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Summary Working principle microscopes
- Instelling
- Universiteit Antwerpen (UA)
This document includes the working principles of microscopes seen in microscopy part of biomedical imaging.
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Working principle of microscopes1. Darkfield microscopy
Only refracted light is visualized. Light is sent in at such an angle that it does not
fall within the NA so that you will normally only see a black background. If the
object is optically active, the light that falls within the NA will break and so you
will see an image on a black background.
Concept:
A small annulus is placed through which the light is directed and it falls sharply
through the sample. It will fall outside the lens so you will see a black image.
However, part of the light beam will be scattered toward the lens due to the
presence of the sample and that light will be captured.
2. Zernike phase contrast microscopy
An annulus is also used to direct a light beam. A phase plate is placed behind the
object. If no sample is present, the light will incident on the central part of the
black plate and you will see a white image. When the light is refracted, the light
will also pass through the edges of the phase plate. Since these edges are thicker
it will slow down the light leading to phase differences. This will cause the light
beams to interfere (destructively and constructively). You will be able to translate
these differences into amplitude differences so you will now see an image (= halo
effect).
3. Fluorescence microscopy.
This is an inverted microscope.
Problem:
Light source with white light of all wavelengths. So we are going to have to
capture excitation from it and capture emission. This is done by filters.
- Shortpass filter: allows light to pass through up to a certain wavelength
- Bandpass filter: allows light to pass between 2 specific wavelengths
- Longpass filter: lets light through from a certain wavelength
Diascopic arrangement:
Light falls in on an excitation filter and will pass only light we want for the
fluorochrome. Goes incident on the sample and will emit fluorescence in all
directions. Some of this will be captured and sent through the emission filter and
onto the camera. However, this is largely scattered light and we want to avoid
this as much as possible. Therefore, an episcopic arrangement will be used.
Episcopic arrangement:
That is, excitation will be in 1 direction and emission will rotate 90°. A dichroic
mirror that can distinguish between 2 colors will be used.
- Filter cube: provides the perfect separation between excitation and
emission light by the presence of an excitation filter, dichroic mirror and
emission filter
o Dichroic mirror: white light will shine on the excitation filter which
will select only the wavelength at which that the fluorescent object
is excited. The transmitted light collides with the mirror (45°) where
short wavelengths are reflected and long ones are transmitted. If the
object fluoresces this is going to happen in all directions. As the
wavelength is changed (longer) it is going to pass through the
mirror, be filtered by the emission filter and thus reach the eye.
1
, o Emission filter is placed as multiple fluorochromes may be present in
the sample which may have different stokes shift or emission
maximum.
- Strong lamps required
- Objective: determine magnification and resolution of the microscope
o The greater NA, the greater the angle, the greater the brightness
o The larger NA, the more light you can capture (104) NA scales by
the 4th power with the intensity you capture
o The more you magnify, the smaller the image you can see, the less
light you capture
- Camera: wide field microscopy convert light into electrons (the more
pixels, the more resolution)
The disadvantage is that you cannot distinguish between what is below and
above because everything is in 1 plane. The solution to this is by using a confocal
microscope.
4. Confocal microscopy
You can make sections (optical sectioning) of a sample in 3D without damaging
the sample. This will make the outline fluoresce without the inside fluorescing.
You will be able to visualize details much better with this by creating more
contrast. One will thus illuminate point by point (laser that provides excitation =
point illumination) and detect = point detection (pinhole). During detection, a
distinction is made between what is in-focus and out-of-focus.
- Pinhole (= aperture) ensures that the point we detect comes primarily from
the light that is in-focus (the smaller the aperture, the smaller the optical
section you are mapping).
Since you scan point by point, a camera is not used as a detector but a
photomultiplier tube (= PMT: captured photons from individual points are
multiplied so that they will form a strong signal). You will have to be able to move
this beam which will limit the speed (compared to wide field microscopy).
Solution = spinning disk microscopy
5. Spinning disk microscopy
Here you can expose and scan points simultaneously to speed up the process.
However, these points must be parallelized and far enough apart otherwise you
risk bleed through.
Disadvantage:
- Many points have to be exposed at the same time which will reduce the
intensity. A solution to this is by using a 2nd spinning disk that captures
the light.
- Crosstalk and bleed through: because there are several pinholes, there is a
risk that a point that is exposed has a fluorescence that is detected
elsewhere.
o This is so with thick samples so people only use thin samples
6. Near-field microscopy
Can only be used if the sample is close to the source. Near-field is divided into 2
parts:
- NSOM = near-field scanning optical microscopy.
o This involves bringing a light probe close to the sample (less than
the wavelength of the light source)
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