Tutorial: 2

TEM vs. light microscope

TEM Light microscope

• Utilizes a beam of electrons instead of visible light • Uses visible light to illuminate specimens
for imaging
• Limited resolution
• Offers much higher resolution due to the shorter
• Offers lower magnification
wavelength of electrons
• Easy to use
• Provides significantly higher magnification
• Sample preparation is easy.
• Complex and Costly
• They are non-destructive to biological samples,
• Specimen preparation for electron microscopy is
allowing for observation of living organisms.
time-consuming and often involves harsh processes
like fixation, dehydration, and sectioning.
• They are non-destructive to biological samples,
allowing for observation of living organisms.
Electron micrograph of an animal cell
1. Describe the ultrastructural features of mitochondria, chloroplasts, and peroxisomes that allow biologists to
identify and distinguish them in electron micrographs of eukaryotic cells.

• Biologists can distinguish mitochondria by the presence of cristae and the double membrane,
chloroplasts by the thylakoid membrane system, and peroxisomes by their single membrane and
characteristic contents.
2. What are the advantages and disadvantages of light microscopes? What are the advantages and disadvantages
of electron microscopes?

• Light microscopes are user-friendly and versatile but have limited resolution, while electron
microscopes offer unparalleled resolution and magnification but are costly and require complex sample
preparation.
• Light microscope can image live cells and dead cells, while electron microscope cannot image live
specimen.
3. These are two images of parts of a eukaryotic cell. Which structures can you identify in this
image? What type of microscopy was used to take each image?
4. You’re working in a research lab and your supervisor has just given you a new batch of human cells to study. Your supervisor tells you to determine the
morphology (shape) and number of mitochondria in these cells at rest and they want you to determine if either of these two factors change over the course of
mitosis. Describe three or four experiments you could perform to answer these questions (one experiment does not need to answer both questions, you could
perform an experiment to answer one question and another experiment to answer another question).

Experiments in the context of specific protein targets and their applications:

 Fluorescence Microscopy with a GFP-Tagged Protein: Protein Target: For specific labeling of mitochondria, they can choose
a protein tagged with GFP (Green Fluorescent Protein) that is localized exclusively to the mitochondria. An example of such a protein is
Mitochondrial Matrix Targeting Sequence (MTS)-GFP.
Application: This experiment allows for live imaging of mitochondrial dynamics during both the rest phase and mitosis. GFP-tagged
proteins will emit green fluorescence in the mitochondria, providing real-time visualization of mitochondrial morphology and distribution.
 Immunofluorescence Microscopy: Protein Target: Antibodies can be directed against mitochondrial proteins like Tom20
(Translocase of the outer mitochondrial membrane 20), which is a mitochondrial outer membrane protein. Tom20 antibodies can be used to
specifically stain mitochondria.
 Western Blotting: Protein Target: Western blotting can be employed to determine the relative levels of mitochondrial proteins. A
commonly used mitochondrial marker is Cytochrome c, a protein found in the mitochondrial intermembrane space.
 Subcellular Fractionation Using Equilibrium Density-Gradient Centrifugation: Subcellular fractionation can isolate
mitochondrial fractions from the rest of the cell components. This can be followed by Western blotting to determine the relative levels of
mitochondrial proteins. Alternatively, the isolated fractions can be examined using electron microscopy to study mitochondrial morphology
in detail.
Argonaute is a family of proteins that plays a crucial role in the process of RNA interference
(RNAi) and related gene regulatory pathways in eukaryotic organisms. These pathways are
essential for controlling gene expression and are involved in various cellular processes, including
development, defense against viruses, and maintaining genome stability.
5. Your supervisor in the lab has just asked you to figure out if the protein you’ve been studying, Argonaute, is
present in the nuclei of kidney, liver, and or heart muscle cells. Outline all the steps you would take to answer
this research question.

The outlined experimental steps aim to investigate whether the Argonaute protein is present in the nuclei of primary cell
cultures derived from various organs.
• Establish Primary Cell Cultures: Cultivate primary cell cultures from different organs of interest. These cultures will
serve as the source of cells for subsequent experiments.
• Subcellular Fractionation: Fractionate the cells to isolate the nuclei from the rest of the cellular components. This can
be done using a method such as differential centrifugation.
• Determine Nuclei Purity: western blot or microscopy.
Western Blot: Prepare protein extracts from the isolated nuclei and perform a Western blot using antibodies against a
nuclear marker protein (e.g., Lamin A/C or Histone H3). If the nuclear marker is present but cytoplasmic or membrane
proteins are absent, it suggests the isolated nuclei are relatively pure.
Microscopy: Alternatively, use fluorescence microscopy to visualize the isolated nuclei. Stain the nuclei with a
nuclear-specific dye (e.g., DAPI) and observe them under a microscope. Check for the absence of contaminating
cytoplasmic material.

• Western Blot for Argonaute: If Argonaute is detected in the nuclear fraction, it suggests that Argonaute is present in the
nuclei of these cells.
• Immunofluorescence Microscopy: Alternatively, perform immunofluorescence microscopy on the isolated nuclei to
determine the cellular localization of the Argonaute protein. If the Argonaute protein is detected within the nuclei
(colocalization with nuclear staining), it indicates its presence in the nucleus.
 6. You are an undergraduate student volunteering in a research lab over the summer. The PhD student who is supervising you
has just performed an organelle fractionation. They wanted to isolate pure nuclei, pure ER, and pure mitochondria. In order to
verify that the fractions are pure, they performed a western blot on each of the four fractions. They used 6 different antibodies
that each recognize different proteins that should localize to only one organelle in the cell. They don’t have time to analyze
this result, so they hand this result to you. Your job is to interpret this result. What can you tell the PhD student about the
purify of their fractions? You may need to look up some of these proteins as we have not gone over them yet in class.

 Histone is a nuclear protein. The nucleus is pure because the nuclear fraction
only contains histone protein.
 GAPDH is a cytoplasmic protein. The cytoplasm fraction is pure because it
only contains GAPDH. It is not contaminated by any other organelles.
 NADH dehydrogenase is a mitochondrial protein. It is the first component
of the electron transport chain. NADH dehydrogenase is found in only the
mitochondrial fraction, indicating that mitochondria are not contaminating
the nuclear fraction, the cytoplasmic fraction, or the ER fraction.
 Catalase is a peroxisome protein. Catalase is also found in the mitochondrial
fraction. This indicates that the mitochondrial fraction is contaminated
with peroxisomes.
 Calreticulin is an ER protein. Calreticulin is only found in the ER fraction,
indicating that fragments of the ER are not contaminating any other fractions.
 EGFR is a plasma membrane protein. EGFR is also found in the ER fraction.
This indicates that the ER fraction is contaminated with plasma membrane
vesicles.