Lecture 1 & 2: Advances In Microscopy Flashcards
1
Q
History: Leeuwenhoek original microscope (ca. 1673)
A
- created simple microscopes
- single lens design
- 1st person to see bacteria, protists, spermatozoans etc.
2
Q
Robert Hooke’s microscope
A
- using the microscope he discovered cells in 1655 leading to his cell theory. (His cell theory said all animals & plants are based on cells).
- used a compound microscope (optical/light microscopy, uses 2 sets of lenses (objective & eyepiece) to achieve high magnification)
3
Q
Advances/ Forms of microscopy
A
- Light (or optical) microscopy (LM)
- Electron microscopy (TEM, SEM)
- Scanning Probe Microscopy (SPM)
4
Q
Microscopy types
A
- Optical (light microscope): uses visible light to illuminate & magnify specimens. Simple to use, inexpensive, visualise samples in real-time without extensive sample preparation. Suitable for thin sections with sizes ranging from micrometers to millimetres.
- TEM (transmission electron microscopy): At top of microscopes have a piece of metal thats coiled around & put charge through, it creates electrons, electrons then pass down the column to the specimen. electrons pass through specimen, interacting with its structure & resulting electron transmission pattern used to generate image with high R. Offers extremely high R, allowing visualisation of subcellular structures, nanoparticles, & atomic-scale details.
- SEM (scanning electron microscopy): Similar arrangement as TEM but looking at surface features of material, secondary electrons impinge on detector, signals from detector appear on screen & shows what u looking at (not looking through specimen, just on surface). Detected electrons used to generate a high-R image of specimens surface topography. Provides 3-D images, with high R, can visualise surface features, textures & morphology. Used for cells, tissues, microorganisms etc.
5
Q
Microscopy types
A
-
SPM (scanning probe microscopy): utilise a sharp probe to scan a sample surface to generate high-R images, at nanometre scale. SPM techniques:
a) AFM (atomic force microscopy): A stylus either touches specimen or close to specimen, it records forces & is converted to image. measures interaction forces between a sharp probe tip & surface of a sample. Used for polymers, biomolecules, nanoparticles etc.
b) STM (scanning tunneling microscopy): measures tunneling current between a conductive probe tip & surface of a conductive sample. (When a sharp metal tip is brought very close to a conductive sample surface, electrons can tunnel through the small gap between tip & sample, by measuring tunneling current, which is highly sensitive to distance, can map out surface topography with atomic-scale resolution).
c) SNOM (scanning near-field optical microscopy): uses sharp probe tip to scan across sample surface & the near field signal is measured & used to generate image. (Overcomes limitation of diffraction of light by using a nanometer-scale probe to scan sample surface in close proximity)
6
Q
Resolution & equation
A
- (if have lots of magnification (e.g. x200) but little resolution will not see clearly)
- ability to discriminate between 2 points in the same field as distinct
- not same as magnification
- higher the resolution, finer the details that can be distinguished.
- resolution (R) = (1.22 λ) / (2 x N.A.) = (1.22 λ) / (2n sin θ)
- theoretical factors that affect R:
N.A. = Numerical Aperture (measure of the cone of light that an optical system can gather or emit, higher N.A. = better R) (e.g. x4 magnification is closer to specimen, x100 is further away, that cone of light affects R)
n = refractive index of medium (higher n = stronger refraction of light rays = better R)
θ = (called theta) half the angle of cone of light entering the objective
λ is the wavelength of light in (nm). (We can vary but don’t usually with ordinary microscopes)
7
Q
Resolution
A
- best R we can achieve is ca. (about) 200 nm (theoretical limit), achieving this in practice is challenging
- R is limited by optical flaws (aberrations (result in distortions or blurring of the image) etc in glass) present in materials used to manufacture lenses
8
Q
Different types of objectives have different Resolutions
A
- higher magnification (M) = better R
- microscopes by Nikon sell 3 diff levels of objectives:
1. Plan Achromat: (basic objective). - 100x M = 0.22 μm R. (In context of bacteria (approx 2 to 1 μm in diameter) using x100 M u should see bacteria) but if use 4x M which = 3.75 μm R, u will not see them as discrete particles.
2. Plan Fluorite (glass made of fluorite): 100x M = 0.21 μm R.
3. Plan Apochromat: 100x M = 0.20 μm R. Slightly better R but expensive. However, the field is flatter & the edge of the field does not go out of focus, the contrast & sharpness of the image is better.
(data by Nikon)
9
Q
Resolution: wavelength of light
A
- visible light is approx 500-550 nm (white light) wavelength
- R is linked to wavelength of light
- if drop wavelength of light e.g. 360 nm improves R.
- so can use microscopes with different light sources to improve R. But not commonly used & doesn’t make a huge difference to R.
10
Q
Electron Microscopes (EM) have greater Resolution
A
- as EM uses an electron beam not a light source
- reducing wavelength is main THEORETICAL way to improve R.
- the greater resolving power of electron microscopy results from properties of electrons. the wavelength of electron in the beam depends on accelerating voltage (V) applied (speed of electrons).
- λ ((Å) in Angstroms (unit of length)) = square root of 150/V
- practical limit of R of TEMs is ca. 5 Å
- for SEM it is ca. 5 nm.
(1 Å is equal to 0.1 nm)
11
Q
Techniques used in LIGHT MICROSCOPY
A
- phase contrast microscopy
- differential interference contrast microscopy
(Both 1. PCM and 2. DICM enhance contrast but use diff optical mechanisms & so have diff effects on depth of field)
- fluorescence & immunofluorescence
- laser scanning confocal microscopy
12
Q
- PHASE CONTRAST MICROSCOPY
A
- invented by Dutch physicist, Frits Zernike, in 1934
- employs an optical mechanism to translate small variations in PHASE (timing or position of light waves as they pass through diff parts of specimen, when light passes through specimen it interacts with cells or structures inside causing light waves to be slightly out of sync or in different phases) into corresponding changes in AMPLITUDE (refers to magnitude/strength of oscillation of the wave, determines intensity or brightness of the light, in PCM amplitude of light wave remains relatively constant as passes through specimen) which can be visualised as differences in image CONTRAST (difference in intensity or brightness between different parts of the image, high contrast means distinct differences in brightness between features in specimen) in living material
- useful for living animal cells that have little or no contrast unless stained
- PCM is a way of creating contrast in an image that doesn’t have contrast otherwise there
- but when use PCM there is a phase halo around cell/structure (white line ring around structure/cell), it isn’t real, it is a defect of the method
13
Q
- PHASE CONTRAST MICROSCOPY
A
Structure of microscope:
- phase-plate: in the objective. Phase ring around it that sits in the objective.
- condenser: underneath the specimen on the stage is a condenser. In condenser is another phase-plate (or annulus). There is hole in it.
The 2 phase-plates have to be aligned on top of eachother. If they not in line u don’t see PHASE.
14
Q
- PHASE CONTRAST MICROSCOPY
A
- diff objectives tend to have diff size phase-rings
- phase ring (in the objective) becomes smaller as M increases
- rings in condenser are diff sizes too
15
Q
- DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY
A
- also known as Nomarski
- similar to PCM
- but adds a polariser filter (ensures light waves oscillate in specific direction)
- also adds another prism (above objective) called 2nd Wollaston Prism, after passing through specimen the split beams of light are recombined by the prism before reaching the eyepiece. (there is another prism below condenser called 1st Wollaston Prism, this prism splits the polarised light into 2 beams with slightly diff paths)
- relies on interference of 2 light beams to create contrast in specimen
16
Q
- DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY
A
Advantages:
+ higher R than PCM
+ lack of phase halo
+ 3D like appearance (isn’t real 3D)
Disadvantages:
– expense
– difficult to set up
17
Q
Depth of field in PCM & DICM
A
- depth of field is shallow in PCM, but better, sharper appearance with better R
- relatively greater depth of field in DICM, clearer
18
Q
FLUORESCENCE & IMMUNOFLUORESCENCE
A
FLUORESCENCE
- powerful imaging technique
- utilizes fluorescent molecules (fluorophores or fluorochromes) to visualize specific structures or molecules within a sample
- a specimen is labeled with fluorescent molecules that emit light when excited by a specific wavelength of light
- emitted light is then captured by a detector to generate image
IMMUNOFLUORESCENCE
- specific application of fluorescence microscopy used to detect & visualize specific proteins or antigens within cells or tissues
- antibodies labeled with fluorescent molecules are used to selectively bind to target proteins or antigens within the sample
-the bound antibodies are then visualized using fluorescence microscopy, enabling precise localization & visualization of target molecules within sample
