27. Visualizing Life – Dyes and Stains
TLDRThe video discusses fluorescence and its value for studying biology. It explains how fluorophores absorb and emit light, with emission at longer wavelengths, allowing visualization of cellular components. Antibodies produced against specific targets enable fluorescent tagging of proteins in fixed cells. DNA microarrays exploit fluorophore labeling of complementary DNA strands to detect sequences related to diseases. Overall, fluorescence provides unique, vivid signals to examine complex biological systems.
Takeaways
- 😀 Fluorescence involves exciting a molecule to a higher energy state with light, which then emits light at a lower energy, longer wavelength when relaxing back to ground state
- 💡 Common fluorescent dyes like ethidium bromide and DAPI can visualize DNA but some are toxic, leading to improved dyes
- 🔬 Antibodies from animal immune systems enable targeting and visualizing specific proteins in cells with fluorescence
- 🌈 The emission wavelength of a fluorophore is always a longer, lower energy wavelength than its excitation wavelength
- 👀 Fluorescence provides unique, bright signals to visualize molecules in cells that lack intrinsic fluorescence
- 🚀 Combining different colored fluorescent labels on antibodies enables visualizing multiple targets simultaneously
- 🧪 DNA microarrays with fluorescent probes can detect thousands of specific DNA/RNA sequences on a microscope slide
- 🔍 Fluorophores' fluorescence is highly dependent on molecular environment, allowing sensing biochemical changes
- ❗ Fixed, permeabilized cells are required for antibody labeling because antibodies can't cross live cell membranes
- 📈 Injected antigens trigger immune cells to produce antibodies with binding sites specific to that target
Q & A
What is the difference between luminescence and fluorescence?
-Luminescence is the general term for light emission not associated with heat. Fluorescence is a type of luminescence that involves a molecule absorbing light energy and then emitting light energy at a longer wavelength when it returns to the ground state.
Why is ethidium bromide toxic to cells?
-Ethidium bromide is toxic because it intercalates into DNA between the base pairs. This can interfere with essential cellular processes like DNA replication and transcription.
How do DAPI and Hoechst dyes bind to DNA differently than ethidium bromide?
-Unlike ethidium bromide which intercalates, DAPI and Hoechst dyes bind in the minor groove of DNA. This does not disrupt replication as much so these dyes are less toxic.
What is a polyclonal antibody versus a monoclonal antibody?
-A polyclonal antibody contains multiple antibody molecules that recognize different parts of an antigen. A monoclonal antibody contains identical antibody molecules that recognize a single epitope on an antigen.
Why must cells be fixed and permeabilized to use antibody labeling?
-Cells must be fixed and permeabilized because antibodies are too large to cross the plasma membrane on their own. This allows the antibodies access to intracellular proteins.
What does the BDJ system allow for in terms of antibody diversity?
-The BDJ system allows for combinatorial rearrangement of small pieces of DNA to produce a highly diverse array of antibodies without requiring a dedicated gene for each one.
How can fluorescence be used with DNA microarrays?
-With DNA microarrays, known DNA sequences are spotted onto a slide. Fluorescently-labeled DNA can be hybridized to these spots to detect the presence and quantity of complementary sequences.
Why is fluorescence useful for studying biological systems?
-Most biological molecules do not intrinsically fluoresce, so adding external fluorescent labels gives unique, measurable signals that stand out against background noise.
What dictates the excitation and emission wavelengths of a fluorescent molecule?
-A molecule can only be excited by light matching its absorption spectrum. Emission always occurs at a longer, lower energy wavelength than excitation due to energy loss during the excited state.
How can antibodies raised in one species be used to study cells from another species?
-As long as the antigen sequence differs between species, antibodies raised against the antigen from one species can recognize and bind to that same antigen when introduced to cells from another species.
Outlines
🧪 Overview of fluorescence and luminescence
The professor provides an introduction to fluorescence and luminescence. She explains that luminescence is light emission without heat, while fluorescence involves excitation of a molecule by light which then emits light of a longer wavelength when returning to ground state. She also discusses types of luminescence like chemiluminescence and bioluminescence.
🌈 The electromagnetic spectrum and fluorescent dyes
The professor relates fluorescence to the electromagnetic spectrum, noting that excitation occurs at shorter wavelengths while emission is observed at longer wavelengths. She then discusses common fluorescent dyes used in biology like ethidium bromide for DNA staining and intercalation.
💉 Biological tools for cellular monitoring
The professor introduces antibodies as biological tools for recognizing and monitoring proteins and other cellular components through fluorescence. She provides background on B cells and antibody structure and diversity.
🐰 Producing antibodies in animals
The professor explains how antibodies are produced by injecting animals like rabbits with human antigen samples, allowing the immune system to generate antibodies that can then be fluorescently labeled and used to detect cellular proteins and structures.
🔬 Using antibodies for fluorescence microscopy
The professor demonstrates how different fluorescently-tagged antibodies can be used to label components like actin and tubulin in fixed, permeabilized cells, allowing visualization by fluorescence microscopy. This highlights proteins' distributions.
🧬 DNA microarrays
The professor concludes by introducing DNA microarrays - microscope slide-sized chips that can display fluorescence from up to 40,000 DNA probes, allowing highly multiplexed genetic analyses and disease profiling.
Mindmap
Keywords
💡Fluorescence
💡Luminescence
💡Wavelength
💡Antibody
💡Ethidium bromide
💡HOECHST
💡Microarray
💡B cells
💡Fixed cells
💡Emission
Highlights
Fluorescence is the emission of light not associated with heat, unlike a burning flame
Higher energy light has shorter wavelengths, lower energy light has longer wavelengths
Ethidium bromide fluoresces bright orange when intercalated into DNA
DAPI and Hoechst are improved DNA binding dyes that are less toxic than ethidium bromide
Antibodies are agents of the human adaptive immune system that can be used to recognize proteins
Antibodies have constant and variable regions, the variable regions bind to the antigen
A polyclonal antibody recognizes different parts of an antigen, a monoclonal antibody recognizes a single epitope
Fluorescently labeled antibodies can visualize proteins like actin and tubulin in fixed, permeabilized cells
Fluorophores give unique fluorescence signals compared to intrinsic cellular fluorescence
DNA microarrays use fluorophore-labeled DNA probes to detect thousands of sequences
Fluorescence is used to label DNA, antibodies, and other biological molecules to study cells
Cardinal rule of fluorescence - excite at shorter wavelength, emit at longer wavelength
Luminol chemiluminescence used in forensics to detect blood at crime scenes
Bioluminescence like in glowing plankton comes from ATP reactions with luciferase
Fluorophores change properties based on molecular environment like solvent exposure
Transcripts
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