Podcast
Questions and Answers
Differentiate between magnification and resolution in microscopy.
Differentiate between magnification and resolution in microscopy.
What is the definition of resolution? What is a formula that can tell us resolution?
What is the definition of resolution? What is a formula that can tell us resolution?
Which is more important - resolution or magnification? Why?
Which is more important - resolution or magnification? Why?
Why do transmitted light microscopes have white backgrounds? Why do emitted microscopes have a dark background? Answer using a light diagram.
Why do transmitted light microscopes have white backgrounds? Why do emitted microscopes have a dark background? Answer using a light diagram.
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Why is a dichroic mirror essential for fluorescence microscopy? What is its function?
Why is a dichroic mirror essential for fluorescence microscopy? What is its function?
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Visible light in brightfield is coming from the sample.
Visible light in brightfield is coming from the sample.
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Light field microscopy harnesses the power of electrons to view images
Light field microscopy harnesses the power of electrons to view images
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GFP is not useful in brightfield microscopy.
GFP is not useful in brightfield microscopy.
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You are a scientist studying a particular protein in a cell. You get your fluorescence microscope set up and look at your cell - but you can't see anything, it's all dark! What went wrong? What could you do to correct this?
You are a scientist studying a particular protein in a cell. You get your fluorescence microscope set up and look at your cell - but you can't see anything, it's all dark! What went wrong? What could you do to correct this?
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What is a fluorophore? Why is it important in fluorescence microscopy?
What is a fluorophore? Why is it important in fluorescence microscopy?
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Compare and contrast immunolabelling and GFP engineering for fluorescence microscopy.
Compare and contrast immunolabelling and GFP engineering for fluorescence microscopy.
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Why can GFP not be used in electron microscopy?
(The specific reason is not important, learning why might just make it easier to remember!)
Why can GFP not be used in electron microscopy?
(The specific reason is not important, learning why might just make it easier to remember!)
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Compare and contrast light field and electron microscopy. What are the sub-atomic particles used to generate an image in each technique?
Compare and contrast light field and electron microscopy. What are the sub-atomic particles used to generate an image in each technique?
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You are a scientist investigating the structure of microtubules in the cross-section of a cell. Which microscopy technique, brightfield, fluorescence, SEM, or TEM, would you use?
You are a scientist investigating the structure of microtubules in the cross-section of a cell. Which microscopy technique, brightfield, fluorescence, SEM, or TEM, would you use?
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What are the different "light" sources in light field and electron microscopy?
What are the different "light" sources in light field and electron microscopy?
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Why is the sample dark in TEM?
Why is the sample dark in TEM?
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What is a situation where you would choose fluorescence microscopy over brightfield microscopy? SEM over TEM? Brightfield over TEM?
What is a situation where you would choose fluorescence microscopy over brightfield microscopy? SEM over TEM? Brightfield over TEM?
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Imagine you are a scientist studying a eukaryotic cell. What features of the cell could you see with each microscopy technique we discussed in class?
Imagine you are a scientist studying a eukaryotic cell. What features of the cell could you see with each microscopy technique we discussed in class?
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Discuss the...
- Advantages/Disadvantages
- Light path
- Type (brightfield vs electron, type of brightfield)
- Sample preparation
- Size range
....of each microscopy technique we discussed in class
Discuss the...
- Advantages/Disadvantages
- Light path
- Type (brightfield vs electron, type of brightfield)
- Sample preparation
- Size range
....of each microscopy technique we discussed in class
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Study Notes
Magnification vs Resolution
- Magnification refers to the ability to enlarge an object's image.
- Resolution describes the ability to distinguish between two closely spaced objects.
- Resolution is more critical than magnification because high magnification without good resolution results in a blurry, meaningless image.
- Resolution is the minimum distance between two objects that can be distinguished as separate entities.
- Resolution is determined by the wavelength of light used and the numerical aperture of the lens.
Resolution Formula
-
Resolution (d) = 0.61λ / NA
- λ = wavelength of light
- NA = numerical aperture of lens
Light Microscopy Backgrounds
- Transmitted Light Microscopes have a white background because light passes through the sample and into the objective lens.
- Emitted Light Microscopes have a dark background because the light emitted from the sample is captured by the objective lens.
Dichroic Mirror
- Dichroic mirror is essential in fluorescence microscopy.
- It reflects specific wavelengths of light and transmits others.
- It directs excitation wavelengths towards the sample and reflects emitted fluorescence towards the detector.
Fluorescence Microscopy
- GFP (Green Fluorescent Protein) is not useful in brightfield microscopy because it does not emit visible light when illuminated.
- GFP needs excitation light at specific wavelengths.
- Visible light is emitted by the sample in brightfield microscopy.
Fluorescence Troubleshooting
- If a fluorescence microscope shows no signal, the sample might not have been properly labeled or the fluorophore may be quenched or photobleached.
- It is possible that the fluorophore is not being illuminated by the correct excitation wavelength.
- To fix this, ensure the sample is correctly labeled and the excitation wavelength is compatible with the fluorophore.
Fluorophores
- Fluorophores are molecules that absorb light at specific wavelengths and emit longer wavelengths.
- They are crucial for fluorescence microscopy as they allow visualization of specific structures or molecules within the cell.
Immunolabelling vs GFP Engineering
- Immunolabelling uses antibodies linked to fluorophores to visualize specific proteins within the cell.
- GFP engineering involves genetically modifying the organism to express a protein tagged with GFP.
- Immunolabelling is versatile and can be used to label a wide variety of targets.
- GFP engineering allows the visualization of protein expression and localization in living cells.
Electron Microscopy
- The limitation of GFP in electron microscopy is that electrons cannot excite fluorophores.
Light Field vs Electron Microscopy
- Light field Microscopy uses visible light to illuminate the sample.
- Electron Microscopy uses a beam of electrons to illuminate the sample.
- Light field microscopy has a resolution limit of around 200 nm.
- Electron microscopy has a resolution limit of about 0.1 nm.
Microtubule Structure Investigation
- For investigating microtubules, Transmission Electron Microscopy (TEM) would be the ideal technique.
Light Sources
- Light field microscopy uses visible light from a lamp or LED source.
- Electron microscopy uses a beam of electrons accelerated by an electron gun as the light source.
- TEM uses a specially designed electron gun.
TEM Sample Darkness
- Samples appear dark in TEM because most electrons pass through the sample and are collected by a detector.
- The image is generated from the electrons that are scattered by the sample.
Microscopy Applications
- Fluorescence microscopy should be used over brightfield microscopy when visualizing specific molecules or structures in a complex environment.
- SEM should be used over TEM when investigating surface details of a sample.
- Brightfield microscopy should be used over TEM when studying live cells or when a lower resolution is sufficient.
Eukaryotic Cell Features
Brightfield Light Microscopy
- Advantages: Simple setup, relatively inexpensive, versatile for examining live or fixed samples.
- Disadvantages: Limited resolution, low contrast for transparent specimens.
- Light path: Visible light passes through the sample and is focused by lenses onto the eye or a camera.
- Type: Transmitted light microscopy
- Sample preparation: Simple, often involves staining to increase contrast.
- Size range: Larger than 200 nm, depending on the objective used.
Fluorescence Microscopy
- Advantages: High specificity, excellent signal-to-noise ratio, allows visualization of specific molecules and structures within living cells.
- Disadvantages: Requires specialized equipment, can be challenging to set up, photobleaching can occur.
- Light path: Excitation light is directed at the sample, and emitted fluorescence is captured by the objective lens.
- Type: Emitted light microscopy
- Sample preparation: Requires labeling with fluorescent probes (e.g., GFP, antibodies)
- Size range: >= 200 nm, depending on the objective used.
Scanning Electron Microscopy (SEM)
- Advantages: Produces high-resolution 3D images of the sample surface, excellent surface detail.
- Disadvantages: Requires special vacuum conditions, cannot be used on living cells.
- Light path: A focused electron beam scans the sample surface, and secondary electrons are emitted and detected.
- Type: Electron microscopy, using secondary electrons.
- Sample preparation: May require metal coating for best contrast, sample must be fixed and dehydrated.
- Size range: > 1 nm, depending on the specific SEM design
Transmission Electron Microscopy (TEM)
- Advantages: Highest resolution microscopy technique, allows visualization of internal structures.
- Disadvantages: requires special high-vacuum conditions, delicate sample preparation, cannot be used on living cells.
- Light path: A focused electron beam passes through the sample, and transmitted electrons are collected by a detector.
- Type: Electron microscopy, using transmitted electrons.
- Sample preparation: Requires complex processing, including fixation, embedding, and sectioning
- Size range: > 1 nm, depending on the specific TEM design.
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Description
This quiz explores the fundamental differences between magnification and resolution in microscopy. Understand how these two concepts contribute to image quality and clarity in scientific observations.
