Introduction to Microscopy Flashcards

1
Q

What is microscopy

A

Using microscopes to view objects/specimens that are not visible to the naked eye.

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2
Q

Name the parts of a microscope

A
  • Light source
  • Light conditioning system
  • Specimen
  • Objective
  • Detector
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3
Q

How is the light microscopic specimen prepared

A
  • use a coverglass of thickness 0.17mm
  • Sample is surrounded by embedding medium
  • use a glass slide
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4
Q

Explain how life imaging microscopy works

A

A living specimen is placed in a box to be examined with a microscope.

Prevent focus instability

The specimen must be kept alive so the temperature, CO₂ and O₂ levels are monitored and maintained.

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5
Q

What problems may arise from using microscopes to examine specimens for different timescales

A
  • When only measuring for seconds, there may be problems with artifacts in multichannel/4d imaging
  • when measuring from hours to days there may be problems with the viability of the specimen, also the positioning of the capture may change
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6
Q

Describe the triangle of frustration for signal detection

A

We can satisfy 2 of the three parameters when doing a measurement:
- Temporal resolution 1
- spatial resolution 2
- sensitivity 3

All detections have their benefits and limitations, what is best depends on the application requirments

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7
Q

What is spatial resolution

A

The size of the smallest unit of an image capable of distinguishing objects.

Where the bigger the pixel area the smaller the resolution and vice versa

Takes more time to capture an image with more resolution

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8
Q

What is Intensity resolution?

A

It refers to the number of intensity levels used to represent the image.

Where a higher bit value means larger intensity levels. (Shades of grey)

Takes more time to capture an image with more intensity

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9
Q

What are the markings on objective lens’

A
  • Magnification
  • Application
  • Coverslip Thickness
  • Numerical aperture/ Immersion medium
  • Working distance
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10
Q

How does aperture determine resolution

A
  • The higher the numerical aperture the better the resolution power of the objective.

RESOLUTION ≠ MAGNIFICATION

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11
Q

How is the function of light and lenses in a light microscope

A

To illuminate and magnify

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12
Q

How can light microscope be used to study cell histology

A
  • look for proteins of interest using antibodies
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13
Q

How can light microscope be used to study cell morphology

A
  • look for intact and denatured collagen
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14
Q

How can light microscope be used to study cell histology

A
  • study the differentiation of cardiomyocyte-like cells derived from adipocytes
  • study cell migration
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15
Q

What are the parts involved in electron microscopy

A
  • Electron source
  • Electron beam
  • specimen
  • Electromagnetic lens
  • Viewing screen
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16
Q

What are the types of electron microscopes

A
  • Transmission EM
  • Scanning EM
17
Q

What is the difference between Bright field and Fluorescence mode microscopy

A
  • Different light source
  • Fluorescence uses a filter cube whereas bright field uses a normal Wollaston prism
18
Q

How does fluorescence work

A
  • Fluorochrome absorbs energy and emits it at a different wavelength which shows up as a different colour
19
Q

What is photo bleaching

A

Bleaching of fluorochromes: due to high-intensity illumination the fluorophores might permanently lose their ability to emit light

20
Q

How can fluorescent proteins be used: i.e. GFP

A
  • Naturally found in light-producing cells of cnidarians
  • The proteins can be fused with other proteins and introduced in cells via transfection.
  • Allows us to tag organisms for live studies
21
Q

What is the difference between confocal and widefield fluorescence microscopy

A
  • A confocal setup uses a dichroic mirror, detector pinhole aperture and a photomultiplier detector
  • a wide field setup uses CCD or Ocular to view the samples