Microscopy Lecture 2 2024 PDF

ECB6 Chapter 1 pg. 6-13 and MBoC Chapter 9  Introduction to Microscopy 1. Introduction Why microscopy Magnification versus Resolution 2. Light microscopy conventional fluorescence confocal  super-resolution 3. Electron microscop...

ECB6 Chapter 1 pg. 6-13 and MBoC Chapter 9  Introduction to Microscopy 1. Introduction Why microscopy Magnification versus Resolution 2. Light microscopy conventional fluorescence confocal  super-resolution 3. Electron microscopy (EM) transmission EM scanning EM 4. Atomic force microscopy  Learning outcomes 1. At what scale do cells, organelles, and macromolecules exist? 2. Be able to compare light and electron microscopy. How do they work and what are the pros and cons of each. 3. What different methods are used to generate contrast in light versus electron microscopy? How are samples prepared in each case? 4. What is fluorescence microscopy and what different types of fluorochromes can we use for observing subcellular structures and proteins? 5. Be able to identify microscopy techniques by their images. Likewise, be able select an appropriate microscopy technique for a given application.  Discovery of cells cork In 1665, microscopes were a newly invented ‘leading edge’ technology. This new invention led to the discovery of cells by Robert Hooke Microscopy is still the main technique available to study the organization of cells and tissues Cells are measured in microns Atoms: < 1 nm (10 Å) Smaller organelles: Big protein complexes: nm range Macromolecules: Avg. prokaryote cell: 1-10 µm Large organelles: 1-10 µm Avg. eukaryote cell: 10-100 µm Only the largest cell types are visible with the naked eye (mm)  Two kinds of microscopy Light microscopy Electron microscopy Cell Wall Ribosomes E. coli in the light microscope E. coli in the electron Good for viewing cells at low resolution microscope Lets you see the shape of cell, larger When you need to see cells and subcellular organelles might be visible structures at high resolution Resolve details up to 0.2 µm apart Resolves details up to 0.2 nm apart Liver cells under light microscope What features can we see?  Liver cell under electron microscope 1 2 3 5 4 What features can we see?  Magnification vs Resolution Magnification is the number of times an image's size is enlarged. Resolution measures the ability to distinguish two details in an object. An example of “empty” magnification An example of “useful” magnification Light microscopy Electron microscopy Section 1 μm thick Adjacent section 0.25 μm thick Magnification X 4500 Magnification X 4500 A focused beam of electrons beam is much narrower than a beam of light, so it provides superior resolution and we can go to higher magnification without losing detail. Maximum resolution for a light microscope is limited by the wavelength nature of light Structures smaller than 0.2 m = about half the wavelength of visible light) cannot normally be resolved, points closer than this cannot be separated and appear as a single blur.  Light microscopy Electron microscopy Sufficient to observe cell For observing cells, shape or large organelles organelles, macromolecules at low resolution at high resolution  Conventional light microscope 10X Up to 100X Total 1000X “Compound” because there are 2 lenses  Conventional light microscopy Light must be able to pass through the sample Opaque samples (e.g. tissues) must be thinly sliced (sectioned) so light passes through Whole mounts of translucent protozoa Stem section of Arabidopsis plant  Preparing tissue sections for light microscopy Fixatives Fixatives penetrate the cell to immobilize proteins and preserve cell structure Common fixatives are alcohol, acetone, and glutaraldehyde or formaldehyde because they penetrate the tissue quickly reactive Aldehydes cross-link cell proteins, aldehyde keeping cell structure as close as possible to that in the living cell. Preparing tissue sections for light microscopy Fixation Dehydration Embedding to Solvent create a block of (e.g. xylene) wax or plastic Tissue in a solid block tissue in wax or embedding medium of wax or plastic plastic Sectioning with a microtome Operates on a “meat slicer” principle Sections ≈ 1-20 μm thick MBOC Fig. 9-10 Stains are often used to improve contrast in light microscopy Most cells and tissues are colorless making details hard to see Different components of a cell can be selectively stained Stains are most often used with fixed cells Section of onion root tip Section of Arabidopsis stem stained with iron haematoxylin stained with toluidine blue When imaging live cells, optical techniques are generally used to improve contrast 1886 1932 1952 (Nomarski) A. Conventional bright field microscope – specimen illuminated with white light. Classic, but many samples absorb very little visible light and making details hard to see (i.e. poor contrast) B. Phase contrast microscope – converts differences in the refractive index in different parts of the cell (e.g. nucleus vs cytoplasm) into differences in light intensity that are visible to the eye. Creates a halo effect A. Differential interference contrast optics – Two closely-spaced beams of polarized light illuminate the sample. Interference between the light beams generates contrast when the refractive index changes steeply over a short distance (e.g. at the edges of structures). Creates a pseudo-3D effect. The phase of light is shifted when it passes through the more dense parts of a cell Phase Contrast and Differential Interference Contrast (DIC) light microscopes amplify differences in phased light, increasing contrast. Figure 9-4 and Figure 9-7 Molecular Biology of the Cell (© Garland Science 2008)  What kind of microscopy was used to create contrast in these cells? Put your answers in the chat  Specific molecules can be located in cells by fluorescence microscopy Fluorescent Dyes Immunofluorescence Fluorescent in situ Fluorescent Labelling Hybridization (FISH) Protein Tags Fluorescent dyes that Fluorochromes Fluorescently labelled anti- Proteins of interest bind strongly to coupled to an antibody sense nucleic acid probes can be fused to GFP components such as can be used to can be hybridized to or RFP to study their DNA or lipids to to “stain” specific specific RNA or DNA location or movement reveal their proteins or “epitopes” sequences in a cell to in a living cell distribution in a cell in a cell locate them Fluorescent probes are called fluorochromes A fluorochrome Dyes absorbs and is excited by light at one specific wavelength, and emits Proteins light at a longer specific wavelength as it returns GFP, to it ground state CFP, YFP and RFP Because fluorochromes emit light, they can allow objects smaller than 0.2 µm to be “seen” in the cell Excitation wavelength Emission Figure 9-14 Molecular Biology of the Cell (© Garland Science 2008) wavelength  Fluorescence microscopy Filter 2: Detector 2 receives only  of light emitted by fluorochrome 1 Filter 1: sample illuminated with λ of light that excites a fluorochrome Figure 9-13 Molecular Biology of the Cell (© Garland Science 2008)  Fluorescent dyes can “light up” specific macromolecules in a cell Dyed objects A show up in bright colour on a black background Specific dyes may B be used for: -identification -co-localization Tobacco cells stained with a fluorescent dye (DAPI) that binds specifically to DNA  Immunofluorescence Labelling Fluorochrome attached Fluorochrome to secondary attached antibody to a primary antibody INDIRECT IMMUNOFLUORESCENCE DIRECT STAINING IMMUNOFLUORESCENCE STAINING Amplifies the signal  Immunofluorescence labelling mouse 3T3 cells (fibroblasts) -tubulin 1º antibody (green) DAPI (blue) Immunofluorescence can be used in combination with other, non-antibody methods of fluorescent staining  Fluorescence in situ Hybridization (FISH) Shows the location of specific genes or mRNAs in a cell A cell from amniotic fluids that is positive for trisomy Chr 21 by FISH (red) Anti-sense nucleic acid probe is synthesized with bases that incorporate a fluorescent tag Probe and target DNA are denatured Probe is hybridized to target DNA Fluorescent tag is observed under a Another application: fluorescence microscope Detection of cancer markers  Proteins can be tagged in living cells using GFP gene X promoter Gene X GFP gene X promoter GFP Gene X zebrafish Jellyfish green fluorescent Shows the location of proteins protein (GFP) in living plant cells yeast Works in any cell or organism that can take up foreign DNA Combining spectral variants of GFP and RFP from corals can allow up to several labelled proteins to be studied together in a cell eGFP RFP merge e.g. co-localization studies A common problem with fluorescent A confocal microscope fixes microscopy is lack of clarity due to this problem by taking “optical out of focus light sections” along the Z-axis. These are stacked to make a conventional confocal clear image Figure 9-20 Molecular Biology of the Cell How a confocal laser scanning microscope works Figure 9-24 Molecular Biology of the Cell  All-in-focus images are digitally reconstructed from the Z-stack 1 2 3 1 2 3 Reconstructed image Figure 9-22 Molecular Biology of the Cell (© Garland Science 2008) Newer super-resolution confocal microscopes can now digitally break the 0.2 µm resolution barrier for light microscopy Microtubules (green) and clathrin-coated pits (red) Courtesy of Xiaowei Zhuang Laboratory, Harvard University  Decision Tree for Microscopy Low mag/low resolution imaging High mag/high resolution imaging Living cells Light Live or fixed cells Electron cannot be imaged 3D images 2D images Bright field Fluorescence Scanning Transmission Whole cells Florescent probes (dyes, EM EM antibody-linked, or Thin sections for tissues Thin sections of whole cells incorporated into anti-sense surfaces Stains provide contrast in nucleic acid probes) used or tissues -whole cells fixed cells or tissues with fixed or permeabilized virus particles, protein cells -freeze-fractured Optical methods (phase cells complexes, other GFP or variants to monitor macromolecules contrast, DIC) improve contrast when imaging behavior of proteins or gene expression in living cells Atomic Force living cells Microscopy Confocal -surfaces, biofilms Z-stacks to improve resolution -molecules in solution  Which images are electron microscopy? Skin epithelial cells imaged using three different techniques Transmission electron microscope (TEM) A beam of electrons focused by a magnet is transmitted through an ultra-thin specimen Magnification up to 1,000,000 X Resolution up to 0.2 (2 Å) Plant epidermis microstructure Plasma dark areas are Membrane “electron dense” Cell wall Mitochondria The image is formed by electrons that pass through the specimen.  Preparing tissue sections for TEM Two fixatives OsO4 Ultra thin sections Heavy metal salts are used to stain the tissue  Ultrastructure of a cell is clearly visible Osmium tetroxide reacts with double bounds in lipids to fix and stain membrane Heavy metal salts used for staining create areas that are “electron dense” Thin section of a yeast cell Specific macromolecules can be localized by immunogold labeling 1) 1° antibody to specific protein 2) 2° antibody attached to a gold particle Four proteins localized to kinetochore of yeast mitotic chromosomes using different sized gold particles Negative staining is commonly used visualize particles and macromolecules viruses ribosomes DNA cytoskeletal proteins protein complexes Heavy metal salts applied to the sample darken the background contrasting with the biological particles to create a “negative” image Scanning electron microscope (SEM) beam of electrons scans the sample Backscatter or secondary emitted electrons emitted by the specimen are collected to generate the image Creates an image of the surface of the sample The SEM produces high resolution surface images that look three-dimensional Light SEM TEM Not quite as sensitive as TEM. The maximum resolution is between 3 nm and 20 nm depending on the instrument  Conventional SEM The tissue is fixed with glutaraldehyde (proteins) and OsO4 (membranes) Water is removed by “critical point drying” leaving behind a carbon skeleton that can withstand the electon beam under low vaccuum The surface is coated with a thin layer of heavy metal (e.g. gold-palladium) to make it conductive An Arabidopsis floral meristem Freeze-fracture EM exposes the Cryo EM can determine molecular internal surfaces of a cell structures at atomic resolution Chloroplast thylakoid membranes Microtubules Rapidly frozen, split with a glass knife, Rapidly frozen, imaged using low dose of surface details revealed by coating electrons under very high accelerating with platinum-carbon voltage Atomic Force Microscopy Ideal for looking at surface topology of biofilms or macromolecules in solution or in a membrane ATP synthase Atomic Force Microscopy: how it works Probe is a tiny moveable cantilever (beam) that moves over the surface of a hydrated sample (doesn’t touch) Deflections are recorded and turned into surface images AFM can also be used to pick up and move single molecules that adsorb strongly to the tip, to measure the mechanical properties New methods for microscopy are developed all the time green-endosomes red-transport vesicles First color images produced by an electron microscope Adams et al. 2016 Cell Chemical Biology 23, 10  Self-quiz What kind of microscopy technique was used to generate the following images?? IMAGE 1 IMAGE 2 C. elegans (nematode) developing embryo IMAGE 3 100 nm IMAGE 4 IMAGE 5 IMAGE 6 IMAGE 7 IMAGE 8 IMAGE 9 IMAGE 10 160,000 x magnification.  Next class A review of building blocks of the cell Assigned reading ECB Chapter 2 and 4