Super-Resolution and Electron Microscopy

Super-Resolution and Electron Microscopy
Detailed Notes + 10 MCQs

STED • SIM • STORM/PALM • TEM • SEM • Cryo-Electron Microscopy

Exam Focus: These notes are useful for CSIR-NET Life Sciences, GATE Biotechnology, DBT, ICMR, CUET-PG, MSc Biotechnology, MSc Life Sciences and other competitive examinations.

📑 Index / Table of Contents

1. Introduction to Advanced Microscopy
2. Super-Resolution Microscopy
3. Diffraction Limit of Light Microscopy
4. STED – Stimulated Emission Depletion Microscopy
5. SIM – Structured Illumination Microscopy
6. STORM – Stochastic Optical Reconstruction Microscopy
7. PALM – Photoactivated Localization Microscopy
8. TEM – Transmission Electron Microscopy
9. SEM – Scanning Electron Microscopy
10. Cryo-Electron Microscopy
11. Principle of Electron Microscopy
12. Comparison of STED, SIM, STORM/PALM, TEM, SEM and Cryo-EM
13. Advantages and Limitations
14. Important Exam Points
15. Common Exam Traps
16. 10 Practice MCQs
17. Quick Revision

🔬 1. Introduction to Advanced Microscopy

Microscopy is one of the most important analytical approaches in biology. Traditional light microscopy allows researchers to visualize cells, tissues, microorganisms and many cellular structures. However, conventional light microscopy has a fundamental limitation imposed by the wave nature of light. Very small structures cannot always be distinguished from one another because of the diffraction limit.

Modern microscopy has therefore developed in two major directions. The first is the development of super-resolution optical microscopy, which allows researchers to obtain spatial information beyond the classical diffraction limit of conventional light microscopy. The second is electron microscopy, in which electrons rather than visible light are used to form images.

The topics covered in this chapter are particularly important because they represent different strategies for obtaining higher-resolution structural information.

• STED uses stimulated emission depletion.
• SIM uses structured illumination and computational reconstruction.
• STORM uses stochastic activation and localization of individual molecules.
• PALM uses photoactivatable/photo-switchable fluorescent molecules.
• TEM uses transmitted electrons to visualize internal ultrastructure.
• SEM scans the specimen surface with an electron beam.
• Cryo-EM examines rapidly frozen biological samples in a near-native hydrated state.

High-Yield Concept: STED, SIM, STORM and PALM are optical super-resolution methods, whereas TEM, SEM and cryo-EM are electron microscopy approaches.

🌟 2. Super-Resolution Microscopy

Super-resolution microscopy refers to a group of techniques that overcome or circumvent the limitations imposed by conventional optical microscopy. These techniques allow biological structures that are closer together than the classical diffraction-limited resolution to be spatially distinguished.

Why is super-resolution necessary?

Many biologically important structures are very small. Molecular complexes, membrane proteins, cytoskeletal components, synaptic structures and protein clusters can have dimensions below the resolution achievable using ordinary far-field light microscopy.

• Protein clusters may be very closely spaced.
• Membrane structures can be extremely thin.
• DNA-protein complexes may be smaller than the classical optical resolution.
• Synaptic structures may contain molecular organization at nanometre scales.
• Organelle-associated proteins may be separated by distances below the conventional optical limit.

Conceptual illustration: conventional microscopy can blur closely spaced objects, while super-resolution techniques can resolve them more effectively.

📐 3. Diffraction Limit of Light Microscopy

The resolution of conventional optical microscopy is limited by diffraction. When light passes through an optical system, a point object does not form a perfect mathematical point in the image. Instead, it produces a diffraction pattern.

The central bright region of this diffraction pattern is commonly associated with the Airy disk. When two objects are sufficiently close, their diffraction patterns overlap and they can no longer be distinguished as separate structures.

d ≈ λ / (2NA)

where d represents the minimum resolvable distance, λ is the wavelength of light and NA is the numerical aperture.

Important implications

• Shorter wavelengths generally provide better spatial resolution.
• Higher numerical aperture improves resolution.
• Conventional fluorescence microscopy is still limited by diffraction.
• Super-resolution methods use specialized optical or computational strategies to overcome or circumvent this limitation.

Remember: Super-resolution microscopy does not simply mean using a stronger magnifying lens. The key objective is to obtain information about structures that cannot be spatially separated by conventional diffraction-limited imaging.

🔴 4. STED – Stimulated Emission Depletion Microscopy

STED stands for Stimulated Emission Depletion Microscopy. It is a super-resolution fluorescence microscopy technique based on the controlled suppression of fluorescence around the focal region.

Basic principle

In conventional fluorescence microscopy, molecules within the illuminated region can become excited and emit fluorescence. STED adds a second laser beam, called the STED or depletion beam.

The depletion beam is shaped so that it suppresses fluorescence around the central focal region. The central area where fluorescence is not depleted remains available for detection.

A common STED configuration uses a doughnut-shaped depletion beam. The center of the doughnut contains very low or approximately zero depletion intensity, while the surrounding region receives strong depletion illumination.

Sequence of events in STED

1. The excitation beam excites fluorophores.
2. The depletion beam surrounds the central excited region.
3. Stimulated emission removes excited-state molecules from the surrounding region.
4. Only a very small central region remains capable of producing detectable fluorescence.
5. The resulting effective fluorescent spot can be much smaller than the conventional diffraction-limited spot.

Conceptual STED diagram showing excitation and doughnut-shaped depletion.

Advantages of STED

• Provides very high spatial resolution.
• Can be used for fluorescence imaging.
• Can reveal molecular organization below the classical diffraction limit.
• Can provide useful information about dynamic cellular structures.
• Does not require stochastic reconstruction in the same way as STORM/PALM.

Limitations of STED

• Requires specialized laser systems.
• High depletion intensity can increase photobleaching or photodamage.
• Instrumentation can be expensive.
• Fluorophore properties are important.
• Imaging conditions can be technically demanding.

Exam Keyword: STED = Stimulated emission depletion + doughnut-shaped depletion beam.

🟦 5. SIM – Structured Illumination Microscopy

SIM stands for Structured Illumination Microscopy. It is a super-resolution technique that uses a patterned or structured illumination pattern to interact with fine spatial details in the specimen.

Basic principle

In ordinary microscopy, some high-frequency spatial information from the sample cannot be directly detected because it lies outside the accessible optical frequency range.

SIM illuminates the sample using a known structured pattern, commonly a periodic pattern. Interaction between the illumination pattern and the sample creates moiré-like information that shifts otherwise inaccessible spatial information into the detectable frequency range.

Multiple images are acquired using different orientations and phases of the illumination pattern. Computational algorithms then reconstruct a super-resolution image.

Major steps in SIM

1. Illuminate the sample with a structured pattern.
2. Change the orientation and/or phase of the pattern.
3. Acquire multiple images.
4. Process the image data computationally.
5. Reconstruct an image containing enhanced spatial-frequency information.

SIM uses structured illumination and computational reconstruction to recover additional spatial-frequency information.

Advantages of SIM

• Can provide super-resolution using relatively gentle illumination compared with some high-intensity methods.
• Can be applied to many fluorescent samples.
• Useful for live-cell imaging.
• Produces relatively fast image acquisition compared with some single-molecule localization methods.
• Requires computational image reconstruction.

Limitations of SIM

• Resolution improvement is generally more modest than the most extreme localization approaches.
• Multiple raw images are required for reconstruction.
• Image artifacts can occur if acquisition or reconstruction is poor.
• High-quality structured illumination is required.

🟣 6. STORM – Stochastic Optical Reconstruction Microscopy

STORM stands for Stochastic Optical Reconstruction Microscopy. It is a single-molecule localization microscopy technique.

The central concept is that fluorescent molecules are not all allowed to emit strongly at the same time. Instead, only a sparse subset of fluorophores is activated or allowed to fluoresce at a particular moment.

Why is stochastic activation important?

If many nearby fluorophores emit simultaneously, their diffraction-limited spots overlap and cannot be individually localized. STORM solves this problem by separating the emission events in time.

Basic STORM workflow

1. Only a small subset of fluorescent molecules is activated.
2. Each active molecule produces a diffraction-limited spot.
3. The center of that spot can be estimated mathematically with high precision.
4. The molecules are switched off or become inactive.
5. A different subset is activated.
6. Thousands of localization events can be collected.
7. All localizations are computationally combined to construct a super-resolution image.

STORM obtains super-resolution by separating fluorescence events in time and localizing individual molecules.

🟠 7. PALM – Photoactivated Localization Microscopy

PALM stands for Photoactivated Localization Microscopy. It is another single-molecule localization technique and shares an important concept with STORM.

PALM commonly uses genetically encoded photoactivatable or photoswitchable fluorescent proteins. Individual molecules are activated in small subsets, localized, and then combined computationally into a super-resolution image.

STORM vs PALM

Feature	STORM	PALM
Full form	Stochastic Optical Reconstruction Microscopy	Photoactivated Localization Microscopy
Main concept	Stochastic activation/localization	Photoactivation/localization
Typical labels	Fluorescent dyes and suitable probes	Photoactivatable/photo-switchable fluorescent proteins
Common use	Molecular organization and membrane structures	Localization of genetically encoded proteins

Exam Point: STORM and PALM are both single-molecule localization microscopy methods. PALM is especially associated with photoactivatable fluorescent proteins.

⚛️ 8. Principle of Electron Microscopy

Electron microscopy uses a beam of electrons instead of visible light to generate an image. Electrons have a much shorter de Broglie wavelength than visible light when appropriately accelerated. This allows electron microscopes to achieve much higher spatial resolution than conventional optical microscopes.

Major components

• Electron source or electron gun.
• Electromagnetic lenses.
• Specimen chamber.
• Electron beam system.
• Vacuum system.
• Detector or imaging system.

Because electrons interact strongly with matter and because the electron beam travels through a controlled vacuum, specimen preparation is considerably different from ordinary light microscopy.

Key distinction: Light microscope → photons/light.
Electron microscope → electrons.

🔵 9. TEM – Transmission Electron Microscopy

TEM stands for Transmission Electron Microscopy. In TEM, electrons pass through a thin specimen and the transmitted electron signal is used to produce an image.

Basic principle

A high-energy electron beam passes through a very thin section of the specimen. Different regions of the specimen scatter electrons to different extents. The resulting differences in transmitted electrons create image contrast.

What does TEM show?

TEM is particularly useful for studying internal ultrastructure. It can reveal fine cellular structures such as membranes, organelles, ribosomes and other subcellular components when appropriate specimen preparation is used.

Typical TEM workflow

1. Fixation of the biological sample.
2. Dehydration.
3. Embedding in a suitable resin.
4. Preparation of ultrathin sections.
5. Placement of sections on grids.
6. Contrast enhancement using suitable electron-dense stains when appropriate.
7. Electron beam transmission through the specimen.
8. Image formation and detection.

TEM uses transmitted electrons to provide high-resolution information about internal ultrastructure.

Advantages of TEM

• Very high spatial resolution.
• Excellent for cellular ultrastructure.
• Can visualize very small structures.
• Useful for organelles, membranes and macromolecular assemblies.

Limitations of TEM

• Samples usually require extensive preparation.
• Specimens must be sufficiently thin for transmission.
• Traditional preparation can introduce artifacts.
• High vacuum conditions are generally required.
• Live biological samples cannot ordinarily be observed directly in conventional TEM.

🟢 10. SEM – Scanning Electron Microscopy

SEM stands for Scanning Electron Microscopy. Instead of passing electrons through the specimen as in TEM, SEM scans a focused electron beam across the specimen surface.

The interaction between the electron beam and specimen generates signals, including secondary electrons and backscattered electrons. These signals are detected and used to construct an image.

What does SEM primarily show?

SEM is particularly useful for examining surface morphology and topography. The resulting images often have a three-dimensional appearance because surface relief influences the detected signal.

Applications

• Cell surface morphology.
• Microorganism surface structures.
• Plant surface structures.
• Tissue surfaces.
• Materials and nanostructures.
• Surface topography.
• Biomaterial characterization.

SEM scans the surface with an electron beam and detects emitted electron signals.

TEM vs SEM

Feature	TEM	SEM
Full form	Transmission Electron Microscopy	Scanning Electron Microscopy
Electron interaction	Electrons transmitted through thin sample	Electron beam scans specimen surface
Main information	Internal ultrastructure	Surface morphology
Sample requirement	Very thin sections or suitable thin specimens	Surface must be prepared for scanning
Image appearance	Usually 2D projection of internal structures	Strong surface/topographical appearance

❄️ 11. Cryo-Electron Microscopy

Cryo-electron microscopy (cryo-EM) is a group of electron microscopy approaches in which biological samples are rapidly frozen and examined at cryogenic temperatures.

A major goal is to preserve biological structures in a hydrated state that is closer to their native condition than conventional chemical fixation and dehydration methods.

What is vitrification?

In cryo-EM, a thin layer of aqueous sample can be rapidly frozen so that the water forms a glass-like, non-crystalline state called vitreous ice. This process is known as vitrification.

Avoiding crystalline ice is important because ice crystals can damage biological structures and interfere with imaging.

Major steps

1. Prepare the biological sample.
2. Apply a small volume to an EM grid.
3. Blot excess liquid to create a thin film.
4. Rapidly plunge-freeze the sample under suitable conditions.
5. Water becomes vitrified rather than forming large ice crystals.
6. Keep the sample at cryogenic temperature.
7. Image the sample using an electron microscope.
8. Process images computationally.

Cryo-EM workflow: sample preparation, vitrification and imaging under cryogenic conditions.

Why is cryo-EM important?

• Can preserve biological samples in a near-native hydrated state.
• Reduces the need for conventional chemical fixation and dehydration in many workflows.
• Useful for macromolecular structure determination.
• Important for structural biology.
• Can be applied to proteins, protein complexes, viruses and cellular structures.

Single-particle cryo-EM

In single-particle cryo-EM, many copies of a purified macromolecule are frozen in different orientations in vitreous ice. Individual particle images are identified computationally and grouped according to similar orientations and structural information.

Large numbers of particle images can then be aligned and computationally averaged to reconstruct a three-dimensional structure.

Advantages of cryo-EM

• No requirement for crystallization in single-particle cryo-EM.
• Suitable for large macromolecular complexes.
• Can preserve samples in vitreous ice.
• Three-dimensional structures can be reconstructed computationally.
• Extremely high structural resolution can be achieved for suitable specimens.

Limitations

• Instrumentation is expensive.
• Sample preparation can be technically demanding.
• High-quality vitrification is essential.
• Radiation damage remains an important consideration.
• Computational processing can require substantial resources.

📊 12. Complete Comparison of Advanced Microscopy

Technique	Main Principle	Sample/Label	Main Information	Key Feature
STED	Stimulated emission depletion	Fluorescent labels	Molecular/cellular structures	Doughnut-shaped depletion beam
SIM	Structured illumination + reconstruction	Fluorescent samples	Cellular and molecular organization	Patterned illumination
STORM	Single-molecule stochastic localization	Fluorescent probes	Molecular organization	Sequential sparse activation
PALM	Photoactivation/localization	Photoactivatable fluorescent proteins	Protein localization	Single-molecule localization
TEM	Electron transmission through sample	Thin specimens	Internal ultrastructure	Very high resolution
SEM	Scanning electron beam over surface	Prepared specimen surfaces	Surface morphology	Topographical information
Cryo-EM	Electron microscopy of vitrified specimens	Rapidly frozen hydrated samples	Macromolecular/cellular structure	Near-native preservation

🔬 13. Super-Resolution Optical Microscopy vs Electron Microscopy

Feature	Super-Resolution Optical Microscopy	Electron Microscopy
Radiation	Light	Electrons
Examples	STED, SIM, STORM, PALM	TEM, SEM, Cryo-EM
Fluorescent labelling	Often used	Not the basic imaging principle
Live-cell potential	Some methods can image living cells	Conventional EM generally requires non-living prepared samples
Main strength	Molecular specificity and cellular imaging	Very high structural resolution
Sample environment	Can often work under aqueous physiological conditions	Usually vacuum; cryo methods use cryogenic conditions

⚖️ 14. Advantages and Limitations

STED

• Advantage: Very high spatial resolution.
• Advantage: Direct fluorescence imaging.
• Limitation: Requires powerful specialized illumination.
• Limitation: Photobleaching and phototoxicity may be important.

SIM

• Advantage: Useful for live-cell fluorescence imaging.
• Advantage: Relatively fast compared with many localization methods.
• Limitation: Requires computational reconstruction.
• Limitation: Reconstruction artifacts can occur.

STORM/PALM

• Advantage: Very high localization precision.
• Advantage: Can provide molecular-scale organization.
• Limitation: Requires many sequential imaging events.
• Limitation: Reconstruction and labeling strategies can be demanding.

TEM

• Advantage: Excellent internal ultrastructural resolution.
• Advantage: Useful for organelles and membranes.
• Limitation: Requires thin samples.
• Limitation: Conventional preparation can introduce artifacts.

SEM

• Advantage: Excellent surface morphology.
• Advantage: Strong topographical appearance.
• Limitation: Does not directly provide the same internal ultrastructural information as TEM.

Cryo-EM

• Advantage: Near-native hydrated sample preservation.
• Advantage: Powerful structural biology technique.
• Advantage: Can provide three-dimensional reconstructions.
• Limitation: Expensive and technically demanding.
• Limitation: Requires careful vitrification and cryogenic handling.

🎯 15. Important Exam Points

Super-resolution

• Super-resolution methods overcome or circumvent the conventional diffraction limit.
• STED uses stimulated emission depletion.
• SIM uses structured illumination.
• STORM uses stochastic single-molecule localization.
• PALM uses photoactivation and localization.

STED

• Think: Doughnut-shaped depletion beam.
• Stimulated emission depletes fluorescence around the center.
• Only a smaller central region remains fluorescent.

SIM

• Think: Patterned illumination.
• Multiple images are acquired.
• Computational reconstruction is essential.
• Spatial-frequency information is shifted into the detectable range.

STORM

• Think: Stochastic activation.
• Individual molecules are localized.
• Many localization events form the final image.

PALM

• Think: Photoactivatable fluorescent proteins.
• Single molecules are activated and localized.

TEM

• Think: Through the specimen.
• Shows internal ultrastructure.
• Requires thin specimens.

SEM

• Think: Surface scanning.
• Shows surface morphology and topography.
• Uses signals generated by electron-beam/specimen interactions.

Cryo-EM

• Think: Rapid freezing + vitrification.
• Water is preserved in a vitreous state.
• Useful for near-native structural preservation.
• Single-particle cryo-EM can generate 3D structures computationally.

⚠️ 16. Common Exam Traps

• Trap 1: STED is not based on stochastic single-molecule localization. It uses stimulated emission depletion.
• Trap 2: SIM does not depend on a doughnut-shaped depletion beam. That feature is characteristic of STED.
• Trap 3: STORM and PALM are related single-molecule localization methods.
• Trap 4: TEM is mainly associated with internal ultrastructure, whereas SEM is mainly associated with surface morphology.
• Trap 5: Cryo-EM does not mean simply cooling a conventional sample. Proper rapid freezing and vitrification are critical.
• Trap 6: Vitrification means formation of a glass-like amorphous state rather than damaging crystalline ice.
• Trap 7: Electron microscopy uses electrons rather than visible photons.

❓ 17. 10 MCQs – Practice Test

Select one answer for each question and click Check Answer. The explanation will appear immediately.

1. Which technique uses stimulated emission depletion to obtain super-resolution?

A. SIM B. STED C. SEM D. TEM

Correct Answer: B. STED
STED stands for Stimulated Emission Depletion Microscopy. A depletion beam suppresses fluorescence around a small central region, producing an effectively smaller fluorescent spot.

2. Which super-resolution technique uses structured illumination patterns?

A. SIM B. STED C. TEM D. SEM

Correct Answer: A. SIM
SIM stands for Structured Illumination Microscopy and uses patterned illumination followed by computational reconstruction.

3. Which technique is based on stochastic single-molecule localization?

A. STORM B. Bright-field microscopy C. SEM D. TEM

Correct Answer: A. STORM
STORM separates fluorescence events temporally and localizes individual fluorophores before reconstructing the final super-resolution image.

4. PALM is particularly associated with:

A. Photoactivatable fluorescent proteins B. Electron transmission C. Surface electron scattering only D. Crystalline ice formation

Correct Answer: A. Photoactivatable fluorescent proteins
PALM commonly uses photoactivatable or photoswitchable fluorescent proteins and localizes individual molecules.

5. Which electron microscopy technique is most directly associated with internal cellular ultrastructure?

A. SEM B. TEM C. SIM D. STORM

Correct Answer: B. TEM
TEM transmits electrons through a thin specimen and is widely used for visualizing internal cellular ultrastructure.

6. SEM is primarily useful for studying:

A. Surface morphology B. Nuclear DNA sequence C. Protein amino acid sequence D. RNA sequence

Correct Answer: A. Surface morphology
SEM scans the specimen surface and detects signals generated by electron-beam interactions, making it particularly useful for surface topography.

7. In cryo-EM, vitrification is used primarily to:

A. Produce crystalline ice B. Preserve water in a glass-like non-crystalline state C. Increase sample temperature D. Stain the sample with fluorescent dye

Correct Answer: B. Preserve water in a glass-like non-crystalline state
Rapid freezing can produce vitreous ice, helping preserve biological structures without large crystalline ice formations.

8. Which of the following pairs is correctly matched?

A. STED – stimulated emission depletion B. SIM – electron transmission C. TEM – surface scanning only D. SEM – structured illumination

Correct Answer: A. STED – stimulated emission depletion
STED is based on stimulated emission depletion. SIM uses structured illumination, TEM uses electron transmission and SEM scans the specimen surface.

9. Which technique commonly involves repeated localization of sparse subsets of fluorescent molecules?

A. STORM B. TEM C. SEM D. Bright field

Correct Answer: A. STORM
STORM obtains high-resolution information by separating fluorescent emission events and localizing individual molecules over many imaging cycles.

10. Which microscopy method is particularly associated with near-native hydrated sample preservation?

A. Conventional SEM B. Cryo-EM C. Bright-field microscopy D. Phase contrast only

Correct Answer: B. Cryo-EM
Cryo-EM uses rapid freezing and vitrification to preserve biological samples in a hydrated state that can be closer to their native structural condition.

📝 18. Quick Revision Notes

⭐ One-Line Memory Tricks

STED → Stimulated emission depletion → Doughnut beam

SIM → Structured illumination → Patterned light + reconstruction

STORM → Stochastic → Single-molecule localization

PALM → Photoactivation → Fluorescent protein localization

TEM → Transmission → Through the specimen → Internal ultrastructure

SEM → Scanning → Surface → Topography

Cryo-EM → Cryogenic freezing → Vitrification → Near-native structure

Ultra-Short Revision Table

Technique	Remember This
STED	Doughnut-shaped depletion beam
SIM	Structured illumination + computational reconstruction
STORM	Stochastic single-molecule localization
PALM	Photoactivatable fluorescent proteins + localization
TEM	Electrons pass through thin specimen; internal ultrastructure
SEM	Electron beam scans surface; surface morphology
Cryo-EM	Rapid freezing + vitrification + cryogenic imaging

🎓 Final Summary

Super-resolution and electron microscopy have transformed the study of biological structures. Conventional fluorescence microscopy is limited by diffraction, which restricts the ability to distinguish structures separated by very small distances. Super-resolution microscopy provides strategies for extracting information beyond this conventional limit.

STED achieves super-resolution by using stimulated emission depletion. A doughnut-shaped depletion beam suppresses fluorescence surrounding the focal center, effectively reducing the size of the fluorescent region.

SIM uses structured illumination. The specimen is illuminated with a known pattern and multiple images are collected. Computational reconstruction combines these images to recover additional spatial information.

STORM and PALM are single-molecule localization methods. Instead of allowing all fluorescent molecules to emit simultaneously, fluorescence events are separated in time. Individual molecules can then be localized with high precision and thousands of localization events can be combined to form a super-resolution image.

TEM and SEM use electrons rather than visible light. TEM transmits electrons through a thin specimen and is particularly useful for internal ultrastructure. SEM scans the surface and is particularly useful for surface morphology and topography.

Cryo-EM uses rapidly frozen biological specimens. Vitrification preserves water in a glass-like non-crystalline state and helps maintain biological structures in a hydrated condition. Cryo-EM has become an extremely important tool in structural biology, especially for determining structures of large macromolecular complexes.

🔥 Most Important Exam Comparison:

STED → Stimulated emission depletion
SIM → Structured illumination
STORM → Stochastic localization
PALM → Photoactivation/localization
TEM → Internal ultrastructure
SEM → Surface morphology
Cryo-EM → Vitrified near-native sample

📚 Important Terms to Remember

• Diffraction limit
• Airy disk
• Super-resolution
• Stimulated emission depletion
• Depletion beam
• Structured illumination
• Moiré pattern
• Spatial frequency
• Single-molecule localization
• Photoactivation
• Photo-switching
• Electron beam
• Electromagnetic lens
• Ultrastructure
• Secondary electrons
• Backscattered electrons
• Vitrification
• Vitreous ice
• Single-particle analysis
• Three-dimensional reconstruction