Introduction to Cell Biology & Biochemistry: Microscopy - PDF

Introduction to Cell Biology & Biochemistry Visualising Cells & Tissues: Microscopy Dr Andrea Knight  What is Microscopy? Experimental technique used to study the morphology (size, shape, structure) and properties of cells, tissues and small organisms that cann...

Introduction to Cell Biology & Biochemistry Visualising Cells & Tissues: Microscopy Dr Andrea Knight  What is Microscopy? Experimental technique used to study the morphology (size, shape, structure) and properties of cells, tissues and small organisms that cannot otherwise be seen by the eye  Types of Microscopy Electron Optical Uses a series of Scanning Probe electromagnetic/electrostatic Uses a series of glass lenses to lenses to magnify and focus Uses a mechanical probe to feel the magnify and focus light (photons) electrons surface of the specimen  Optical (Light) Microscopes Are Used To: Illuminate specimens Magnify specimens Generate contrast Resolve features Capture images (eye, camera) University of Queensland  Example Uses of Light Microscopy Determine the presence/absence/distribution of certain types of cells in a sample/tissue/organism E.g. blood smear and white blood count Hegde et al 2019 DOI: 10.1007/s11042-018-7107-x  Example Uses of Light Microscopy Determine the stage of cancer biopsies to inform of treatment strategy Serafino et al 2014 DOI: 10.18632/oncotarget.1571  Example Uses of Light Microscopy Determine the presence/absence/distribution of subcellular structures: E.g. Determine number of cells in a sample undergoing mitosis – mitotic index Ibrahim et al 2022 DOI: 10.1136/jclinpath-2021-207742  Example Uses of Light Microscopy Determine the presence/absence/distribution of particular molecules and proteins (with the aid of dyes/stains IHC and FIHC) Immunohistochemistry (IHC) H&E staining Oil Red O = lipid/fat droplets Use antibodies to detect specific proteins Hematoxylin = nucleus ; eosin = cytoplasm Bethune et al 2008 DOI: 10.1371/journal.pone.0001614 Balber et al 2019DOI: 10.3389/fendo.2019.00324 cellsignal.co.uk  Principal of Optical Microscopy Optical microscopy uses a series of lenses to magnify and focus light (photons) Light from the light source is focused onto the specimen by lenses in the condenser Light interacts with the specimen, and this light is collected by the objective and ocular lenses which are arranged to focus the image of the illuminated specimen onto the eye or camera  How Does a Light Microscope Work? Light is focused onto the sample using the condenser and the image of the sample is then magnified through a series of lenses within the objectives which is then viewed through the eye piece or camera All optical/light microscopy have the same basic components light source and controls on/off and adjust intensity diaphragm and condenser to focus light onto the sample stage on which to place the sample controls to move the stage and focus controls course and fine focus adjustments objectives to magnify the sample an eyepiece to view the sample digital camera and computer to capture images  Light… What is Light? Visible light = electromagnetic radiation that can be detected by the human eye andor.oxinst.com Multiple Lenses to Magnify & Focus Light Chromatic Aberration Spherical Aberration olympus-global.com  Magnification = Objective + Ocular Magnification = combination of eyepiece and objective 4x = object appears 4x larger than it actually is Eyepiece = 10x Objective = 4x Total magnification = 40x Eyepiece = 10x Objective = 10x Total magnification = 100x 20x = object appears 20x larger than it actually is star.mit.edu/CellBio/animations/index/html  Magnification & Resolution Magnification Object appears bigger than it is Resolution Ability to distinguish detail Ozcan Lab @ UCLA  Resolution Resolution = Minimum distance at which two distinct points of a specimen can still be seen Low Resolution Medium Resolution High Resolution Circular apertures are used to guide and focus light in microscopes, an even though the lenses used are optimally focused, there is still diffraction of light Light is waves of photons, diffraction describes the bending of these waves through an opening In microscopy, the diffraction appears as a bright point of light surrounded by concentric rings Diffraction of waves through an opening Airy Disk: pattern of diffraction observed in microscopy  Airy Disks and Resolution Low Resolution Medium Resolution High Resolution Wavelength, Optical Diffraction & Resolution Narrow wavelength, narrow diffraction pattern = higher resolution Long wavelength, long diffraction pattern = lower resolution  Numerical Aperture & Resolution Light passes through the sample and enters the objective as an inverted cone. The the angular aperture of the light cone is determined by the focal length of the object The higher the numerical aperture, the greater the light collecting ability of system, resulting in greater resolution and brightness of generated image Numerical aperture (NA) = measure of the light-collecting and resolving power of a lens system (objective) NA = 0.25 NA = 0.36 NA = 0.95 microscopyu.com  Refraction & Resolution Refraction = bending of light between two different media (e.g. air and water) Caused by change of speed of photons moving between the two media Photons move quicker through air than they do water Microscopy photons move through multiple media: air, sample, glass (lenses, sample slide)  Refractive Index & Resolution A way to increase the resolving power of a microscope is to use immersion liquids between the objective and cover slip The immersion liquid is chosen so that the lens of the objective, coverslip/specimen all have the same refractive index Therefore, the photons move at a similar speed throughout all the media and less light is refracted. More light is collected by the objective, increasing resolution and brightness of the sample Air Oil microscopyu.com  Light Microscopy: Limits of Resolution Wavelength, optical diffraction, refraction, and numerical aperture all combine to define the limits of resolution Anything smaller than 200 nm will not be resolved using light microscopy Ability to resolve two molecules of AF450 in a specimen: (λ = 450nm) r = (0.61 x λ)/NA r = (0.61 x 450)/1.3 r = 211 nm 211nm Ability to resolve two molecules of AF647 in a specimen: (λ = 647nm) r = (0.61 x λ)/NA r = (0.61 x 647)/1.3 r = 303 nm 303nm  Fluorescent Molecules: Fluorophores Absorb (excitation) and emit light of specific wavelengths Thermofisher.com Shorter wavelength Longer wavelength Rino et al 2008 DOI: 10.1387/ijdb.072351jr  Fluorescence Microscopy Specific excitation and emission spectra of fluorophores combined with the lasers used as the light source and beam splitters and filters used to detect specific wavelengths in the microscope enable multiplexed images to be obtained DAPI used to stain DNA Alexa Fluor 488 conjugated antibody used to stain microtubules mCherry fusion protein with actin used to show actin localisation Actin filaments: red MTs: green Nucleus: blue N2A cell Spinning disk max projection. @cytoskeletown Yellow indicates co-localisation between green & yellow  Fluorescence Microscopy Used to detect presence, localisation, and amount of fluorescent molecules in a sample Lasers used as the light source and beam splitters and filters used to detect specific wavelengths Emitted light at 530nm Fluorescein Excite sample at 450nm  Epifluorescence vs. Confocal Fluorescence Microscopy Epifluorescence Microscopy Confocal Fluorescence Microscopy Entire sample is flooded with light Specific part of sample exposed to light and a pinhole used to focus emitted light In-focus light Out-of-focus light Out-of-focus light Captures both in-focus and out-of-focus light, but Light that is out-of-focus is blocked, therefore a good depth-of-field sharp image is obtained, but shallow depth-of-field Z-stacks Strozyk 2017 DOI: 10.1002/adfm.201701626 Live-cell Imaging Light Microscopy: Limit of Resolution = 200nm  Beyond 200nm… Electron Microscopy Transmission Electron Microscopy (TEM) Scanning Electron Microscopy (SEM) Cryo-Electron Microscopy (Cryo-EM) 62nm Scanning Probe Microscopy Atomic Force Microscopy (AFM) Scanning Tunnelling Microscopy (STM) 124nm Scanning Probe Electrochemistry (SPE)  Principal of Electron Microscopy Electron microscopy uses a series of electromagnetic &/or electrostatic lenses to magnify and focus electrons Conceptually most similar to stereo-light microscopy Conceptually most similar to light microscopy Requires detection of emission of secondary electrons Used to image thin samples, electrons pass from the surface of the specimen through the sample to project an image Used to image thicker samples  Electron Microscopy & Cryo-EM SEM of white and brown adipocytes: scale = microns Cryo-EM of Insulin Receptor: scale nm TEM of white and brown adipocytes: scale = microns Gutmann et al 2019 DOI: 10.1083/jcb.201907210 Principal of Scanning Probe Microscopy Kuzentsov & McPherson 2011: https://doi.org/10.1128/MMBR.00041-10  Self-directed Research – Reading How are histopathology samples prepared for staining? What are the basics of sample isolation, fixation, embedding, sectioning, H&E staining? A good starting point: https://histology.siu.edu/intro/tissprep.htm What is live-cell imaging? What considerations do researchers need to take into account for experiments? A good starting point: https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning- center/molecular-probes-school-of-fluorescence/imaging-basics/sample-considerations/live-cell-imaging.html#procon For those interested… Nikon (a leading microscope manufacturer) has an excellent website that goes into more detail on various microscopy techniques: microscopyu.com As do Zeiss (another leading microscope manufacturer): zeiss-campus.magnet.fsu.edu  Learning Outcomes Outline the basic principals of microscopy Outline the principals of optical, electron, and scanning probe microscopy Assess the usefulness and limitations of information obtained with different types of microscopy Compare how samples are prepared for various types of microscopy, and explain how these preparations affect whether the technique can be used for viewing living cells and tissues