These notes explain the basics of Microscopy relevant to the field of Medicine.
The learning objectives are:
1. Know the basic concepts regarding optic/electron microscopy
2. Understand the main techniques in cellular and molecular biology (+ applications in biomedicine)
3. Understand the val...
Overview
• Concepts of resolution and contrast in microscopy
○ The regular light microscope.
• Electron microscopy
• Fluorescence microscopy
• Immunohistochemistry
-There are 2 main issues regarding RLMs:
1. Cell size, as they are usually too small for view.
○ Solution: Electron Microscopes
2. Cells have translucid structures, making distinctions between structures difficult.
○ Solution: Contrast Techniques
▪ Differential Staining
▪ Special Optical Systems
-Magnification: Up to 1500x The microscopic view is affected by…
1. Quality of the sample
a. eg. Preservation of tissue
2. Quality of the Microphone
- That sharpness is actually the resolution 3. Light Wavelength being used
• LR (human eye): d=0.1 mm
• LR (RLM): d=0.22 μm Objects that are closer to each other or are
smaller than 0.22 μm can't be observed!!
• Hence, RLMs can distinguish • Hence, RLMs CANNOT distinguish
○ Cells ○ Cell Membrane
▪ Nucleus ○ Ribosomes
▪ Mitochondria ○ Viruses
▪ Lysosomes
○ Bacteria
• LR can be defined as the quotient between the wavelength of light (λ) used multiplied by constant ,
divided by the numerical aperture (NA) of the objective.
• Visible light λ falls in between 400-700 nm.
○ Therefore, in LR calculations the average wavelength of light used is 550 nm
For optimal results, the LR of a light microscope is approximately 0.2 μm
Unit 1 Page 1
, For optimal results, the LR of a light microscope is approximately 0.2 μm
IMPORTANT FOR UNITS TO REMAIN CONSISTENT IN THE FORMULA!
• When light waves hits the edges of an object,
these deviate and collide with those in a parallel
trajectory. This interference explains why edges
can't be sharply defined but blurry.
○ Close small objects would be seen as one.
○ If a shorter wavelength is used, the
deviation would be lower, making the
microscopic image more sharp to the eyes
Contrast can be obtained in 2 ways.
1. Modifying the λ that crosses different cellular structures.
(differential staining)
○ Chemical dyes can modify λ, so when a sample is
stained with one, it allows one to see different cellular
structures in different colors, causing contrast.
▪ Combining dyes can lead to a better contrast as
the mix prevents the transmission of certain λ
while allowing other through.
□ Haematoxylin-Eosin staining is useful for
diagnosis.
Haematoxylin shows cellular nuclei
(in blue)
Eosin shows cytoplasm (in pink)
▪ However, staining kills cells (as it´s toxic to
them)
2. Modifying the wave amplitude
○ A train of waves is emitted. Crossing an unstained
tissue will cause a shift (delay) in the waves. Denser
areas will suffer greater shifts than less dense areas.
○ A Phase Contrast Microscope is equipped with an
optical system that transforms phase shifts of
amplitude (brightness) differences, leading to a
grayscale image.
▪ Differences of light are enhanced allowing
contrast and it doesn't kill cells.
□ This is useful when studying properties
like movement.
Unit 1 Page 2
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