Question 4 of 6: Imaging, Identification and Measurement of Biological Materials

Question 4: Imaging, Identification and Measurement of Biological Materials (20 marks)

Question text not reproduced: the examination questions are © Engineers and Geoscientists BC. Open the official past paper (linked at the top of this page) to read the question, then follow the worked solution below.

(a) Confocal microscopy is a fluorescence light-microscopy technique that achieves optical sectioning — sharp, thin (sub-micron to a few micron) in-focus images of a single plane within a thick, otherwise-opaque specimen — by scanning a focused laser spot across the sample and passing the emitted fluorescence back through a small pinhole aperture placed at a plane confocal with the focal plane of the objective lens. Light emitted from in-focus regions passes through the pinhole to the detector, while out-of-focus light (from planes above and below the focal plane) is defocused at the pinhole plane and is largely blocked, dramatically improving contrast and axial resolution compared with conventional widefield fluorescence microscopy, where out-of-focus light blurs the whole image. By recording a stack of such optical sections at successive depths, a three-dimensional reconstruction of the specimen can be built computationally. Confocal microscopy is used wherever fine spatial detail is needed within thick or living specimens: imaging fluorescently labelled (immunofluorescent or genetically tagged, e.g. GFP) proteins and organelles within intact cells or tissue sections, co-localization studies of two or more labelled molecules, live-cell time-lapse imaging of dynamic processes, and 3-D reconstruction of tissue architecture.

(b) Electron microscopy (EM) replaces the light beam of a conventional microscope with a beam of accelerated electrons, whose much shorter wavelength gives resolving power roughly a thousand-fold better than light microscopy — down to the nanometre or sub-nanometre scale, sufficient to resolve individual macromolecular complexes, viruses and membrane ultrastructure that are far below the diffraction limit of visible light. Two main modes exist: transmission electron microscopy (TEM), in which electrons pass through an ultra-thin, heavy-metal-stained section of fixed, dehydrated specimen to reveal internal ultrastructure (organelle membranes, cytoskeletal filaments, viral capsids); and scanning electron microscopy (SEM), in which a focused electron beam rasters across a metal-coated specimen surface and detects backscattered/secondary electrons to build a high-resolution image of surface topography. Because the specimen must be chemically fixed, dehydrated and (for TEM) sectioned or (for SEM) coated, and must sit in a high vacuum during imaging, EM cannot image living, hydrated specimens. EM is used to resolve ultrastructural detail beyond the reach of light microscopy: organelle fine structure, membrane architecture, cytoskeletal organization, viral and macromolecular-complex morphology, and surface topography of cells, tissues or biomaterials.

(c) Cell viability refers to the fraction of cells in a population that are alive and functionally intact — possessing an intact, selectively permeable plasma membrane, active metabolism (e.g. ATP production, enzymatic activity), and, where relevant, the capacity to proliferate — as distinct from cells that are dead or dying (with compromised membranes, arrested metabolism, or undergoing apoptosis/necrosis). Viability is typically assessed by functional assays that require a living, metabolically active or membrane-intact cell: membrane-exclusion dyes (e.g. trypan blue, propidium iodide, which only enter cells with a compromised membrane), metabolic assays (e.g. MTT/resazurin reduction, which require active mitochondrial enzymes), or proliferation assays. Of the two techniques above, confocal microscopy CAN assess viability, because it images live, hydrated, fluorescently labelled cells in near-real time under conditions compatible with continued cell function — a live/dead fluorescent assay (e.g. calcein-AM, which is only converted to a fluorescent product by active intracellular esterases in live cells, imaged alongside propidium iodide, which only labels the nuclei of membrane-compromised dead cells) can be imaged directly by confocal microscopy to score living versus dead cells in a population, even in 3-D tissue. Electron microscopy CANNOT be used to assess viability, because EM sample preparation itself (chemical fixation, dehydration, and imaging under high vacuum) kills the specimen and freezes it in a single fixed, static ultrastructural state; EM can only be applied to specimens that were already fixed at a known moment, so it can visualize the anatomical/structural consequences of cell death (e.g. chromatin condensation in apoptosis, organelle swelling in necrosis) after the fact, but it cannot report on whether a given cell is currently alive and functional, because "currently" has no meaning once the specimen is fixed.