Visualizing Cells: Chapter 9 PDF

Molecular Biology of the Cell Seventh Edition Visualizing Cells: Chapter 9 Not for sale or distribution; property of York University This lecture will cover: Im...

Molecular Biology of the Cell Seventh Edition Visualizing Cells: Chapter 9 Not for sale or distribution; property of York University This lecture will cover: Important concepts in microscopy Types of Light Microscopy Using fluorescent molecules in cell biology Types of Electron Microscopy What you should be able to do: Describe/explain each Compare critically (how they differ, advantages/disadvantages) Choose optimal type of microscopy given a particular situation/need Light Microscopy Bright-field shining white light Phase-contrast phase-shifting of wavelengths phase-shifting and Differential Interference interference of light Dark-field light is emmitted at an angle and scattered by cell parts Fluorescence fluorecent cell parts chrome to detect With Bright-field Microscopy most cells and cell structures have very little contrast Bright-field microscopy uses unmanipulated transmitted light BFM generates a dark image on a light backround -- using unmanipulated transmitted light Since most of the cell is mafe up of water, the light is just passing through, it is not disrupted -- most organelles are not visible using BFM because they don't absorb enough light to generate contrast relative to the backround - little contrast - little detail - some magnification - some resolution Source: https://www.thermofisher.com/ca/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of- fluorescence/imaging-basics/fundamentals-of-fluorescence-microscopy/how-fluorescence-microscopy-works.html  Processing of tissue samples is required for Making tissue sections can help to generate more contrast/detail staining Fixation: covalent cross-linking locks glutaraldehyde forms covalent bonds with proteins into place. free amino groups of our proteins to lock them into place Embedding: wax permeates and then solidifies to harden and stabilize tissue. Sectioning: tissue is thinly sliced with a microtome for observation under a light microscope. after these processes, the sample could potentially be damaged, killed, or portray a distorted image Molecular Biology of the Cell (© Garland Science 2008) Cell components can be stained to create contrast and make them visible Urinary Collecting Duct Plant Root hematoxylin and eosin (H&E) Is this the perfect solution? Hematoxylins stains the ECM (extracellular matrix) Fast green stain - produces a dark blue dye - stains cellulose in plant cells - has an affinity for negative charged molecules - DNA, RNA, acidic amino acids Eosin stains the nuclei - produces a red dye - has an affinity for basic components - basic amino acids (lysine, arginine) The stains allow individual cellular components to stand out which provides more detail about the cell compared to the image using BFM depending on your purpose of examining a sample, you may need the detail the staining provides Phase Shifts in Light Phase Contrast microscopy In phase waves are percieved as light Out of phase waves are percived dark -- they can somewhat cancel each other out Phases can be affected differently as light passes though different parts of the sample (different densities) When light is in different phases, they can interfere with eachother combining different phases of light to manipulating light, expoliting generate contrast differences in light behaviour can generate contrast Fig. 9.3, Molecular Biology of the Cell Light passing through a specimen can be absorbed, slowed, transmitted, reflected, scattered… unmanipulated out of phase light passing waves combine through the to create contrast sample BFM PCM Fig. 9.6, Molecular Biology of the Cell Phase Contrast Microscopy converts small differences in phase into contrast to generate an image Bright Field Phase Contrast ▪ Generates a dark image Visualize living cells of an object over a light Not all structures are visible background (what can you see /not see?) BFM can be used for live PCM can show more in camparision to cells but are hard to see BFM but it is still not detailed Fig. 9.7, Molecular Biology of the Cell  Paramecium ▪ exploits differences in refractive index Can see between the cytoplasm and the - nucleus surrounding medium or between - cillia different organelles. can be used to view live cells and cellular organelles Didinium eating Paramecium https://www.youtube.com/watch?v=rZ7wv2LhynM Copyright © 2020 W. W. Norton & Company Differential Interference Microscopy manipulates Phase Differences to visualize edges or boundaries Used to view unstained living cells produces a 3D appearance (optical slice) DIM uses phase shifting and interference of light Both PCM and DIM are used to view Used to visusalize live cells living cells and can view unstainsatained cells Figure 9-7  DFM produces an image using scattered light ▪ Dark-field optics enable microbes to be visualized as halos of bright light against darkness. ▪ Allows the detection of objects that are unresolved by bright-field microscopy Very narrow cells (0.1 mm), such as Treponema pallidum Produces a bright image on a dark backround Cell components, such as flagella Copyright © 2020 W. W. Norton & Company Which type of light microscope do you think would be most useful and why? hard to visualize specific parts/ molecules/proteins of the cell when using light microscopy Limitations of light microscopy Solutions: dyes, fluorescent probes, digitization, different types of microscope Specific Molecules/Ions Can Be Located in Cells by Fluorescence Microscopy labelled different RNAs with fluorescently labelled probes -- each probe can only detect a specific RNA target Identification of different RNA in the developing Drosophila embryo Electrons can absorb photons and move to an excited state. Excited electrons can release energy as light when they return to the ground state: fluorescence when a photon encounters an electron, the photon provides the electron with energy to move the electron from the ground state to the excited state -- the excited electrons release the energy as light when they get back to the ground state -- emmited as fluorescence Fluorescent probes will be excited by one wavelength and emit another, longer, wavelength. fluorophore is some How do fluorescent probes work? fluorescent chemical compound that re-emits light upon light excitation ▪ The specimen absorbs light of a specific wavelength, then emits light fluorescence at a longer wavelength a specimen will absorb light at a specific wavelength (excitation wavelength) and it will emit light at a longer wavelength (emmision wavelength Figure 9-10a Fluorescent probes are Fluorescent probes are used to detect certain excited at a higher structures in samples by energy and emit light at absorbing and emitting light a lower energy Fluorescent molecules absorb higher energy (shorter wavelength) photons and emit lower energy (longer wavelength) photons. rhodamine B DAPI absorbs GFP absorbs absorbs green light at the UV blue light and and fluorese level and fluorese red - stains fluorese blue - green - stains various stains DNA, proteins structures nuclei Figure 9-12 Molecular Biology of the Cell (© Garland Science) Fluorescence Microscopy ▪ the specimen absorbs light of a defined wavelength and then emits light of lower energy, thus longer wavelength; that is, the specimen fluoresces. ▪ e.g. can be used to view marine bacteria and gut bacteria Fluorescence Microscopy allows you to visualize fluorescent molecules. Fluorescence microscopes excite the fluorescent molecules with the excitation wavelength, then focus the emitted fluorescent light to your eye. By Howard Vindin CC BY-SA 4.0 Figure 9-10  How do you make cells/molecules fluorescent? Some things are naturally fluorescent (autofluorescence) e.g. Chlorophyll Source: Photosynthesis lab manual York University Source: https://commons.wikimedia.org/wiki/User:Dietzel65 This file is licensed under the Creative Commons Attribution-Share Alike 4.0 International license.  Fluorophores for Labeling ▪ fluorophore is a fluorescent chemical compound. Its cell specificity can be determined by: – Chemical affinity – Labeled antibodies – DNA hybridization – Gene fusion reporter Copyright © 2020 W. W. Norton & Company  Fluorescent Dyes can stain specific structures e.g. DAPI (4′,6-diamidino-2- phenylindole) – binds and labels DNA Dyes such as DAPI emit blue -- bind to specif regions of our DNA -- ATregions -- impermient (need higher concentrations of it to eneter a live cell) cant permeate through a semi permeable membrane Source: Hieronim Golezyk http://www.golczyk.cal.pl/golczyk.htm  Immunocytochemistry uses antibodies and ICC = labelling a cell using fluorescent probes SA is antibodies SA is carrying markers/ more PA directed against fluoreescent dye -- general antigen A marker is recognizing PA and Antibodies amplifies are the signal immune proteins that work against invading antigens PI binds to antigen A Antigen/target molecule Antibody binds specifically Can be quantitative or to the antigen qualitative (concentrations of antigen) Figure 9-15 Molecular Biology of the Cell (© Garland Science) The downside of immunocytochemistry Usually cells must be fixed (dead), permeabilized and often partially extracted (leads to artifacts) Fixed cells (cells that have been chemically prepared) provide only a snapshot of what is going on or present in the cell. Sometimes it is easy to tell from a snapshot photo what just happened But not always Green Fluorescent Protein (GFP) GFP gene and protein isolated from jelly fish Aequoria victoria GFP is a tag that can be used to visualize proteins in live cells  History: Green Fluorescent Protein (GFP) The Nobel Prize in Chemistry 2008 Osamu Shimomura, Martin Chalfie, Roger Y. Tsien Douglas Prasher cloned/sequenced the gene for GFP cellular marker Roger Tsien constructed many GFP mutants  Green Fluorescent Protein (GFP) chromophore responsible for GFPs fluorescence is shielded from quenching by aqueous solvent chromaphore composed of 3 AA at the center of the 42 Å long and 24 Å in structure diametersheilded from quenching from any aqueous solvent serine-tyrosine-glycine light handling machinery 11 beta strands, pleasted sheet arrangement, beta barrel arrangement (beta can), 5 alpha helixes -- structural GFP can be taken and a protein of intrest, and can be recombined -- generates a chromphore can fusion protein be added to protein of intrest Figure 9-16 Molecular Biology of the Cell (© Garland Science) and GFP will start  The gene for GFP can be inserted and expressed in GFP can prokaryotic and eukaryotic cells be engineere d to the protein of intrest GFP is only active in certain nuerons GFP expressed under a neuronal promoter in Drosophila Figure 9-17 Molecular Biology of the Cell (© Garland Science) GFP as a tool to study gene expression 1. GFP gene can be fused to any GFP can be used to study the cellular promoter sequence loaction of a protein given that we can express our fusion protein at any level, and by adding GFP the cell can retain its 2. Promoters drive gene expression normal function 3. This indicates where, when and how much of a protein is made  The gene for GFP can be fused with other genes to study the expression and localization of specific proteins. bsp.med.harvard.edu Molecular Biology of the Cell (© Garland Science)  Summary: Green fluorescent protein (GFP) is a bioluminescent protein isolated from jellyfish Aequorea victoria Beta strands form the beta barrel (beta can), chromaphore in the center composed of 3 AA -- structure is important to anable it to fluoresce Ser65–Tyr66–Gly67 Figure 9-16 Molecular Biology of the Cell (© Garland Science)  Variants of GFP the key is to make small mutations that change the stability of the chromophore Making small mutation to GFP -- replase tyrosine with typrophan  Rachel Glinsman Source: https://www.ebi.ac.uk/pdbe/about/news/dressed-fluoresce  Confocal fluorescence microscopes allow you to take optical sections through a cell. In confocal microscopy, a microscopic laser light source scans across the specimen. taking multiple images to generate a 3D representation -- Regular fluorescent Confocal 3-D can take images of image optical section reconstruction subcellular levels Confocal microscopes remove the out of focus light from parts of the cell above and below the focal point, generating an optical section Multiple sections can be digitally combined to create a 3-D image Figure 9-25 Molecular Biology of the Cell (© Garland Science) New techniques generate images with improved resolution Super sensitive digital cameras Averaging multiple images Requires computing power and sophistication low levels limit of of staining, resolution superress = 200 nm olution -- 2nm with improved resolution Seeing cells without Light: solving the resolution problem  Transmission Electron Microscope Transmission EM allows you to visualize molecular structure LOR = 0.002 nm LIGHT ELECTRON MICROSCOPE MICROSCOPE theoretical LOR = resolution 200nm 0.002 nm electrons travels through the electron specimen beam tungsten is the source of Figure 9-40 Molecular Biology of the Cell (© Garland Science)  Transmission Electron Microscopy (TEM) + -high resolution (~1nm) -see lots of internal structure -molecular structure BUT – requires thin sections (25-100nm) ~200 to go through a cell Stained mith an electron- Sample dense material, electrons will needs to pass though the spcimen, be fixed stained areas will appear darker Molecular Biology of the Cell, Fifth Edition (© Garland Science) TEM Different Views of a Single Object Can Be Combined to Give a Three-Dimensional Reconstruction Multiple thick sections of the Golgi apparatus which have been digitally reconstruction White speres = lysosomes Figure 9-47 Sample Processing for Electron Microscope 1. Fixation with glutaraldehyde to cross-link proteins in place used to staballize membranes and proteins 2. Treatment with osmium tetroxide to ___________________ Other heavy-atom salts can be used (lead and uranium) to increase electron contrast 3. Dehydration and embedding in resin 4. Sliced into 50-100 nm thick slices frazen to maintain Often samples are flash-frozen (liquid helium) integrity before the cell of treatment structure Thought to better maintain integrity of cell structure  Transmission Electron Microscopy (TEM) What’s the Downside? Cells fixed Embedded in resin Sectioned Stained with electron dense material Just see where the stain sticks TEM Figure 1-42 Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008)  Transmission electron microscope image of yeast cell Darker areas = electrons were blocked by dense material Lighter areas = electrons passed through the sample because of less density Molecular Biology of the Cell (© Garland Science)  Immunogold Electron Microscopy 1. Detect specific molecules (like immunocytochemistry) 2. Stain thin slices with primary antibody against protein of interest 3. Follow with secondary antibody conjugated with gold-particles 4. Gold-particles are electron dense and can be 5-20 nm in size. 5. Can do dual staining using different secondary antibodies conjugated with different sized-particles  Immunogold localization of Rubisco and a carbonic anhydrase enzyme -CcmM in a cyanobacteria: Nostoc b Fig 1a. Sections of Nostoc were immunolabelled with Rubsico antibody. Gold particles (15 nm) are predominantly localized in the carboxysome. a de Araujo et al., 2014  Scanning Electron Microscopy (SEM) Surface is coated scanning accross the specimen and with heavy metal produce surface informatio Measures quantity of electrons scattered over surface Figure 9-49 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008) Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) SEM TEM Figure 1-42 Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008)  Transmission Electron Microscopy (TEM) Good for visualizing internal features of cell Preparation fixed, stained with electron dense material (e.g. heavy metal), embedded in resin, thinly sliced Electrons (instead of light) pass through specimen and electron stained areas appear dark Resolution up to 0.3 nm Scanning Electron Microscopy (SEM) Good for visualizing external features Heavy metals added to outside of specimen Uses scattered electrons to produce 3D image Resolution 3-20 nm LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE Images of Surfaces Can Be Obtained by Scanning Electron Microscopy Scanning electron microscopy (SEM) can provide a high resolution 3 dimensional surface images. However, the resolution is not as great as TEM. Stereocilia from a hair cell A) SEM B) Light microscopy C) TEM  X-Ray Diffraction Analysis ▪ X-ray data undergo digital analysis to generate sophisticated molecular models. ▪ For samples that can be crystallized, X-ray diffraction makes it possible to fix the position of individual atoms in a molecule. A beam of X-rays is shot at a crystallized sample. e.g., the anthrax lethal factor – A toxin produced by Bacillus anthracis the pattern allows us to fix of the x- the posion of rays can the atom in a X-rays diffracts at the be used to molecule posiotion of the atoms predict the structure which are used to ma of proteins the protein structure Cells are packed full of molecules! How does everything find its place and do its job(s)?  In the bright-field microscopic image below the dark green areas represent regions where A. Less light travelled through the specimen B. More light traveled through the specimen  The ________ microscope relies on the principle of interference to produce images of the specimen without staining or damaging it. a) Bright-field b) Dark-field c) Phase-contrast d) Transmission electron e) Scanning probe Copyright © 2020 W. W. Norton & Company  All of the following statements apply to scanning electron microscopy EXCEPT: a) The specimen is usually fixed and embedded. b) The embedded specimen is cut into thin sections with a microtome. c) It cannot be used to view live specimens. d) It provides 3D images of the specimen. Copyright © 2020 W. W. Norton & Company  You want to study the structure of a particular protein in bacteria. Which technique would you use? a) Light microscopy b) Electron microscopy c) Ultracentrifugation d) X-ray crystallography e) Cryo-electron tomography Copyright © 2020 W. W. Norton & Company  Summary: The Light Microscope Detection vs. Magnification vs. Resolution Magnification: apparent “visual” increase in the size of an object. There is no limit to magnification. Resolution: “visual” separation of the individual components of an object, which previously appeared as one; quality of the image. Resolution limit: is reached when additional magnification does not separate further detail. The result is simply an enlarged blurry image. Detection: able to visualize light signals from objects much smaller than 0.2 mm, including a single molecule. However, objects appear like they are 0.2 mm in size (due to diffraction).