Microscopy

Microscope
• Microscopes are specialized optical instruments designed to
produce magnified images of specimens that are too small to
be seen with the naked eye
• It is a very powerful tool for understanding the structure and
function of tissues and is widely used in biomedical science
courses, as well as in research and diagnostic laboratories
• The ability to identify structures and specific cell types has
been greatly aided by different staining techniques and by
immunohistochemistry.
Types of Microscopes

• Light Microscope
• Electron Microscope:
• Transmission Electron Microscope - TEM
• Scanning Electron Microscope - SEM
Light Microscopes

https://microbenotes.com/light-microscope/
Scanning Electron Microscope
https://microbenotes.com/transmission-electron-microscope-tem/
Principles of Imaging Using Light and Electron
Microscopy for imaging
• Light can be bent when it passed through a transparent material.
• The incident ray enters into the material and is bent towards the
normal as it passes towards the normal to emerge as the refracted
ray.
Imaging of the Electron Microscope
• An electron beam can be bent as it passes through a series of
electromagnets
• The electron beam passes through the
object in its path and is focused to
form an image.
Parts of a Light Microscope
Principles of Light Microscopy
• Electric bulbs or light emitting diodes (LEDs) are used to
produce a beam of light which is illuminates and is
focused on the sample.
• The transmitted light passes through a set of objectives,
along the tube through the eyepiece into the eye.
• Parallel light entering a lens from a small object is
brought to a sharp focus at a point behind the lens,
then the eyepiece allows a magnified real image to be
formed below the eyepiece
Light path of a Microscope
 How to Use the Microscope
• Turn on the light source
• Adjust the voltage and filters to a suitable illumination level.
• Focus and centre the condenser and adjust the field iris to illuminate
the area under the section and the objective.
• Select a low power objective, set the slide on the stage and carefully
rotate to focus whilst ensuring that the objective does not touch the
section.
• While observing though the eyepiece, focus back to the correct position
using coarse and fine focus controls
 Using the Microscope
• If the microscope has independent eyepieces, focus the fixed
eyepiece using the focusing controls, then focus the independent
eyepiece
• Scan the section, first at low power and then switch to a higher power
objective, having located the tissue on the slide.
• When closing down a microscope, the steps above should be reversed
so that the instrument is left with the lowest power objective in
position and the light voltage set at a low value.
 Bright Field Microscopes
• This is so-named because the microscopic field is bright while objects
being viewed are dark
• Light directed at the image is absorbed to form an image
• Unstained specimen have poor contrast
• Stained specimen show excellent contrast
• Ideal for studying stained bacteria, cells and tissues.
• Light source is from tungsten or halogen source
 Inverted Microscopes
• This is modified for special use to allow viewing of cells in flasks,
welled plates or other deep containers.
• It allows viewing of structures that do not fit between the objective
lens and the stage of standard microscopes
Inverted Microscope

Light Source

Objective lenses
 Dark Field Microscope

• This employs the use of a bright specimen and a dark background

• Light that is not scattered by the specimen bypasses the objective,
making the field/background dark.
• Can see very small objects but resolution is variable
• High contrast
• Good for unstained, live, and motile specimen
 Dark Field Microscopy

The algae Hydrodictyon reticulatum viewed with

darkfield microscopy. Photo by Dr. D. Folkerts, AU.
 Phase Contrast Microscope

• This is a technique that is useful for viewing unstained

/live cells
• Converts differences in refractive index in a specimen to
differences in image brightness
• Provides high contrast and good resolution
showi
Figure showing Phase contrast microscopy. The two micrographs show the same culture of live
cells (unstained) using standard illumination (a) and phase contrast illumination (b). Very little is
visible under normal illumination because all parts of the cells are equally transparent. Phase
contrast microscopy converts differences in the phase of the light passing through the cell into
differences in amplitude, so that some subcellular detail, including the nuclei, are visible.
 The thin tissue section in Figure 1(a) reveals
a human basal cell carcinoma stained with
eosin and hematoxylin to generate color
contrast in brightfield imaging mode. Figure
1(b) shows living HeLa cells in a plastic
tissue culture vessel imaged with phase
contrast. A fixed culture of Indian Muntjac
cells mounted in an aqueous medium are
presented in differential interference
contrast image in Figure 1(c). A fresh tissue
section of mouse heart muscle bathed in
aqueous saline solution is displayed in the
Figure 1(d) panel, where contrast is
generated using Hoffman modulation
contrast, an oblique illumination technique.
The brilliant bright-on-dark contrast
observed with darkfield illumination is
shown in Figure 1(e) using an Obelia
hydroid specimen. Rabbit skeletal muscle
fibers (Figure 1(f)) are among the biological
specimens that are birefringent and
demonstrate contrast in polarized light.

https://zeiss-campus.magnet.fsu.edu/articles/basics/contrast.html
 Fluorescent Microscope
• This uses ultraviolet light for resolution e.g. mercury or
xenon arc lamp
• Specific fluorescence dyes absorb short light waves
• are utilized to determine special structures especially
proteins in tissues
• These proteins are made to fluorescence and this can be
observed.
• High resolution and contrast images are formed
• It is used in histochemical diagnosis and biomedical research
 Fluorescence Microscopy
Fluorescent image showing metaphase in lung newt cell.
image by Rieder and Hughes, NY State Dept. Health, Albany, NY.
Endothelial Cells
Polarizing Microscope
• Observes specimen that are visible due to their birefringent (have
double refraction; velocity of light refracted is not same in all
directions) character
• Specimen is placed between two polarizers crossed at an angle of 90°
to each other in the condenser and objective
• Creates a bright image with a dark background
• Used for study of anisotropic and birefringent structures of the cell
e.g cell membrane, cilia, pigments, flagella, crystals and minerals etc.
 Polarizing Microscope
Sugar Crystal Collagen fibers in breast cancer
tissue

Shribak, M. Polychromatic polarization microscope:

https://www.leica-microsystems.com/science- bringing colors to a colorless world.Sci Rep 5, 17340 (2015).
lab/galleries/polarizing-microscope-image-gallery/ https://doi.org/10.1038/srep17340
Ultraviolet Light microscope
• This microscope utilizes quartz optics
• The lens are made up of quartz which has the ability to utilize light
from sunlight
• Resolution with UV light is higher than with normal light.
• It is used in the study of RNA and DNA
Differential Interference Contrast Microscopy
• Resembles phase contrast but is more sensitive and gives a higher
resolution, high NA and high contrast.
• Produces 3D images by detecting changes in the refractive index of
the specimen
• Good for unstained specimen, live mounts and can see membranes
within the cell
 Differential Interference Contrast (DIC)

Rabbit muscle image

https://micro.magnet.fsu.edu/primer/virtual/dic/index.html
Confocal Laser Scanning Microscope
• This uses a laser focused on the specimen which scans it in a
horizontal plane
• Light from the scanned plane reaches the detector and the scanned
image is digitally recorded and 3D images created
• High resolution and sharp images are created
• Eliminates background interference
Confocal Laser Scanning Microscope

a
Mouse Intestine
https://micro.magnet.fsu.edu/primer/virtual/confocal/index.html
Scanning Electron Microscopy (SEM)
• Specimen are placed in a vacuum and an electron beam scans back
and forth over it
• Electrons that bounce over the metal coated specimen surface are
collected, converted to a digital image and displayed on a monitor
• A 3D image is produced
• Gives information about the external topography of a specimen
• Much higher resolution and magnification than in a light microscope
Scanning Electron Microscope images

Grasshopper https://micro.magnet.fsu.edu/primer/java/electronmicr
oscopy/magnify1/index.html
Transmission Electron Microscope (TEM)
• The electron beam is passed through a tissue sample and is absorbed
or deflected by heavy metal stains to cast shadows onto a film or
phosphorescent plate.
• A 2D image is produced
• Reveals ultrastructure
• High magnification and resolution is obtained
https://www.majordifferences.com/2016/08/difference-between-sem-and-tem.html
TEM of SARS-CoV-2
https://microbenotes.com/transmission-electron-microscope-tem/