; 100,000,000 times
Use minuscule, pointed, electronic probes to magnify more than 100,000,000 times
Probe Microscopes
Two Types of Probe microscopes
-Scanning tunneling microscopes (STM)
- Atomic force Microscopes (AFM)
Detects the surface structure of the objects based on the tunnel effect of the quantum mechanics.
STM (Scanning Tunneling Microscopes)
Comparison between AFM and Electronic Microscopes
- Optical and electron microscopes can easily generate 2D images of sample surface. 1000X for optical microscope and a few hundred thousands for an EM
- These microscopes cannot measure the Vertical dimension (z-direction) of the sample, the height/depth of the surface features.
- AFM uses a sharp tip to probe the surface features by RASTER scanning
- Measurement of AFM is made in Three dimensions, the Horizontal (X-Y plane) and the vertical Z dimention
