#super-resolution microscopy

Microscopy

Which microscopy is best for detecting single molecules?

Total internal reflection fluorescence excels for single-molecule sensitivity near plasma membrane. A laser totally reflects at glass-water interface, generating an evanescent field decaying within 100-200 nanometers above coverslip, selectively illuminating fluorophores in basal membrane while excluding bulk cytoplasm fluorescence. This extreme background suppression dramatically improves signal-to-noise, enabling detection of individual GFP-tagged receptors, vesicle fusion events and motor protein stepping. Confocal excites deeper volumes causing out-of-focus noise, while DIC and phase contr

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Microscopy

Which microscopy uses photoactivatable probes for super-resolution imaging?

Direct stochastic optical reconstruction microscopy belongs to single-molecule localization super-resolution methods. It relies on photoswitchable or photoactivatable fluorophores that transiently flip between dark and bright states in special buffers. At any moment only a sparse subset emits, so individual point spread functions do not overlap and can be fitted with nanometer precision. Thousands of frames accumulate localizations building a composite super-resolved image with 20-nanometer resolution. This blinking behavior enables resolving nucleosome arrangements, membrane receptors and vir

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Microscopy

Which of the following microscopy techniques uses Moiré fringes for super-resolution imaging?

Structured Illumination Microscopy enhances resolution by projecting patterned light onto sample, generating Moiré fringes where illumination and specimen spatial frequencies interfere. These interference patterns encode high-frequency structural information normally beyond diffraction limit into detectable low-frequency moiré. Multiple images acquired with shifted and rotated gratings are mathematically reconstructed to double lateral resolution to ~100 nm and improve axial resolution. SIM requires only conventional fluorophores, low laser power, and works with live cells, making it valuable

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Microscopy

Which of the following achieves resolution of 10-30 nm and uses photoactivatable proteins?

PALM utilizes genetically encoded photoactivatable fluorescent proteins like mEos and Dronpa. Initially dark proteins are sparsely photoactivated by low-intensity violet light, imaged, then photobleached, ensuring only few molecules emit simultaneously. Gaussian fitting of each spot provides ~20 nm localization precision. Repeating this activation-imaging cycle builds a super-resolved map from thousands of localizations. Achieving 10-30 nm resolution, PALM is particularly suited for live-cell studies of molecular clustering, because photoactivatable proteins can be fused endogenously and track

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Microscopy

Which super-resolution microscopy technique uses a depletion laser to confine excitation to a nanometer scale region?

STED microscopy breaks diffraction by superimposing two synchronized lasers. A diffraction-limited excitation beam is overlapped by a doughnut-shaped depletion beam that drives stimulated emission at periphery, quenching fluorescence everywhere except central zero-intensity spot. This confined emissive region shrinks to 20-70 nm laterally, enabling super-resolution imaging of living cells. Unlike stochastic localization methods requiring extensive computational reconstruction, STED achieves resolution through targeted deterministic de-excitation, providing rapid scanning without heavy post-pro

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details