The document outlines different types of microscopes, primarily classifying them into light and electron microscopes based on their energy source for image generation. Light microscopes include simple and compound types, as well as various techniques such as fluorescence microscopy, which uses fluorescent markers to detect specific proteins in cells. Electron microscopes, such as transmission and scanning electron microscopes, utilize electron beams for high-resolution imaging of specimen surfaces and are valuable for studying cellular structures.

1. Types of microscope
(based on source of energy to generate an image)
1. Light microscope –in which light is used to detect the small object
2. Electron microscope – in this, short beam of electrons are used to generate
the image.
Light microscope is again broadly classified into two categories
(based on the number of lenses)
1. Simple microscope- it consists of only one biconvex lens
1. Compound microscope- consist of two separate lens systems such as
objective and ocular (eye piece)

2. The Light Microscope
• many types
– bright-field microscope
– dark-field microscope
– phase-contrast microscope
– Differential interference contrast microscope (DIC)
– fluorescence microscopes

3. Fluorescence Microscopy
Fluorescent molecules absorb light at one wavelength and emit it at
another, longer wavelength.
If we illuminate such a compound at its absorbing wavelength
and then view it through a filter that allows only light of the emitted
wavelength to pass, it will glow against a dark background
This microscope is similar to an ordinary light microscope except that the
illuminating light is passed through two sets of filters-
one to filter the light before it reaches the specimen and
One to filter the light obtained from the specimen.
The first filter passes only the wavelengths that excite the particular
fluorescent dye,
while the second filter blocks out this light and passes only those
wavelengths emitted when the dye fluoresces

4. Pholobleaching
Continuous illumination of fluorophoros leads to loss of fluorescence from the
sample resulting in fading of the image

5. Application
Fluorescence microscopy is most often used to detect specific proteins or
other molecules in cells and tissues by fusion with GFP, YFP/RFP.
A very powerful and widely used technique is to couple fluorescent dyes
(fluorescein,, rhodamine)) to antibody molecules, that bind selectively to the
particular macromolecules they recognize in cells or in the extracellular
matrix.
Cell in Mitosis stage
Spindles microtububles with
a green Fluorescent antibody
Centromeres with a
Red fluorescent antibody
DNA- DAPI

6. Electron Microscopy
Uses short beam of electrons and magnetic condenser lenses to
produce the image. It is possible to resolve objects as small as
0.001 nm.
transmission electron microscope (TEM)
scanning electron microscope (SEM)

7. transmission electron microscope (TEM)
TTEM uses electrons as energy source
and much lower wavelength to get
good resolution.
In TEM, beam of electron are
transmitted through the specimen,
magnified and focused by an
objective.
Drawback
Sample preparation is laborious and
time consuming process
Application: tomographic reconstruction of small cells or thin section of
larger cells

8. scanning electron microscope (SEM)
A scanning electron microscope (SEM) directly
produces an image of the three dimensional structure
of the surface of a specimen.
TEM uses the electrons that have passed through the
specimen to form an image,
the SEM uses electrons that are scattered or emitted
from the specimen's surface.
Thus only surface features can be examined and used
to study whole cells and tissues rather than
subcellular organelles
A beam of electrons is produced at the top of the
microscope by an electron gun. The beam travels
through electromagnetic fields and lenses, which
focus the beam down toward the sample. Once
the beam hits the sample, electrons and X-rays
are ejected from the sample and they are
collected by detector converted into a signal. This
produces the final image.

9. Application of SEM
Produces images in 3D format