MICROSCOPY ADDITIONAL COURSE MATERIAL 2 Approaches to Problems in Cell Biology Biochemistry-You can define a enzyme reaction and then try to figure what does it, when, where and under what control Genetics- You can make a mutation and then try to figure out what you mutated Cell Biology- You can visualize a process and try to

understand it- for instance cell division was one of the earliest Today- there are no distinctions. You cannot be just one thing, or be knowledgable about one thing. You need to take integrated appoaches to problems using the appropriate tools when needed. If you limit your approach, you limit your science Three branches of Microscopy 1. Optical 2. Electron 3. Scanning Probe Optical and Electron microscopy measure

refraction, diffraction, and reflection of the source radiation Optical uses white light, fluorescent light, or lasers Electron uses electromagnetic radiation/electron beams Scanning uses a physical probe to interact with the surface of the specimen Imaging Techniques

Technique Image Formed By Lowest Resolvable Unit Optical Microscopy Light Rays Microns (m)m)m)

Confocal Microscopy Coherent Light Source (m)Laser) Microns (m)m)m) Electrons Angstroms (m)) Electrons

Nanometers (m)nm) to Angstroms (m)) Molecular Mechanical Probes Angstroms (m)) Transmission Electron Microscopy (m)TEM) Scanning Electron

Microscopy (m)SEM) Atomic Force & Scanning Tunneling Microscopies (m)AFM/STM) Approx Lower Limit 1 m)m (m)monochromatic light) .1 m)m (m)X-Y Direction)

2 (m)high resolution TEM) 10 nm (m)100 ) 40 (m)theoretical) Units of Measure m)m - Micrometer 1,000,000 micrometers = 1 meter

Strand of hair has a diameter of ~ 20-180 m)m 10-6 nm - Nanometer 1,000,000,000 nanometers = 1 meter 10-9 Wavelength of visible light (m)400-700 nm) - Angstrom 10,000,000,000 Angstroms = 1 meter 10-10 Used to measure the size of atoms/bond lengths Length of a C-H bond in methane is ~1 Angstrom

Microscope Parts A. B. C. D. E. F. G. H. I. J. K.

L. M. Ocular Body tube Stage clip Revolving nose piece Objective Arm Stage Diaphragm Lever to move stage clip Course adjustment

Fine adjustment Light source Base Using the microscope Always observe using the LOWEST POWER objective first. Focus using the COARSE ADJUSTMENT KNOB to bring the object into focus. Bring the object into sharp focus by using the

fine adjustment knob. Focus, and then move to a higher power objective, if needed. Use only the FINE ADJUSTMENT KNOB when using the HIGHEST (longest) POWER OBJECTIVE. Keep both eyes open to reduce eyestrain.

Determine total magnification of the object by multiplying the power of the ocular (10x) the power by the power of the objective. Immersion Oil is a special oil used in microscope work with the highest power objective lenses (ie 100X lens). Immersion oil increases the light-gathering ability of a lens by allowing some of the light rays emerging from the specimen

There are two basic types of immersion oil, Type A and Type B. The only difference between the two is the viscosity. One or two drops of oil are placed on top of the coverslip and the 100X objective lens is brought into position so that it touches the oil and creates a "bridge" of oil between the slide and objective lens. 9 Preparing a slide

Using a pipet or dropper, add a drop of water or another solvent to a clean microscope slide. Then, place the specimen in the water. Place the edge of a coverslip on the slide so that it touches the edge of the water. Slowly lower the coverslip to prevent the formation of air bubbles.

Properties of Light Reflection Diffraction-scattering of light around edges of objects Limits the resolution Refraction- bending of light when changing medium (index of refraction) principle that lenses use to focus light Used in contrasting techniques Interference

light waves can subtract and add Polarization- allowing only light of a particular vibrational plane Refraction Diffraction Refraction Change in the direction of

a wave (m)light) due to a change in speed The straw in the picture looks bent because the light is bending as it moves from the water to the air Refractive Index (m)RI) RI of a material a measure of the speed of light in material RI is the ratio of the velocity of

light in a vacuum to the speed of light in the specified material Incident angle (m)1) is related to the refraction angle (m)2) by Snells Law n1sin(m)1)=n2sin(m)2) Used in calculating focusing power of lenses and dispersion properties of prisms Reflection Reflection is defined as a

change in direction of a wave at an interface between 2 different media so that the waveform returns to the media from which it came Used in focusing light waves to increase transmitted light Limitations light waves diffract at edges-smearing causes

limits resolution = minimum separation of two objects so that they can both be seen Size of specimen Magnification and Resolutions Magnification and Resolutions are different! Magnification can be increased virtually without any limit, resolution can not because it is a function of physical properties of light. Magnifications of about 2000X are the upper limit for light

microscopes. At magnifications above this, resolution does not improve. Resolution is a function of the wavelength of light which is used 17 Summary Types of microscopes Parts of microscopes Units of measure Properties of light

Limitations Magnification and resolution Quiz 2 10.12.2018 Start 11:00 Be on time Meiosis and Microscopy Sizes of Objects Eukaryotic cell- 20m Procaryotic cell-1-2m nucleus of cell-3-5m

mitochondria/chloroplast- 1-2m ribosome- 20-30nm protein- 2-100nm Microscope Objectives complex combinations of lenses to achieve high magnification low optical distortion Low chromatic distortion flat field Contrast Cells are essentially water and so are

transparent In addition to resolution and brightness, you need to generate contrast to see things Two objects may be resolvable by the microscope, but if they dont differ from the background, you cannot see them Contrast can be accomplished with staining or optical techniques Microscope types Brightfield

Stereo Phase contrast Differential Interference Contrast Fluorescence Confocal Electron Transmission Scanning Atomic Force Microscopes Stereo Different images are sent to the two eyes from

different angles so that a stereo effect is acheived. This gives depth to 3D objects Brightfield use a prism to send the light to both eyes light passing through specimen is diffracted and absorbed to make image Staining is often necessary because very low contrast

Bright Field Sample illumination is transmitted as white light and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample. Contrast differences arise because cells absorb or scatter light to varying degree.

Bacterial cell is difficult to see. (not pigmented cell) Staining increase contrast for Bright-Field Microscopy. (Gram Staining) 25 Dyes are organic compounds; affinity for specific

cellular materials Dyes can be used to stain cells and increase their contrast so that they can be more easily seen in the bright-field microscopy. Many dyes used in microbiology are positively charged, and for this reason they are called basic dyes (methylene blue, crystal violet, and safranin) Basic dyes bind strongly to negatively charged cell

components, such as nucleic acids and acidic polysaccharides. 26 Gram staining 27 Differential staining 28 Phase Contrast and Dark Field Microscopy

Staining kills bacteria! Without staining Wet mount preparation.. Some samples can be placed directly under the microscope. However, many samples look better when placed in a drop of water on the microscope slide. This is known as a "wet mount. Phase Contrast: cells differ in refractive index from their surruondings the refractive index is a factor by which light is slowed as it passes through a material

Result: A dark image on a light backround 29 Phase Contrast 30 Phase Contrast Light source

4 2 Differential Interference Contrast or DIC or Nomarski A prism is used to split light into two slightly diverging beams that then pass through the specimen. On recombining the two beams, if they pass through difference in refractive index then one retarded or advanced relative to the other and so they can

interfere. By changing the prism you can change the beam separation which can alter the contrast. Also measures refractive index changes, but for narrowly separated regions of light paths-ie it measures the gradient of RI across the specimen Gives a shadowed 3D effect Optically sections through a specimen 3D imaging Differential Interference Contrast Microscopy (DIC)

That contains polarizer in the condenser to produce polarized light. Light passes through prism that generates two distinct beams These beams traverse the specimen and enter the objective lens where they are recombined into one and create the interference effect. Cellular structure; nucleus of eukaryotes, endospores, granules of bacterial cells appear more 3D 33

Three-dimensional imaging of cells using Differential interference contrast (DIC) 34 3D Imaging Confocal Scanning Laser Microscopy is a technique for

obtaining highresolution optical images with depth selectivity. Computerized microscope Computer can focus the laser on single layers of the specimen Different layers can then be compiled for a threedimensional image Resolution is 0.1 m for CSLM 35

3D Imaging Atomic Force Microscopy (AFM) tiny stylus is positioned extremely close to the specimen so that weak repulsive forces are established between the probe on the stylus and atoms on the surface of the specimen During scanning, the stylus surveys the specimen surface, continually recording any deviations from a flat surface pattern that is generated is processed by a series of

detectors that feed the digital information into a computer which then outputs an image 36 AFM Interference Reflection Microscopy Looks at light reflected off the surface only. By polarizing the light and then analyzing the resultant, can see differences in height

of reflecting surface. If something is closely opposed to the glass surface, then it does not pass through a new medium and when reflected back it is eliminated. Total Internal Reflection Microscopy Light shined on a reflective surface at an appropriate angle will generate an evanescent wave, a wave of energy propagating perpendicular to the surface It only propagates about 100-200nm from

the surface Allows one to visualize events taking place near the membrane (exocytosis, cytoskeleton) Fluorescence Microscopy Fluorescent dye- a molecule that absorbs light of one wavelength and then re-emits it at a longer wavelength Can be used alone or in combination with another

molecule to gain specificity Fluorescence microscopy Principle: The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluophores, causing them to emit light of longer wavelengths (i.e., of a different color than the absorbed light). 41

Dead cells stained with a Fluorescent reagent (fluorescent phalloidin- a fungal toxin) to visualize actin filaments Endoplasmic Reticulum Stained with a synthetic dye that dissolves in ER membranes Cyanobacteria Bright field microscopy Cyanobacteria Contain chlorophyl a and other pigment

E.coli Fluorescent dye DAPI 44 Specimens Live cells or tissuecan you see the structure in a live cell? can you image the cell without damaging it with light? Fixed-try to retain structure intact

Glutaraldehyde- reacts with amines and cross links them-destroys 3D structure of many proteins Formaldehyde-reacts with amines and cross links them slower reaction, reversible, not as extensive Methanol, acetone, ethanol, isopropanol- precipitate material- not as good for retaining structure Rapid freeze (liquid helium)- then fix Summary Sizes of objects

Brightfield microscope 3D imaging AFM Fluorescence microscopy Specimens Quiz 2 10.12.2018 11:00 Meiosis and Microscopy Be on time Co-localization of Proteins

FRET- Fluorescence Resonance Energy Transfer If the emission wevelength of one probe overlaps with the excitation wavelength of another probe you can get resonance energy transfer Non-radiative transfer- the energy is transferred directly from molecule to molecule The two molecules need to be within 10 nm because the energy transfer falls off with the

6th power of distance You excite with the donor wavelength and measure emission at the recipient wavelength Co-localization of proteins FLIM-Fluorescence Lifetime Imaging When a probe is excited briefly, the rate of decay of fluorescence is different for each probe-so if you have different probes in the cell you can characterize them based upon lifetime FRET-FLIM- measure the decay of the donor during FRET

Confocal Microscopy Fluorescence microscope Uses confocality (a pinhole) to eliminate fluorescence from out of focus planes Minimum Z resolution=0.3m Because you can optically section through a specimen, you can determine the localization of probes in the Z dimension You can also build 3D (4D) models of structures and cells from the data

Laser scanning confocal Uses a laser to get a high energy point source of light The beam is scanned across the specimen point by point and the fluorescence measured at each point The result is displayed on a computer screen (quantitative data) Spinning Disk confocal Microscope Illuminates the whole field simultaneously

with a field of points Captures images of the whole field at once with a camera Much faster than LSCM Can be viewed through eyepieces Two photon confocal microscopy A fluor like fluorescein normally absorbs a photon of about 480nm and emits one at about 530nm If fluorescein absorbs two photons of 960nm near enough to each other in time so that the first does not

decay before the second is absorbed, it will fluoresce- 2 photon fluorescence Confocal microscope with a laser that emits picosecond pulses of light instead of a continuous beam is used Advantage 960nm light penetrates farther into biological specimens The density of light is very high at focal point, but low elsewhere, so damage to cell is less You dont need a second pinhole because excitation only happens at the focal point Second harmonic Imaging

Uses same instrument as 2-photon microscope If you shine 960nm light on a non-fluorescent sample, interaction of the light with certain structures will cause it to be converted to 480nm light Works mostly with polarizable materials like filaments How do we get fluorescent probes into cells Kill the cell and make the membrane

permeable Live cells Diffusion: some can cross membrane Microinjection- stick and tiny needle through membrane Trauma: rip transient holes in membrane by mechanical shear (scrape loading) or electrical pulse (electroporation) Lipid vesicles that can fuse with membrane Transfect with fluorescent protein vector Loading Cells (Alberts 4-59)

Types of Probes Some change intensity of fluorescence depending on pH or [Ca++] Some bind specific structures ER actin Golgi Plasma membrane Mitochondria Fluorescently labeled purified protein

Antibodies Microinjected Fluorescent Tubulin in a live cell Immunofluorescence localization of proteins in dead/fixed cells You can purify almost any protein from the cell (Biochemistry) Make an antibody to it by injecting it into a rabbit or mouse (primary antibody) Use the antibody to bind to the protein in the fixed cell

Fixed cells can be made permeable so antibodies can get into interior Use a fluorescent secondary antibody (anti-rabbit or mouse) to localize the primary antibody Anti-tubulin Immunofluorescent localization of microtubules Green Fluorescent Protein (GFP)- An Ongoing Revolution in Cell Biology

Protein from fluorescent jellyfish The protein is fluorescent Now cloned, sequenced and X-ray structure known If you express it in a cell, the cell is now fluorescent! Use a liver promoter to drive gene expression, and you get a fluorescent liver! All cells in the liver make GFP which fills the Liver specific promoter cytoplasm with

fluorescence. GFP gene DNA GFP Protein on Liver Fuse the DNA sequence of a protein to the DNA sequence of GFP and the cell will express it and make a fusion protein which has two domains. Wherever thatLiver

protein is in the cell, you will see DNA protein gene GFP gene fluorescence! Liver protein GFP protein Protein How to get around the problem of resolution? Invent the Electron Microscope

Uses electrons instead of light to form an image Wavelength of electron decreases as velocity increases so accelerated electrons have a very short wavelength compared to visible light You need to use magnets as lenses to focus the beam Sample Preparation for EM Must be done in vacuum for electron gun to work Cant have water in vacuum! Dry tissue does not have enough density to scatter electrons so you have to replace it with something dense. Procedure

Fix Tissue (glutaraldehyde or osmium) Dehydrate and embed with plastic Stain with Osmium, lead etc. or make metal replica For TEM- Section (0.02-0.1m thick)- so you only look at very thin section For SEM- No sectioning- you only see the outer surface What you see is the scattering of electrons by the metal. There is no biological material left! a) Transmission Electron Microscopy (TEM) A "light source" at the top of the microscope emits the electrons that

travel through vacuum in the column of the microscope. Instead of glass lenses focusing the light in the light microscope, the TEM uses electromagnetic lenses to focus the electrons into a very thin beam. The electron beam then travels through the specimen you want to study. Depending on the density of the material present, some of the electrons are scattered and disappear from the beam. 66

Whereas the resolving power of a high quality light microscope is about 0.2 micrometer, the resolving power of high quality TEM is about 0.2 nanometer TEM is used to examine cells and cell structure at very high resolution and magnification. Special techniques of thin sectioning are needed to prepare specimens before observing them.

You can study small details in the cell or different materials down to near atomic levels. 67 b) Scanning Electron Microscopy (SEM) Type of electron microscope that images a sample by scanning it with a beam of electrons in a raster scan pattern.

If only the external features of an organism are to be observed, thin sections are unnecessary. In SEM the specimen is coated with a thin film of a heavy metal, such as gold Electrons scattered from the metal coating are collected and activate a viewing screen to produce an image Electron micrographs are black and white

images. SEM close-up view of a Wolf Spider 68 Immuno-electron microscopy You cant see antibodies in the EM You can attach dense particles to antibodies to make them visible Allows you to visualize the localization of specific proteins in the EM

Very hard to do! Summary Co-localization of proteins How to get fluorescence probes into the cell? GFP Electron microscopy SEM TEM Quiz 2 10.12.2018 11:00

Meiosis and Microscopy Be on time

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