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. , , and preserve and enhance cellular features, while enable dynamic imaging of living cells. These methods provide invaluable insights into cell biology and function.
Microscopy Techniques
Light vs electron microscopy techniques
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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)
employs a beam of electrons to magnify samples, achieving higher resolution compared to light microscopy
(TEM) provides detailed internal structure of thin sections (organelles, viruses)
(SEM) reveals surface topography of specimens (cell surfaces, nanostructures)
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
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, , and co-localization studies (multiple fluorescent labels)
techniques overcome the diffraction limit of light microscopy (∼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)
(SIM) employs patterned illumination to extract high-frequency information (cytoskeletal structures, nuclear pores)
(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 (, electron microscopy)