Day 3+4 part 4 microscopy (not on the exam)

Environmental Biotechnology

🌈 Why We Use Fluorescent Staining

Under a normal light microscope, biological samples often appear transparent and lack contrast. To make structures visible, scientists use fluorescent stains that emit light in different colors. Example: A stained mouse brain can show neurons in many distinct colors — yellow, green, blue, red — each marking different structures.

Fluorescence lets us see:

Multiple cellular components at once
Fine structures that white light alone cannot reveal

💡 What Is Fluorescence?

Fluorescence is a property of molecules and light — it happens when a molecule absorbs light at one wavelength and emits it at a longer wavelength.

🔬 Atomic-level explanation

An electron absorbs a photon (light particle) → it gets excited to a higher energy level.
The electron loses a bit of energy as vibration.
It then returns to its ground state, emitting a photon of lower energy (longer wavelength).

⚡ Key point: The emitted light always has lower energy (longer wavelength) than the absorbed light. This shift is called the Stokes shift, and it lets us separate excitation and emission light by color.

🎨 Example of Excitation and Emission

For a given fluorescent dye:

It may absorb (get excited) at ~480 nm (blue-green light)
And emit at ~510 nm (green light)

So, you shine blue light in ➜ green light comes out. This shift is how we tell excitation and emission apart.

🧭 How Fluorescence Microscopes Work

A fluorescence microscope uses filters and mirrors to control which light enters and leaves the sample.

Components:

Excitation filter 🎯 – Selects the exact wavelength to excite the fluorophore (e.g., blue light).
Dichroic mirror 🪞 – Reflects excitation light down onto the sample. – Lets the emitted light (of longer wavelength) pass upward to the detector.
Emission filter 🎥 – Blocks leftover excitation light. – Only allows emitted fluorescence to reach your eyes or camera.

This setup ensures:

Only the correct light hits the sample.
Only fluorescence reaches the detector.

Together, these parts form a filter cube, the core optical block of fluorescence microscopes.

🧪 How We Introduce Fluorescence into Samples

We can attach fluorescent color to our target structures in three main ways:

1️⃣ Chemical stains

Simple dyes that bind directly to certain molecules.

Example:Hoechst (DAPI) 🧬

Binds specifically to double-stranded DNA
Emits blue fluorescence when bound
Excited by UV light Used to label nuclei in eukaryotic cells or entire bacterial cells.

2️⃣ Antibody labeling

Used when there is no direct chemical stain for a structure.

Process:

A primary antibody binds to a target protein (e.g., actin, ion channels).
A secondary antibody (fluorescently labeled) binds to the primary antibody.

This amplifies signal and saves money — you can use the same fluorescent secondary antibody with many different primaries.

💎 Advantage: High specificity 💸 Disadvantage: More complex and costly labeling steps

3️⃣ Genetically encoded fluorescence (e.g., GFP)

Instead of staining, we make cells produce fluorescent proteins themselves.

🪸 The story of GFP (Green Fluorescent Protein)

Douglas Prasher discovered the gene from jellyfish 🪼.
He cloned and sequenced it but couldn’t get funding — his lab shut down.
He sent the sequence to Martin Chalfie, who expressed it in C. elegans.
Later, Chalfie, Shimomura, and Tsien shared the Nobel Prize (2008) for their GFP work.
Prasher eventually rejoined science — a happy ending.

🌟 GFP and Its Variants

GFP forms a barrel-shaped protein with an internal chromophore that emits green light. Through mutations, scientists created a rainbow palette of fluorescent proteins:

🟢 eGFP (enhanced GFP)
🔴 mCherry
🟣 CFP, YFP, RFP, etc.

Each has unique properties:

Brightness
Photostability
Expression efficiency
Aggregation tendency (e.g., mCherry can clump)
Background autofluorescence (less in red than green)

This allows multi-color labeling in tissues — like every neuron in a worm brain glowing in different colors.

⚠️ Functional Considerations

Adding fluorescent tags is not neutral — they’re large proteins that can:

Alter the function of the target protein
Change aggregation or degradation patterns
Affect folding or localization, depending on whether you tag the N- or C-terminus

Researchers must test whether tagging changes the protein’s normal behavior.

🧩 Summary Cheat Sheet

Concept	Key Idea	Example / Detail
Fluorescence	Molecule absorbs short λ, emits longer λ	Blue in → Green out
Microscope setup	Filters + dichroic mirror separate excitation/emission	Blue excitation filter → Green emission
Chemical stain	Direct dye binding	Hoechst binds DNA (blue)
Antibody labeling	Fluorophore on antibody	Primary + fluorescent secondary
GFP tagging	Genetic fluorescent marker	GFP, mCherry, YFP
Limitations	Tag may alter protein behavior	Aggregation, misfolding, interference

Quiz

Score: 0/30 (0%)

Q0. What is the main advantage of using fluorescent staining in microscopy?

It reduces background noise

It allows visualization of specific structures in otherwise transparent samples

It increases the sample’s size

It changes the refractive index of the specimen

Q1. What is the physical process underlying fluorescence?

Absorption of low-energy light and emission of higher-energy light

Emission of the same wavelength as excitation

Absorption of light followed by emission of lower-energy (longer wavelength) light

Refraction of light through the sample

Q2. What is the 'Stokes shift' in fluorescence?

The loss of energy between excitation and emission leading to longer emission wavelength

The energy gain after emission

The increase in intensity during excitation

The phase shift of polarized light

Q3. Which component in a fluorescence microscope reflects excitation light and transmits emitted light?

Excitation filter

Dichroic mirror

Emission filter

Objective lens

Q4. Why are filter cubes used in fluorescence microscopy?

To magnify images

To separate and control the excitation and emission wavelengths

To maintain sample temperature

To focus the beam on the sample

Q5. In a fluorescence microscope setup, what is the role of the excitation filter?

It blocks all light except the desired excitation wavelength

It amplifies emitted light

It detects fluorescence

It filters emitted light

Q6. Which dye binds specifically to double-stranded DNA and fluoresces blue under UV light?

Rhodamine

Hoechst (DAPI)

FITC

mCherry

Q7. Why is antibody labeling commonly used in fluorescence microscopy?

It is cheaper than chemical staining

It allows specific detection of proteins or structures for which no direct dye exists

It eliminates the need for filters

It provides 3D imaging capability

Q8. What is the function of the secondary antibody in indirect immunofluorescence?

It binds directly to the target protein

It binds to the primary antibody and carries the fluorescent tag

It amplifies the excitation light

It removes background autofluorescence

Q9. What was Douglas Prasher’s main contribution to fluorescence research?

He developed confocal microscopy

He discovered the gene encoding GFP

He synthesized fluorescent dyes

He invented antibody conjugation methods

Q10. Which organism was first used to express GFP in research?

Drosophila melanogaster

Escherichia coli

Caenorhabditis elegans

Mus musculus

Q11. Which property of mCherry makes it different from GFP?

It fluoresces blue

It forms aggregates and emits red light

It binds directly to DNA

It is not a protein

Q12. Why might GFP tagging affect protein function?

Because GFP changes the protein’s charge and mass

Because GFP is a large protein that can interfere with folding or localization

Because GFP emits toxic wavelengths

Because GFP competes with actin filaments

Q13. What is an advantage of using genetically encoded fluorescent proteins over chemical stains?

They do not require excitation light

They can be expressed directly by living cells

They are brighter than all dyes

They automatically stain DNA

Q14. Why is it beneficial to have a palette of different fluorescent proteins (e.g., GFP, YFP, mCherry)?

To study temperature effects

To mark multiple structures or cell types simultaneously

To reduce signal intensity

To visualize movement in black-and-white

Q15. Fluorescence always emits light with a shorter wavelength than excitation light.

True

False

Q16. The dichroic mirror in a fluorescence microscope both reflects and transmits light depending on wavelength.

True

False

Q17. Hoechst dye fluoresces even when not bound to DNA.

True

False

Q18. The emission filter allows both excitation and emission light to pass to the detector.

True

False

Q19. Antibody labeling can be used to detect specific proteins or cellular structures.

True

False

Q20. Douglas Prasher received the Nobel Prize for GFP discovery.

True

False

Q21. Martin Chalfie demonstrated GFP expression in C. elegans.

True

False

Q22. GFP and mCherry are examples of chemical dyes.

True

False

Q23. Using a secondary antibody with a fluorophore reduces cost because it can be reused for multiple primary antibodies.

True

False

Q24. The Stokes shift refers to a decrease in emission energy compared to excitation energy.

True

False

Q25. In fluorescence, excitation light always has a longer wavelength than emitted light.

True

False

Q26. Fluorescent tags can interfere with the target protein’s normal function.

True

False

Q27. The dichroic mirror is unnecessary if the sample emits strong fluorescence.

True

False

Q28. Fluorescence microscopy allows imaging of multiple fluorophores simultaneously by using different filter sets.

True

False

Q29. GFP can be placed at either the N-terminal or C-terminal end of a protein for tagging.

True

False