Microscopy Flashcards
History of microscope: Antoni van Leeuwenhoek ?
Microscope was held against the light
→ Light passes through the object
• Technique regularly used in modern light microscopes
Full history of microscopy ?
- 1590 Hans Janssen invents the microscope
- 1665 Robert Hooke observes “cells“
- 1676 Antoni van Leeuvenhoek observes microorganisms, spermatozoa, blood
- 1839 Theodor Schwann and Matthias Schleiden claim all living things are made of cells
- 1858 Rudolf Virchow concludes that all cells come from preexisting cells
Explain Light Microscopy ?
• Magnification up to 1000 times
• Resolution: 0.2 μm
• Limitation by wavelike structure of light, not quality of lenses
• 3 requirements for viewing cells in a light microscope:
- Bright light, focussed onto specimen by lenses in the condensor
- carefully prepared specimen to allow light to pass through
- an appropriate set of lenses (objective and eyepiece) arranged to focus an image of the specimen in the eye
Explain Fluorescence microscopy ?
- Fluorophores absorb light at one wavelength and emit light at a different, longer wavelength
- Optical filters can selectively block certain wavelengths of light while allowing others to passthrough
E.g. endothelial cells:
Nuclei: blue
Microtubules: green
Actin filaments: red
Explain Confocal Fluorescence microscopy ?
Confocal microscopy uses a laser beam to focus light at a particular point in the specimen which allows finer resolution of the emitted light
Explain Transmission Electron Microscopy (TEM)?
- Electron beam used instead of light beam
* Resolution: up to < 1 nm
Explain Scanning Electron Microscopy (SEM)?
• Focussed beam of electrons scans surface of sample
• Electrons interact with electrons in atoms of sample
→ produce signals about surface topography of sample
Convert:
1 mm (millimeter)
1 μm (micrometer)
1 nm (nanometer)
• 1 mm (millimeter) = 10-3 m • 1 μm (micrometer) = 10-6 m • 1 nm (nanometer) = 10-9 m
Describe how a microscope works ?
- Light from the lamp is focused on the specimen by the condenser
- Light passing through the specimen is captured by the objective and focused again within the tube
- The ocular lenses focus the image onto the retina
What are objectives ?
Objectives are the primary magnifying lenses of a microscope
Two key properties of an objective are indicated on its side:
The magnification and the numerical aperture
The magnification indicates?
How much bigger the specimen will appear by the activity of the objective alone
The numerical aperture is?
An indicator of the resolution of the objective
Magnification is a ?
Relative measure of the size of the specimen to how it appears to your eye
Microscopes have two magnifying lenses ?
The objective lens and the ocular lens, and the magnification of the combination is the product of the individual magnifications
Resolution (r) is defined as ?
The shortest distance between two points on a specimen that can still be
distinguished by the observer as separate entities
Smaller the value for resolution, the ?
Clearer (better) the image quality
Resolution is directly proportional to
The wavelength of light:
• Thus there is a physical limit to the resolution available with a microscope using visible light (~0.2μm)
• The electron microscope can give finer resolution (to~0.2nm) because the wavelength of electrons is shorter than that of light in the visible spectrum
The numerical aperture (N.A.) of a microscope objective is ?
A measure of its ability to gather light and resolve fine specimen details at a fixed object distance
Resolution is directly proportional to ?
Wavelength (λ) and inversely proportional to N.A., as r =λ/2 N.A
The bigger the N.A. ?
The finer the resolution
Depth of focus (depth of field) is ?
The longitudinal resolving power of an objective
- determines how much vertical depth of the image will be in focus with respect to a particular object plane
- High N.A. → low depth of field
Light moving from glass into air is ?
Bent or refracted
The angle of refraction depends on ?
The ratio of the refractive indices of the two materials
In a microscope, by changing the medium between the specimen and the objective lens, the light?
The light capturing ability of the lens can be improved
This can be done by ?
Placing a drop of water or oil between the slide and lens, immersing the lens in a medium of higher refractive index
This is the principle behind?
An immersion objective
To use the microscope for measurement, we align ?
An arbitrary scale, eyepiece micrometer, with a real scale in the form of a calibration slide placed on the microscope stage
