The document discusses visualization of cells and subcellular components using light and fluorescence microscopes. It provides details on: 1) Light microscopes use lenses to magnify cells and organelles, but resolution is limited due to the wavelengths of visible light. Fluorescence microscopes overcome this by using fluorescent dyes and ultraviolet light to illuminate specific structures. 2) Both microscope types take advantage of differences in refractive index between cellular components to generate contrast, while fluorescence microscopy additionally localizes fluorescent probes to targeted molecules. 3) Key cellular structures like nuclei, mitochondria, and endoplasmic reticulum can be visualized using brightfield or fluorescence microscopy, aiding the study of living and dead cells.

1. VISUALIZATION OF CELL AND SUB
CELLULAR COMPONENTS BY LIGHT
MICROSCOPE AND FLUORESCENT
MICROSCOPE
Ankita Das
210705180008
Supervised by-Dr.GaganKumarPanigrahi
Department of Zoology
School of Applied Sciences

2. CONTENT
• Introduction
• Light Microscopy
• Fluorescent Microscopy
• Visualization of cell and sub cellular
components by light Microscope
• Visualization of cell and sub cellular
components by Fluorescent Microscope
• Reference
• Acknowledgement

3. INTRODUCTION
• Cell Components – A cell is defined as the smallest unit of an organism with a
nucleus.
• A cell consists of three parts :
 The cell membrane
 The nucleus and
 Cytoplasm
• Sub Cellular Components – The sub cellular components of cell include :
 Cytoplasm , Cytoskeleton, Nucleus
 Centrioles , Lysosomes
 Endoplasmic reticulum, Ribosome
 Golgi apparatus ,Mitochondria etc.
• A typical animal cell is 10-20 pm in diameter ,which is about one-
fifth the size of the smallest particle visible to the naked eye.
• Animal cell are not only tiny but they are also colorless and
translucent.

4. LIGHT MICROSCOPE
o In light microscope the lenses used to
focus light on the specimen to produce
an image.
o The magnification varies depending on
the number of lenses.
o Simple light microscope (It has low
magnification because it uses a single lens)
o Compound light microscope (It has higher magnification
compared to the simple microscope because it uses at
least two lenses an objective lens and an eyepiece)
o The lenses are aligned in such a way that we can able to
bend light for efficient magnification of the image.
o Types – Bright field light microscope , Phase contrast light
microscopy, Dark field light microscopy.

5. FLUORESCENCE MICROSCOPE
• In florescence microscope the specimen is
exposes to ultra violet light.
• A dark field condenser provides a black
background which helps fluorescent objects
to glow.
• Usually the specimens are stained with
fluorescent dyes called flourochromes.
• A flourochromes is a fluorescent chemical compound that can re-emit
light, which contains aromatic group, or plane or cyclic molecules with
several pie bonds.
• Types : Internal- are part of molecule.
• eg: Trp amino acid in proteins
• External- molecules that lack internal fluoroscense are linked with other
small fluoresent molecules externally.

6. VISUALIZATION OF CELL AND SUB
CELLULAR COMPONENT BY LIGTH
MICROSCPE
• Both cellular and sub cellular part visible depends on contrast and
resolution also a visible light source.
• Magnification power: It is the ability to enlarge any object viewed in
the microscope . Total magnification power is calculated by
multiplying magnification power of objective lens and eyepiece.
• Resolution : It is the ability of magnifying the image such a way that
we can distinguish two objects that are closer to each other.
• Limit of resolution : that is reciprocal of resolving power.
wave length, Angular aperture , Refractive index
The sub cellular (cell organelles) part have different refractive index
so that when the light passes through the cell each part of the cell
change the path of the light according to their refractive index.
• This create different light rays , so that above the objective lens a
phase plate is used to combine the light rays to generate bright
image.
• Light from a mirror is reflected up through the specimen , or object
to be viewed into the powerful objective lens, which produce first
magnified image which is again magnified by the eyepiece lens,
where eyepiece react as a simple magnifying glass.

7. VISUALIZATION OF CELL AND SUB
CELLULAR COMPONENTS BY
FLUORESCENCE MICROSCOPE
• Light microscope use light in the 400-700nm range- the range through which light
is visible to the human eye, but fluorescence microscopy uses much higher
intensity light.
• Resolution in light microscopy in limited but on the other hand fluorescence
microscope use light produced by fluorophores in sample itself.
• It causes them to give off light with lower energy and longer wavelength which
produces the magnified view rather than the original light source.
• That means that flourescent microscopy uses reflected rather than transmitted
light.
• A fluorescence microscope uses a mercury or xenon lamp to produce UV light,
the light comes into the microscope and hits a dichroic mirror ( a mirror that
reflects one range of wavelength and allows another range to pass through)
which reflects the UV light to the specimen.
• The objective lens collects the fluorescent-wavelength light produced than the
fluoresecent light passes through the dichroic mirror and a barrier filter (that
eliminates wavelengths other than fluorescent), making it to the eyepiece to form
the image.
• We can use Calcein/AM , which binds with calcium so that the living cell shows
fluoresce green under UV light of microscope and the dead cell have no
fluoresce green.
• Also we can use Propidium iodide which binds to DNA in the nucleus and shows
fluoresces red under UV light.

8. Fig- showing how the light Fig - showing how the Fluorescence
microscope works. Microscope works.

9. REFERENCE
• Reuben , S (2010) Classification of
microscope. Ms nucleus
organization.Lesson-2-Page 3.
• Appendix , A (2006) Journal of the
Royal Microscopical Society. Page 716.

10. ACKNOWLEDGEMENT
• My subject teacher : Dr. Gagan Kumar
Panigrahi.
• DR. Yashaswi Nayak, HoD and Dean
SoAS
• All the faculty members of
Depertment of Zoology, School of
Applied Science , CUTM.
• Family and friends.

11. THANK YOU