HC 04: Fluorescence microscopy
Overview - what fluorescence gives you:
Fluorescence microscopy lets you selectively detect specific molecules with high sensitivity and contrast by exciting fluorophores with short-
wavelength light and collecting their longer-wavelength emission.
• Fluorescence is a molecularly specific contrast mechanism — label molecules (dyes, antibodies, fluorescent proteins) to see their distribution
and dynamics.
• Key advantages: high sensitivity (single-molecule possible), spectral multiplexing (multicolor), live-cell imaging (GFP, biosensors), and
dynamic assays (FRAP, FRET, TIRF).
• Main trade-offs: photobleaching, autofluorescence background, possible phototoxicity in live cells.
Why fluorescence? (Applications):
Use fluorescence to map molecular localization, quantify dynamics and interactions, and measure functional signals (ions, voltages), often in
living cells.
• Localisation: tag proteins/structures (GFP fusions, immunofluorescence, organelle dyes — Hoechst, phalloidin, MitoTracker).
• Dynamics: time-lapse of trafficking, assembly/disassembly, diffusion (FRAP), photoactivation and photoswitching experiments.
• Interactions: FRET for nanometer proximities; fluorescence correlation spectroscopy (FCS) for molecular concentrations and diffusion.
• Surface / membrane events: TIRF — excites only <200 nm above coverslip for single-molecule near-membrane studies.
• Ion & biosensors: ratiometric dyes / protein sensors (Ca²⁺, pH, membrane potential) for quantitative readouts.
Photophysics - Jablonski diagram & timing (physical basis):
Fluorescence arises when molecules absorb photons, relax non-radiatively, then emit lower-energy
photons; key states and transitions are summarized by the Jablonski diagram.
• Energy & photon: 𝐸 = ℎ𝑐/𝜆. Shorter λ → higher photon energy.
• Jablonski timescales: absorption (~10⁻¹⁵ s, femtoseconds), vibrational relaxation/internal
conversion (~10⁻¹² s, picoseconds), fluorescence emission (~10⁻⁹ s, nanoseconds).
• Singlet vs triplet: absorption & emission usually occur between singlet states (S ₀ → S₁ → S₀).
Intersystem crossing to triplet (T₁) is slower but important for photochemistry (triplet lifetimes
longer, can produce reactive oxygen species).
• Phosphorescence: emission from T₁ → S₀ (longer lifetimes, µs–s), generally not used for
microscopy.
Absorption & emission spectra, Stokes shift - short summary:
Fluorophores have characteristic absorption (excitation) and emission spectra; the emission is red-shifted relative to excitation (Stokes shift) and
is independent of excitation wavelength.
• Absorption spectrum: wavelengths the fluorophore can absorb (reflects vibrational
sublevels of excited states).
• Excitation spectrum: practical measure of how well excitation at each λ produces
emission at a chosen λ.
• Emission spectrum: distribution of emitted photon wavelengths (result of relaxation to
lowest S₁ then radiative decay). Emission profile is largely independent of excitation λ.
• Stokes shift: Δλ between excitation peak and emission peak; large Stokes shifts
simplify filter design and reduce bleedthrough.
• Mirrored shapes: absorption and emission are often roughly mirror images because
of vibrational level distributions.
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,Bleaching, blinking, quenching - short summary:
Quenching and blinking are (often reversible) pathways that reduce emission via nonradiative processes or temporary dark states, while
photobleaching is irreversible chemical destruction of fluorophores (often oxygen-mediated) after many excitation cycles. (See Fig. 11.13.)
• Quenching (non-radiative deactivation):
o Caused by nearby molecules/ions that open nonradiative decay channels (O ₂, halogens, amines, some polymers).
o Lowers quantum yield without necessarily changing spectra.
o Can be collisional (dynamic) or static (complex formation).
• Blinking (intermittent on/off):
o Fluorophore stochastically enters a long-lived dark state (often a triplet or other nonfluorescent state); off times range ms → minutes.
o Predicted theoretically early (Bohr) but observed experimentally; important for single-molecule/super-resolution (STORM/PALM) where
controlled blinking is exploited.
o Can be modulated by redox agents (oxidizing/reducing buffers) and imaging buffer composition.
• Photobleaching (irreversible):
o Occurs when excited molecules (especially via triplet state) react chemically (often with O ₂), producing covalent modification and loss
of fluorescence.
o Photodynamic bleaching produces reactive oxygen species (¹O₂, O₂•⁻, OH•) that also damage cells.
o The average number of excitation cycles before bleaching depends on fluorophore structure and environment — some dyes bleach
after few photons, others survive millions.
Fluorophore properties - what matters for experiments:
Choose fluorophores by brightness (ε × Φ), photostability, spectral separation, chemical compatibility, and biological suitability.
• Molar extinction coefficient (ε): how strongly the dye absorbs at its excitation λ (units M⁻¹cm⁻¹).
• Quantum yield (Φ): fraction of absorbed photons re-emitted as fluorescence (0–1).
• Brightness: brightness = 𝜀 × 𝛷— the primary practical measure of how bright a label will appear.
• Photostability: resistance to photobleaching (vital for time-lapse and high laser power experiments).
• Chemical properties: solubility (sulfonation increases water solubility), pKa (pH sensitivity), conjugation chemistry (NHS, maleimide),
tendency to aggregate or self-quench.
Common fluorophore classes — short summary:
Fluorophores include small organic dyes (fluorescein, rhodamine, Alexa, Cy dyes), fluorescent proteins (GFP family), quantum dots
(semiconductor nanocrystals), and hybrid self-labeling tags.
• Organic dyes: small, bright, easy conjugation, some very photostable (Alexa, ATTO), but may be hard to load into living cells.
• Alexa / ATTO / Cyanine (Cy) dyes: modern synthetic dyes with high Φ, good photostability, tailored excitation to laser lines (e.g., Alexa488,
Cy3, Cy5).
• Quantum dots: CdSe/ZnS cores; very photostable, narrow emission, broad absorption; require surface coating and conjugation; size-
tunable emission; toxicity & targeting can be issues.
• Fluorescent proteins (FPs): GFP & spectral variants (CFP, YFP, RFP, mCherry, far-red FPs). Genetically encodable, great for live cells, but
typically less photostable and larger than small dyes; chromophore matures autocatalytically (requires O ₂). (Fig. 11.7)
• Hybrid tag: SNAP/CLIP/ HaloTag allow genetic fusion + covalent labeling with synthetic dyes (combines live-cell targeting with bright dyes)
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, Immunofluorescence & probes — short summary:
Immunofluorescence uses antibody–fluorophore conjugates (direct or indirect) to label endogenous proteins; organelle probes and small
molecule biosensors give functional readouts.
• Direct IF: primary antibody directly conjugated with fluorophore (simpler, less
amplification).
• Indirect IF: unlabeled primary + labeled secondary (amplifies signal because
multiple secondaries bind one primary).
• Common probes: Hoechst/DAPI (DNA/nucleus), phalloidin-Alexa (F-actin), WGA
(membranes), MitoTracker (mitochondria), LysoTracker (lysosomes).
• Environmental probes: dyes change intensity or spectrum with ion binding (e.g.,
Ca²⁺ indicators), pH, polarity—used for ratiometric quantitation.
• Quantum dot & nanoprobes: strong photostability for long acquisitions, but size and targeting are considerations.
Instrumentation: epi-illumination, filters, and cubes — short summary:
Fluorescence microscopes use epi-illumination (excitation and emission through the same objective) and a filter set
(exciter, dichroic mirror, emission barrier) to separate excitation and emission wavelengths.
• Epi-illumination advantage: objective serves as both condenser and collection lens — efficient excitation and
collection, especially with high-NA objectives.
• Filter set components:
o Excitation (bandpass) filter: selects wavelengths that excite the fluorophore.
o Dichroic mirror / beamsplitter: reflects excitation to specimen, transmits longer emission to
detector (45°).
o Emission (barrier) filter: passes only emission band to detector, blocks stray excitation.
• Filter cube: physical mount containing matched filters; rotating cubes enable rapid switching between
channels.
• Bleedthrough & spectral overlap: excitation/emission bands may overlap → bleedthrough (~10–15%
typical) — minimize by choosing fluorophores with separated spectra and narrow band filters.
Light sources — short summary:
Choose excitation source to match fluorophore and experiment: arc lamps (mercury/xenon/metal-halide) for broad spectral lines, lasers for
narrow high-power peaks, and LEDs for stable, low-cost illumination.
• Mercury arc: very bright spectral lines (e.g., 365, 405, 436, 546 nm) — great for DAPI, rhodamine; expensive to maintain, short lifetime vs
LEDs.
• Xenon: more continuous output; useful for some dyes (fluorescein, Cy5).
• Metal-halide: bright, continuous, and more stable than mercury for many dyes (good for GFP).
• Lasers: monochromatic, high intensity, essential for confocal, TIRF, and laser-scanning systems; matched to common fluorophores (488,
561, 640 nm).
• LEDs: narrow bands, long life, rapid switching, low heat; increasingly popular for routine epi-fluorescence and live imaging.
• Matching: match source spectrum/lines to fluorophore excitation maxima and to filters for best efficiency.
Objectives & resolution in fluorescence — short summary:
Use high-NA, low-autofluorescence, color-corrected objectives (e.g., plan-apochromat, fluorite) to maximize collection efficiency and resolution;
fluorescence lateral resolution ≈ 0.61λ/NA.
• Objective choice: high NA (1.25–1.49 oil) yields maximum light collection and best resolution; use low-fluorescence glass and appropriate
immersion medium.
• Image brightness scaling: fluorescence photon flux ∝ NA⁴ / M² (so NA has huge impact).
• Resolution for incoherent fluorescence: 𝑑 = 0.61 𝜆/𝑁𝐴(where λ is mean emission wavelength). Axial resolution is ~2–3× worse than lateral.
• Coverslip & immersion matching: correct coverslip thickness and immersion oil minimize spherical aberration and loss of signal.
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Overview - what fluorescence gives you:
Fluorescence microscopy lets you selectively detect specific molecules with high sensitivity and contrast by exciting fluorophores with short-
wavelength light and collecting their longer-wavelength emission.
• Fluorescence is a molecularly specific contrast mechanism — label molecules (dyes, antibodies, fluorescent proteins) to see their distribution
and dynamics.
• Key advantages: high sensitivity (single-molecule possible), spectral multiplexing (multicolor), live-cell imaging (GFP, biosensors), and
dynamic assays (FRAP, FRET, TIRF).
• Main trade-offs: photobleaching, autofluorescence background, possible phototoxicity in live cells.
Why fluorescence? (Applications):
Use fluorescence to map molecular localization, quantify dynamics and interactions, and measure functional signals (ions, voltages), often in
living cells.
• Localisation: tag proteins/structures (GFP fusions, immunofluorescence, organelle dyes — Hoechst, phalloidin, MitoTracker).
• Dynamics: time-lapse of trafficking, assembly/disassembly, diffusion (FRAP), photoactivation and photoswitching experiments.
• Interactions: FRET for nanometer proximities; fluorescence correlation spectroscopy (FCS) for molecular concentrations and diffusion.
• Surface / membrane events: TIRF — excites only <200 nm above coverslip for single-molecule near-membrane studies.
• Ion & biosensors: ratiometric dyes / protein sensors (Ca²⁺, pH, membrane potential) for quantitative readouts.
Photophysics - Jablonski diagram & timing (physical basis):
Fluorescence arises when molecules absorb photons, relax non-radiatively, then emit lower-energy
photons; key states and transitions are summarized by the Jablonski diagram.
• Energy & photon: 𝐸 = ℎ𝑐/𝜆. Shorter λ → higher photon energy.
• Jablonski timescales: absorption (~10⁻¹⁵ s, femtoseconds), vibrational relaxation/internal
conversion (~10⁻¹² s, picoseconds), fluorescence emission (~10⁻⁹ s, nanoseconds).
• Singlet vs triplet: absorption & emission usually occur between singlet states (S ₀ → S₁ → S₀).
Intersystem crossing to triplet (T₁) is slower but important for photochemistry (triplet lifetimes
longer, can produce reactive oxygen species).
• Phosphorescence: emission from T₁ → S₀ (longer lifetimes, µs–s), generally not used for
microscopy.
Absorption & emission spectra, Stokes shift - short summary:
Fluorophores have characteristic absorption (excitation) and emission spectra; the emission is red-shifted relative to excitation (Stokes shift) and
is independent of excitation wavelength.
• Absorption spectrum: wavelengths the fluorophore can absorb (reflects vibrational
sublevels of excited states).
• Excitation spectrum: practical measure of how well excitation at each λ produces
emission at a chosen λ.
• Emission spectrum: distribution of emitted photon wavelengths (result of relaxation to
lowest S₁ then radiative decay). Emission profile is largely independent of excitation λ.
• Stokes shift: Δλ between excitation peak and emission peak; large Stokes shifts
simplify filter design and reduce bleedthrough.
• Mirrored shapes: absorption and emission are often roughly mirror images because
of vibrational level distributions.
1|Page
,Bleaching, blinking, quenching - short summary:
Quenching and blinking are (often reversible) pathways that reduce emission via nonradiative processes or temporary dark states, while
photobleaching is irreversible chemical destruction of fluorophores (often oxygen-mediated) after many excitation cycles. (See Fig. 11.13.)
• Quenching (non-radiative deactivation):
o Caused by nearby molecules/ions that open nonradiative decay channels (O ₂, halogens, amines, some polymers).
o Lowers quantum yield without necessarily changing spectra.
o Can be collisional (dynamic) or static (complex formation).
• Blinking (intermittent on/off):
o Fluorophore stochastically enters a long-lived dark state (often a triplet or other nonfluorescent state); off times range ms → minutes.
o Predicted theoretically early (Bohr) but observed experimentally; important for single-molecule/super-resolution (STORM/PALM) where
controlled blinking is exploited.
o Can be modulated by redox agents (oxidizing/reducing buffers) and imaging buffer composition.
• Photobleaching (irreversible):
o Occurs when excited molecules (especially via triplet state) react chemically (often with O ₂), producing covalent modification and loss
of fluorescence.
o Photodynamic bleaching produces reactive oxygen species (¹O₂, O₂•⁻, OH•) that also damage cells.
o The average number of excitation cycles before bleaching depends on fluorophore structure and environment — some dyes bleach
after few photons, others survive millions.
Fluorophore properties - what matters for experiments:
Choose fluorophores by brightness (ε × Φ), photostability, spectral separation, chemical compatibility, and biological suitability.
• Molar extinction coefficient (ε): how strongly the dye absorbs at its excitation λ (units M⁻¹cm⁻¹).
• Quantum yield (Φ): fraction of absorbed photons re-emitted as fluorescence (0–1).
• Brightness: brightness = 𝜀 × 𝛷— the primary practical measure of how bright a label will appear.
• Photostability: resistance to photobleaching (vital for time-lapse and high laser power experiments).
• Chemical properties: solubility (sulfonation increases water solubility), pKa (pH sensitivity), conjugation chemistry (NHS, maleimide),
tendency to aggregate or self-quench.
Common fluorophore classes — short summary:
Fluorophores include small organic dyes (fluorescein, rhodamine, Alexa, Cy dyes), fluorescent proteins (GFP family), quantum dots
(semiconductor nanocrystals), and hybrid self-labeling tags.
• Organic dyes: small, bright, easy conjugation, some very photostable (Alexa, ATTO), but may be hard to load into living cells.
• Alexa / ATTO / Cyanine (Cy) dyes: modern synthetic dyes with high Φ, good photostability, tailored excitation to laser lines (e.g., Alexa488,
Cy3, Cy5).
• Quantum dots: CdSe/ZnS cores; very photostable, narrow emission, broad absorption; require surface coating and conjugation; size-
tunable emission; toxicity & targeting can be issues.
• Fluorescent proteins (FPs): GFP & spectral variants (CFP, YFP, RFP, mCherry, far-red FPs). Genetically encodable, great for live cells, but
typically less photostable and larger than small dyes; chromophore matures autocatalytically (requires O ₂). (Fig. 11.7)
• Hybrid tag: SNAP/CLIP/ HaloTag allow genetic fusion + covalent labeling with synthetic dyes (combines live-cell targeting with bright dyes)
2|Page
, Immunofluorescence & probes — short summary:
Immunofluorescence uses antibody–fluorophore conjugates (direct or indirect) to label endogenous proteins; organelle probes and small
molecule biosensors give functional readouts.
• Direct IF: primary antibody directly conjugated with fluorophore (simpler, less
amplification).
• Indirect IF: unlabeled primary + labeled secondary (amplifies signal because
multiple secondaries bind one primary).
• Common probes: Hoechst/DAPI (DNA/nucleus), phalloidin-Alexa (F-actin), WGA
(membranes), MitoTracker (mitochondria), LysoTracker (lysosomes).
• Environmental probes: dyes change intensity or spectrum with ion binding (e.g.,
Ca²⁺ indicators), pH, polarity—used for ratiometric quantitation.
• Quantum dot & nanoprobes: strong photostability for long acquisitions, but size and targeting are considerations.
Instrumentation: epi-illumination, filters, and cubes — short summary:
Fluorescence microscopes use epi-illumination (excitation and emission through the same objective) and a filter set
(exciter, dichroic mirror, emission barrier) to separate excitation and emission wavelengths.
• Epi-illumination advantage: objective serves as both condenser and collection lens — efficient excitation and
collection, especially with high-NA objectives.
• Filter set components:
o Excitation (bandpass) filter: selects wavelengths that excite the fluorophore.
o Dichroic mirror / beamsplitter: reflects excitation to specimen, transmits longer emission to
detector (45°).
o Emission (barrier) filter: passes only emission band to detector, blocks stray excitation.
• Filter cube: physical mount containing matched filters; rotating cubes enable rapid switching between
channels.
• Bleedthrough & spectral overlap: excitation/emission bands may overlap → bleedthrough (~10–15%
typical) — minimize by choosing fluorophores with separated spectra and narrow band filters.
Light sources — short summary:
Choose excitation source to match fluorophore and experiment: arc lamps (mercury/xenon/metal-halide) for broad spectral lines, lasers for
narrow high-power peaks, and LEDs for stable, low-cost illumination.
• Mercury arc: very bright spectral lines (e.g., 365, 405, 436, 546 nm) — great for DAPI, rhodamine; expensive to maintain, short lifetime vs
LEDs.
• Xenon: more continuous output; useful for some dyes (fluorescein, Cy5).
• Metal-halide: bright, continuous, and more stable than mercury for many dyes (good for GFP).
• Lasers: monochromatic, high intensity, essential for confocal, TIRF, and laser-scanning systems; matched to common fluorophores (488,
561, 640 nm).
• LEDs: narrow bands, long life, rapid switching, low heat; increasingly popular for routine epi-fluorescence and live imaging.
• Matching: match source spectrum/lines to fluorophore excitation maxima and to filters for best efficiency.
Objectives & resolution in fluorescence — short summary:
Use high-NA, low-autofluorescence, color-corrected objectives (e.g., plan-apochromat, fluorite) to maximize collection efficiency and resolution;
fluorescence lateral resolution ≈ 0.61λ/NA.
• Objective choice: high NA (1.25–1.49 oil) yields maximum light collection and best resolution; use low-fluorescence glass and appropriate
immersion medium.
• Image brightness scaling: fluorescence photon flux ∝ NA⁴ / M² (so NA has huge impact).
• Resolution for incoherent fluorescence: 𝑑 = 0.61 𝜆/𝑁𝐴(where λ is mean emission wavelength). Axial resolution is ~2–3× worse than lateral.
• Coverslip & immersion matching: correct coverslip thickness and immersion oil minimize spherical aberration and loss of signal.
3|Page
