Microscopy techniques are essential tools for exploring the intricate world of cells. From basic light microscopy to advanced super-resolution methods, these approaches allow scientists to visualize cellular structures and processes with incredible detail.

Sample preparation is crucial for successful microscopy. Fixation, sectioning, and staining techniques preserve and enhance cellular features, while fluorescent probes enable dynamic imaging of living cells. These methods provide invaluable insights into cell biology and function.

Microscopy Techniques

Light vs electron microscopy techniques

• Light microscopy utilizes visible light to magnify samples but has limited resolution due to the wavelength of visible light (400-700 nm)
  • Commonly used to observe living cells, histological sections (tissue slices), and stained specimens (H&E, Gram stain)
• Electron microscopy employs a beam of electrons to magnify samples, achieving higher resolution compared to light microscopy
  • Transmission electron microscopy (TEM) provides detailed internal structure of thin sections (organelles, viruses)
  • Scanning electron microscopy (SEM) reveals surface topography of specimens (cell surfaces, nanostructures)
• Fluorescence microscopy uses fluorescent probes or tags to visualize specific molecules or structures
  • Requires a light source to excite fluorophores (GFP, DAPI) and filters to separate excitation and emission wavelengths
  • Enables localization of specific proteins, tracking molecular dynamics (FRAP), and studying protein interactions (FRET)

Confocal and super-resolution microscopy applications

• Confocal microscopy uses a pinhole to eliminate out-of-focus light, improving resolution and contrast
  • Enables optical sectioning, allowing for 3D reconstruction of samples (tissue architecture, organelle distribution)
  • Commonly used for visualizing thick specimens, live-cell imaging, and co-localization studies (multiple fluorescent labels)
• Super-resolution microscopy techniques overcome the diffraction limit of light microscopy ($\sim$200 nm), providing nanometer-scale resolution
  • Stimulated emission depletion (STED) microscopy uses a depletion laser to reduce the effective excitation volume (protein clusters, viral particles)
  • Structured illumination microscopy (SIM) employs patterned illumination to extract high-frequency information (cytoskeletal structures, nuclear pores)
  • Single-molecule localization microscopy (SMLM) localizes individual fluorescent molecules with high precision (protein organization, receptor distribution)

Sample Preparation and Imaging

Sample preparation for microscopy

• Fixation preserves the structure and composition of biological samples
  • Chemical fixation uses fixatives like formaldehyde or glutaraldehyde to cross-link proteins (immunohistochemistry, electron microscopy)
  • Physical fixation employs rapid freezing techniques to immobilize samples (cryo-electron microscopy, freeze-fracture)
• Sectioning produces thin slices of fixed samples for microscopy
  • Paraffin embedding used for light microscopy and immunohistochemistry (tissue sections, histopathology)
  • Resin embedding used for electron microscopy (ultrathin sections, 50-100 nm)
  • Cryosectioning used for fluorescence microscopy and preserving sensitive antigens (immunofluorescence, enzyme histochemistry)
• Staining enhances contrast and highlights specific structures or molecules
  • Histological stains: hematoxylin and eosin (H&E), Masson's trichrome (connective tissue), Periodic acid-Schiff (PAS) for carbohydrates
  • Immunohistochemistry uses antibodies to label specific proteins (biomarkers, signaling molecules)
  • Fluorescent stains: DAPI (DNA), phalloidin (actin), MitoTracker (mitochondria), ER-Tracker (endoplasmic reticulum)

Fluorescent probes in live-cell imaging

• Fluorescent probes are small molecules that bind to specific targets and emit fluorescence
  • Calcium indicators (Fura-2, Fluo-4) measure intracellular calcium dynamics (neurotransmission, muscle contraction)
  • Membrane potential sensors (DiBAC, TMRM) detect changes in membrane potential (neuronal activity, apoptosis)
  • Organelle stains (MitoTracker, LysoTracker) label specific organelles and track their dynamics (mitochondrial transport, lysosomal function)
• Genetically encoded tags are fluorescent proteins fused to a protein of interest via genetic engineering
  • Examples: green fluorescent protein (GFP), red fluorescent protein (RFP), and their variants (YFP, CFP)
  • Allow for specific labeling and tracking of proteins in living cells (protein trafficking, subcellular localization)
  • Fluorescence recovery after photobleaching (FRAP) measures protein mobility and dynamics (diffusion rates, binding kinetics)
  • Förster resonance energy transfer (FRET) detects protein-protein interactions and conformational changes (enzyme activation, receptor dimerization)