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Questions and Answers
What is the key requirement for image formation/relay in microscopy?
What is the key requirement for image formation/relay in microscopy?
- Rays must maintain a constant intensity.
- Rays must diverge significantly.
- Rays must be perfectly parallel.
- Rays from a single point must converge to another point (conjugate planes). (correct)
In the context of microscopy, what does 'NA' stand for?
In the context of microscopy, what does 'NA' stand for?
- Numerical Alignment
- Neutralizing Angle
- Numerical Aperture (correct)
- Nominal Aperture
How does a higher Numerical Aperture (NA) affect the resolution of a microscope objective?
How does a higher Numerical Aperture (NA) affect the resolution of a microscope objective?
- NA has no effect on resolution.
- Higher NA only affects the depth of field.
- Higher NA increases resolution. (correct)
- Higher NA decreases resolution.
What formula relates the speed (c), frequency (ν), and wavelength (λ) of light?
What formula relates the speed (c), frequency (ν), and wavelength (λ) of light?
Which of the following is a key property of waves that affects image formation in microscopy?
Which of the following is a key property of waves that affects image formation in microscopy?
What phenomenon describes the bending of waves around obstacles or apertures?
What phenomenon describes the bending of waves around obstacles or apertures?
What is the key characteristic of 'infinite conjugate detection' in microscopy?
What is the key characteristic of 'infinite conjugate detection' in microscopy?
What is the role of the tube lens in an infinite conjugate microscope system?
What is the role of the tube lens in an infinite conjugate microscope system?
Which of the following parameters is quantified by Numerical Aperture (NA)?
Which of the following parameters is quantified by Numerical Aperture (NA)?
What is the formula for calculating Numerical Aperture (NA)?
What is the formula for calculating Numerical Aperture (NA)?
Which of the following is defined as the 'full width at half maximum' (FWHM) in the context of microscopy?
Which of the following is defined as the 'full width at half maximum' (FWHM) in the context of microscopy?
What is the effect of diffraction on the image formed by a lens?
What is the effect of diffraction on the image formed by a lens?
What is the optical path length when light travels through a medium?
What is the optical path length when light travels through a medium?
Under what conditions does refraction occur?
Under what conditions does refraction occur?
According to Snell's Law, what determines the angle of refraction when light passes from one medium to another?
According to Snell's Law, what determines the angle of refraction when light passes from one medium to another?
What is the relationship between wavelength and resolution in microscopy?
What is the relationship between wavelength and resolution in microscopy?
Consider a lens with a fixed numerical aperture. If the refractive index of the imaging medium is increased, what happens to the resolution?
Consider a lens with a fixed numerical aperture. If the refractive index of the imaging medium is increased, what happens to the resolution?
Why is an oil immersion objective capable of achieving higher resolution than a dry objective with the same numerical aperture value?
Why is an oil immersion objective capable of achieving higher resolution than a dry objective with the same numerical aperture value?
What is the primary factor that determines Abbe's diffraction limit?
What is the primary factor that determines Abbe's diffraction limit?
In total internal reflection (TIR), what condition must be met for light to be completely reflected at the interface between two media?
In total internal reflection (TIR), what condition must be met for light to be completely reflected at the interface between two media?
What is the main principle behind expansion microscopy for improving resolution?
What is the main principle behind expansion microscopy for improving resolution?
What is the effect of increasing the wavelength of light on the size of the Airy disk?
What is the effect of increasing the wavelength of light on the size of the Airy disk?
How does the refractive index of a medium typically vary with the wavelength of light (dispersion)?
How does the refractive index of a medium typically vary with the wavelength of light (dispersion)?
Considering the concept of conjugate planes in microscopy, if the objective lens forms an image at a specific plane, where should you place a pinhole to block out-of-focus light and improve image clarity?
Considering the concept of conjugate planes in microscopy, if the objective lens forms an image at a specific plane, where should you place a pinhole to block out-of-focus light and improve image clarity?
How are the XY PSF and XZ PSF related to the concept of axial resolution in microscopy?
How are the XY PSF and XZ PSF related to the concept of axial resolution in microscopy?
If $E = hv$ is the energy of a photon, where $h$ is Planck's constant and $v$ is the frequency, and $c = v\lambda$ relates frequency and wavelength, what happens to the energy of photons as the wavelength increases?
If $E = hv$ is the energy of a photon, where $h$ is Planck's constant and $v$ is the frequency, and $c = v\lambda$ relates frequency and wavelength, what happens to the energy of photons as the wavelength increases?
Given Snell's Law ($n_1 \sin(\theta_1) = n_2 \sin(\theta_2)$), if light travels from air ($n_1 \approx 1$) into water ($n_2 \approx 1.33$) at an incident angle of 45 degrees, what happens to the angle of refraction ($\theta_2$)?
Given Snell's Law ($n_1 \sin(\theta_1) = n_2 \sin(\theta_2)$), if light travels from air ($n_1 \approx 1$) into water ($n_2 \approx 1.33$) at an incident angle of 45 degrees, what happens to the angle of refraction ($\theta_2$)?
Given that resolution ($r$) is approximately $0.61 \frac{\lambda}{NA}$, and expansion microscopy expands a sample by a factor of $x$, by what factor does the apparent resolution improve, assuming wavelength and numerical aperture remain constant?
Given that resolution ($r$) is approximately $0.61 \frac{\lambda}{NA}$, and expansion microscopy expands a sample by a factor of $x$, by what factor does the apparent resolution improve, assuming wavelength and numerical aperture remain constant?
Flashcards
Image Formation/Relay
Image Formation/Relay
Rays from a point converge to a point.
Infinite Conjugate Detection
Infinite Conjugate Detection
When rays coming from the specimen are parallel when entering the objective lens.
Numerical Aperture (NA)
Numerical Aperture (NA)
Quantifies the amount of light collected by the objective lens. Higher NA, higher the resolution.
Working distance (WD)
Working distance (WD)
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Focal Length (f)
Focal Length (f)
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What is light?
What is light?
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Interference
Interference
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Diffraction
Diffraction
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Refraction
Refraction
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Optical Path Length
Optical Path Length
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Refraction
Refraction
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Index of Refraction (n)
Index of Refraction (n)
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FWHM
FWHM
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Abbe's Diffraction Limit
Abbe's Diffraction Limit
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How to improve resolution
How to improve resolution
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Monochromatic and Collimated Light
Monochromatic and Collimated Light
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Total Internal Reflection
Total Internal Reflection
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Fiber Optics
Fiber Optics
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Lenses
Lenses
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Positive (Converging) Lens
Positive (Converging) Lens
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Negative (Diverging) Lens
Negative (Diverging) Lens
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Study Notes
Image Relay
- Rays from a point must converge to a point, which are conjugate planes, for image formation and relay.
Infinite Conjugate Detection
- Tube length for Nikon is 200 mm.
- Tube length for Olympus is 180 mm.
- Tube length for Leica is 200 mm.
- Tube length for Zeiss is 164.5 mm.
- M = ft / fo; M is magnification, ft is the focal length of the tube lens, and fo is the focal length of the objective.
Numerical Aperture (NA)
- Numerical aperture (NA) quantifies light collection.
- Higher NA results in higher resolution.
- NA is calculated as n × sine, where n is the refractive index of the medium and is the half-angle of the maximum cone of light that can enter or exit the lens.
Focal Length (f) and Working Distance (WD)
- The Numerical Aperture (NA) is calculated as n × sine.
Light as a Wave
- Light can be described as an electromagnetic wave.
- The equation relating speed, frequency, and wavelength is c = νλ, where c is the speed of light, ν is the frequency, and λ is the wavelength.
- Light can also be described as a stream of photons.
- The energy of a photon is given by E = hv.
- The energy of a photon can also be expressed as E = hc/λ.
Key Properties of Waves
- Interference involves the superposition of waves, leading to constructive or destructive interference.
- Diffraction is the bending of waves around corners of an obstacle or aperture.
- Refraction is the change in direction of waves as they pass from one medium to another.
Interference
- Constructive interference increases amplitude.
- Destructive interference decreases amplitude.
Diffraction
- Diffraction involves bending of waves around obstacles.
Diffraction and Airy Disks
- Diffraction patterns are related to Airy disks.
- ~84% of the signal is within the Airy disk.
Point Spread Function (PSF)
- Point spread function (PSF) is the full width at half maximum (FWHM).
Resolution Criteria
- Resolution criteria include Rayleigh limit, Abbe limit, and Sparrow limit.
- Various conventional resolution limits and their definitions exist
- In the Rayleigh convention, the first minimum of one Airy profile overlaps the maximum of the second Airy profile, with the sum of the two profiles showing a distinct dip.
- In the Sparrow criterion, the sum of the two Airy patterns produces a flat intensity profile
- In the Abbe limit, a small dip is still discernible between the two maxima.
Resolution
- Abbe's diffraction limited resolution can be defined in both lateral and axial dimensions.
- Lateral resolution is λ / (2 × NA).
- Axial resolution is (2 × λ) / NA².
Improving Resolution
- Resolution can be improved using electron microscopes.
- Resolution improvement has allowed for the resolution of the lipid bilayer of the plasma membrane, which is less than 5nm.
Improving Resolution with Expansion Microscopy
- Expansion microscopy is another way to improve resolution.
Refraction
- Refraction involves the speed ratio, described as Index of Refraction n, where v = c/n.
- The index of refraction for a vacuum is 1.
- The index of refraction for air is 1.0003.
- The index of refraction for water is 1.333.
- The index of refraction for cytoplasm is 1.35-1.38.
- The index of refraction for glycerol is 1.475 (anhydrous).
- The index of refraction for immersion oil is 1.515.
- The index of refraction for fused silica is 1.46.
- The index of refraction for optical glasses is 1.5-1.9.
- The index of refraction for diamond is 2.417.
- Snell's law is given by n₁ Sin(θ₁) = n₂ Sin(θ₂).
- Mirror law is θr = θ₁.
- Refraction causes light to bend towards the normal in a higher-index medium.
- Refraction depends on wavelength.
Total Internal Reflection
- Total internal reflection occurs when light strikes a medium with a lower index of refraction at an angle greater than θc.
- Fiber optics use total internal reflection.
Lenses
- Lenses work by refraction to focus light.
- Lenses can be positive (converging) or negative (diverging).
- Positive lenses include double convex, plano-convex, and convex meniscus.
- Negative lenses include double concave, plano-concave, and concave meniscus.
Quality of Light
- Light can be monochromatic or polychromatic.
- Light can be linearly polarized or non-polarized.
- Light can be coherent or noncoherent.
- Light can be collimated or divergent.
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