microscopy Flashcards
what is Köhler illumination?
Köhler illumination id the most common form of bright field microscopy, making imaging possible with proper alignment of illumination path components
what does the microscope in Köhler consist of and in what order?
light source => collector lens => field diaphragm => condenser lens => microscope stage with sample => eyepiece
what are the 2 illumination paths?
1) transmitted lights - condenser focuses the light on a sample; objective collects it from the sample
2) reflected light -objective is the condenser focusing light on sample
what are the 2 types of planes?
1) OBJECT / FIELD PLANES = focused images of sample
2) APERTURE PLANES = focused images of light source
what are the light paths like in an UPRIGHT microscope?
transmitted light comes from below the sample, while reflected light is above
what are the 3 most important components of a microscope?
1) OBJECTIVE = collects light from sample
2) OCULAR/EYEPIECE = sends light to recording device or eye
3) CONDESNSER LENS = focuses light on sample
what are the 3 definitions of SPATIAL RESOLUTION?
smallest object size that can be determined by an imaging system
minimum distance between 2 objects needed to tell them apart
highest spatial frequency that can be measured without ambiguity
what is spatial resolution?
It is basically the capability of the microscope to reproduce the finest details.
what does ABBE’S THEORY say?
When light hits a sample, it causes change in phase which results in an interference pattern. The specimen can be treated as a random phase diffraction pattern.
So , in order to generate an image by interference, the objective lens must collect 2 adjacent orders of diffracted magnitude.
what is DEPTH OF FIELD?
Depth of field = distance over z where object appears to be in focus
what is depth of FOCUS?
The depth of focus is the distance over z where the image appears to be in focus
what is the purpose of the tube lens?
The tube lens provides a space in the microscope where the image forming rays are parallel
what is the reticle?
The reticle is used as a visual reference for determining size of objects when viewed through a compound microscope
what is interference?
Interference is an essential wave phenomenon, that occurs when 2 or more waves overlap in space resulting in a combination or interaction of their amplitudes
what is coherence?
COHERENCE refers to overlap of sinusoidal parts of the wave packets
when does no interference occur and when does total interference occur?
No interference occurs when the electric field components are perpendicular to each other, while total interference occurs when the electric field components are parallel.
what are 2 coherent point sources?
Coherent point sources are light sources that emit waves that have a fixed phase relationship with each other
define DIFFRACTION
Diffraction represents the deviation from a “straight” propagation direction of light, “bending” of wavefronts around obstacles. Optical resolution is limited by diffraction.
What does Huygens’ Principle state?
Huygens’ Principle states that each point on a wavefront acts as a second source of secondary wavelets.
A new wavefront represents a combination of secondary wavelets.
what is the condition for full constructive interference?
The phase delta should be equal to 2pi*m.
what does Rayleigh criterion state?
The points are just resolved when the angular maximum of an Airy disk coincides with the first minimum of another Airy disk. (larger a = higher peak)
What are the 5 behaviours of light?
ABSORPTION, EMISSION, TRANSMISSION, REFLECTION, REFRACTION
What are amplitude objects?
Amplitude objects produce amplitude differences in the image
what are phase objects?
Phase objects produce images with low contrast. Thick phase objects are imaged in bright-field microscopy, while thin phase objects have little to no contrast. In phase object microscopy, they convert shifts in light passing into images with brightness differences
Can phases be measured directly?
No, only differences in phase can be measured. Fritz Zernike came with the idea of converting differences in phase to differences in amplitude using interference.
what are the 3 types of waves and what is the relationship between them?
S WAVE = surround/undiffracted = transmitted light
P WAVE = particle wave
D WAVE = diffracted wave (phase shifted by lambda/4)
what are the main important components in PHASE CONTRAST MICROSCOPY?
1) CONDENSER ANNULUS = creates oblique illumination
2) PHASE PLATE = retard or advance the phase of direct oblique rays relative to light scattered from sample
what happens in POSITIVE CONTRAST? what about NEGATIVE CONTRAST?
positive: advances S wave by lambda/4, retards D wave by lambda/4
negative: retards phase of S wave and advances phase of D wave
what do we have to do for maximum contrast?
Condenser annulus and phase plate have to be perfectly aligned for loss of enhanced interference effect
when does destructive interference hapen?
when the total net phase is lambda / 2
what is an alternative for phase contrast microscopy?
what are its main advantages and drawbacks?
DAK FIELD MICROSCOPY: non-diffracted rays / oblique rays are removed from image, so objects appear on a dark background
advantage: it can allow for detection of weakly diffracted light signals, inexpensively implemented, no special objective needed
drawback: lower resolution, information is lost due to excluding low angle rays
What is the concept behind contrast using polarization?
objects change polarization of light
polarization microscopy enhances contrast based upon birefringent materials
based on the ability of polarized light to interact with polarizable bonds of ordered molecules
when is light fully absorbed?
when the angle between the transmission direction of polarizer and analyzer is 90 degrees
What is BIREFRINGENCE?
Birefringence is the phenomenon of double refraction. Waves of 2 different polarizations (extraordinary and ordinary rays) have different indices of refraction and therefore, different outgoing angles.
In polarization microscopy, the birefringent material is used to separate, thus create a phase difference between o-ray and e-ray, which leads to achieving an image by interference at the imaging point.
what is the relationship between absorption and emission?
Absorption of light at a certain wavelength causes emission of light at another wavelength. Absorption typically corresponds to an electronic transition of a valence electron. Electrons basically move to a higher energy state.
what are the 2 types of relaxation?
1) RADIOACTIVE DECAY = emission of a photon
2) NON-RADIOACTIVE DECAY = can take the form of a chemical reaction, giving heat to surroundings
what is photobleaching?
Photobleaching represents less expression of the label due to fluorophore
what is the MOLAR EXTINCTION COEFFICIENT?
The molar extinction coefficient corresponds to lambda of peak absorption
what is a Stokes Shift?
energy change between absorption and emission
what does the ideal fluorescence microscope do?
it excites fluorophore microscope through excitation filter and subsequently collects the light which is emitted through a prism to a camera
what does the FILTER CUBE contain?
1) EXCITATION FILTER = restricts the spectrum of light to match the absorption band of the fluorophore
2) EMISSION FILTER = restricts the spectrum of light to match the emission band of the fluorophore
3) DICHROIC MIRROR = reflection excitation lambdas, transmits emission lambdas
what is the greatest problem of fluorescence microscopy?
overlap of fluorescent signals
what is excitation bleedthrough?
overlap of excitation spectra causes both fluorophores to be excited at the same time
What are 2 types of microscopy in which we have to perform optical sectioning?
TIRF and CONFOCAL
explain TIRF microscoy
it is based on total internal reflection (refracted light at 90 degrees to the normal).
it has really high signal to background ratio
fast acquisition
drawback: it can only reach the part of the sample close to the coverslip; it cannot reach inner part of the sample
if sample is thicker than the depth of field, the image is blurry and optical sectioning is required, then image is reconstructed by stacking up the information obtained through optical sectioning
what is HALFWAVE RETARDATION?
light is retarded by 90 degrees after 1st polarizer
what are the conjugate planes of the field planes / object planes?
suppose an eyelash has fallen into the eyepiece and it is resting on an optic sitting in the plane of the field stop. Is the eyelash in focus when the specimen is in focus?
1) RETINA
2) INTERMEDIATE IMAGE
3) OBJECT / SPECIMEN PLANE
4) FIELD STOP DIAPHRAGM
Yes, the eyelash is in focus because the field stop plane is conjugate to the object/specimen plane.
(conjugate planes refer to corresponding planes in optical path where objects are in focus. This means that when the specimen is in focus, the field stop diaphragm and the specimen plane share the same focal point or distance from the objective lens.
definition of Airy disk
An Airy disk is a patten produced when light passes through a circular aperture, such as a telescope or microscope and is diffracted. the size of it detemines the resolving power of optical systems
what is the difference between absorption and emission?
Absorption typically corresponds to electrons moving to a higher energy state, while emission is the other way around
what is a Stokes shift?
STOKES SHIFT = energy change between absorption and emission
what are the components of a filter cube? (in fluorescence microscopy)
A filter cube contains
1) EXCITATION FILTER - restricts the spectrum of the illumination to match the absorption band of the fluorophore.
2) EMISSION FILTER - restricts the spectrum of the illumination to match the emission band of the fluorophore.
3) DICHROIC MIRROR - transmits emission wavelengths and reflects excitation wavelengths
what is excitation bleedthrough?
overlap of excitation spectra causes both fluorophores to be excited at the same time
how do TIRF work?
TIRF microscopy is based on the total internal reflection phenomenon (refracted light is at 90” angle to the normal). There is a really high signal-to-background ratio but one of the drawbacks is that it can only reach parts of the sample close to coverslip, it cannot really reach to the middle of the specimen. That’s why we need to usually perform optical sectioning.
What is the idea behind CONFOCAL MICROSCOPY?
Confocal microscopy images only one point at a time, scanning the sample point by point by passing through A PINHOLE.
PINHOLE = helps focus on a certain part of the sample and collects them all using a PMT producing a final image
PMT = photomultiplier tube = a ‘single-pixel’ camera that knows the scanner coordinates, thus can reconstruct the image
what is a disadvantage of confocal microscopy?
it is very slow because of having to scan point by point: a mirror is used to change the angle of the incoming light to different spots on the sample.
Descanning also occurs: emission is sent back and again altered by the mirrors to align with the pinhole after the objective
what is the name of the shift between the excitation and emission peaks?
It is called a Stokes shift and it appears because of the energy changes between emission and absorption
It is caused by the fact that excitation energy of the photon is not fully converted into the emission energy of the photon. The fluorophore dissipates energy in the form of processes such as vibration or heat.
how do we find the best excitation laser and relevant wavelengths for the dichroic mirror and emission filter if we want to image a fluorophore?
for the excitation laser wavelength: we have to look at the highest wavelength in the absorption spectrum
for the dichroic cutoff: we have to look at the wavelength where the spectrum starts showing emission (this is to make sure that it efficiently reflects the laser excitation wavelengths but also allows the longer emission wavelength to pass through)
for the emission filter: any lambda after the dichroic cutoff
what can we change in the microscope settings if our signal is weak?
increase laser power, increase camera exposure, narrow filters to reduce bleedthrough
how big does the camera pixel have to be?
Camera pixel needs to sample magnified PSF at Nyquist to get full resolution of the microscope
Camera pixel size needs to be 1/2 of the magnified PSF width
what do we have to add to a Kohler illumination in order to make it a polarization microscope?
we have to add a polarizer and an analyzer
the polarizer should be added close to the collector lens; it causes unidirectional polarized light to go through and reach the sample
the analyzer needs to be placed after the sample, close to the eyepiece; it allows other detect the polarization rotation of light by the birefringent properties of the sample
phase contrast microscope extra components?
condenser annulus and phase plate
the condenser annulus needs to be placed before condenser lens and it creates oblique illumination’
the phase plate needs to be placed after the objective and introduces a specific phase offset (usually lambda/2) to oblique rays.
what is a point spread function?
psf = a 3-dimensional diffraction-limited image that would be obtained from imaging a point source
what happens to the color of the specimen when the analyzer is removed?
the colours produced in the white light microscope disappear when the analyzer is removed. The light passes through the analyzer appears colored because the analyzer is filtering out colors that have had their polarization rotated by 90”.
When the analyzer is removed, there is no filter for polarization and all polarizations of light go into the image.
how do we avoid autofluorescence?
usually, this happens with fluorophores that are excited at small wavelengths. Therefore, we can use higher wavelengths.
high fluorescence background
it can be caused by poor choice of filter sets, such as an emission filter and dichroic that transmits some of the excitation light to the detector.
it can be caused by bleedthrough, which occurs when the signal of interest is contaminated by signals from other fluorophores; either because there is too much overlap of the excitation/ emission spectra
what limits the depthof field in TIRF microscopy?
the depth of field is limited by the penetration depth of the evanescent wave, which is generated when light undergoes total internal reflection. it only extends a very short distance into the sample.
therefore, only fluorophores located within this shallow region near the interface are excited and emit fluorescence.
what type of detector does the spinning disk confocal microscopy use?
it uses cameras to detect the image, instead of point detectors
the spatial resolution is determined by matching the image size to the camera chip so that the camera pixels sample the image. if the pixel size is larger than the distance defined by the rayleigh criterion, the psf is undersampled and resolution is lost
