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Lecture 2

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Biology 2382B
Robert Cumming

Lecture 2: Imaging in Cell Biology How do we look at cells? - Differential interference contrast (DIC)microscopy and florescent microscopy - artificially induced protein has green fluorescent protein (Huntington protein) used as well Time lapsed microscopy (takes image over time) Really cool things can be seen and done w/ microscopy – zombie analogy - Visualizing live cells w/ fluorescent molecules inside them How do we image cells? - Using light microscopy, ways it can used to bring out details (phase contrast, DIC etc.) - Fluorescence microscopy used to make thing inside cell grow - Optimizing ability to see detail using electron microscopy etc. Anton van Leeuwenhoek (1632 – 1723) - 1600s people didn’t know cells existed – only interested in minor details of tissue - Built many simple, single lens microscopes – first person to build microscope big enough to see living cells - First to observe living protazoa and bacteria which he called “animalcules” - Went on to visualize human red blood cells and sperm – dropped water w/ cells on pin – used sunlight and saw lens to see it - With great skill at grinding lenses, naturally acute eyesight and lots of patience he was able to achieve a magnification of 200X - Maximized magnifying and refractive properties of lens used by grinding them - Resolution = details Features of a Modern Compound Microscope Bright-field Microscopy - light source - condenser lens to focus light on specimen - objective lens to collect light after it has passed through specimen - ocular or eyepiece lens to focus image onto eye - typical light microscope magnification is 40 to 1000X - only structures with a high refractive index (ability to bend light) are observable - Conventional light microscope: compound (contains 2 or more lenses that increase the magnification)  Condenser: focuses light from source onto the specimen  Light passes though specimen and collected and focused via the objective lens (magnifying)  Ocular lens = eye piece  Results in magnification processes: objective max. magnification = 100x - Total Magnification = product of 2 magnifying factors  The ability to see cells/ specimens is dependent upon the properties of the specimen itself, and its refractive index  Refraction = ability to bend light  If light goes through unimpeded just see white light  Any light slowed down/ or refracted by specimen…. Resolution of Microscope - Resolution: the ability to distinguish b/w two very closely positioned objects as separate entities - If u mag fuzzy it was still fuzzy – u need to have better resolution - Detail dependent upon resolution of microscope - Resolution of naked eye = 0.2mm - A conventional microscope can never resolve objects/ cellular features that are less than ~0.2µM apart - In light microscopy, because of resolution of microscope we are able to discern finner detail down to bacteria (lmtd. To 0.2 microns) - For viruses and proteins, electron microscopes are used – really small details - Smaller resolution is better Resolution = D - Distance resolved between 2 points - - λ: wavelength of light - Nsin a: numerical aperture(higher is better) - N: refractive index of medium between the specimen and the objective lens - α: ½ angle of light entering objective - The limit of resolution is 0.2 microns=200 nm - Small is good - Refractive index of air is 1 How to make it really small 1. Make denominator bigger by adding oil or water increases refractive index 2. Make denominator bigger by changing alpha (increase angle) – to do w/ how lens is grinded – allowing come of light to be very broad by bringing the objective lens very close to the specimen to increase the angle 3. Make wavelength of light smaller – numerator smaller –decreases Wavelength spectrum used in microscopy - Visible light b/w 300 -700 - Take advantage of wavelength properties of electrons in order to affect wavelength - Obtaining contrast in light microscopy by exploiting changes in the phase of light Two Waves Out of Phase - Certain parts of the cell (i.e. nucleus ) refract light more than other parts - Cellular constituents w/ high refractive properties can slow the passage of a light beam by a quarter wavelength (~ ¼ λ) - In different aspects of cell in how light passes through it - Changes in how light passes through the specimen to increase resolution - Parts of nucleus can reflect light by slowing it down more - Something that passes through a refractive structure - Retards wavelength - slows down – quarter wavelength slowed down - To get most contrast = you can see dark stuff details (light dims in some areas – where waves have destructive interference and cancel each other out - you get darkness - peaks n trough cancel – - High peaks/trough = very bright light - shallow = dim light - If all light goes through = white light – no image though?? Phase Contrast Microscopy - used to examine live “unstained” cells - small differences in refractive index & thickness within the cell are further exploited and converted into contrast visible to the eye - Similar to bright-field only:  Amount of light going through is limited  Concentrate of light generated  Anywhere where light passes through a structure inside the cell slows the light by about a ¼ wavelength  Unimpeded and impeded light is focused through the objective lens: the impeded slowed down light is slowed down again by another ¼ wavelength by the phase plate  Again the waves are recombined at the top, having collectively slowed the light down by ½ a wavelength  Trough and peaks cancel each other, creating dimness  Anywhere where the ½ wavelength is out of sync gives darkness that results in further definition of cells Differential Interference Contrast Microscopy (Normarski microscopy) - used to examine live “unstained”cells - small differences in refractive index & thickness within the cell are converted into contrast visible to the eye - uses polarized light; - DIC microscope is equipped with polarizers. - Defines the outline of large organelles such as nucleus and vacuole and provides better detail of cell edge - Waves of light are 3D Polarity of wavelengths: - DIC takes advantages of this polarity property and uses polarizers to limit the polar nature of light - Can polarize the light (vertically or horizontally) – by recombine the polar forms results in the creation of specific images - A vertical polarizer would only let vertical wavelengths of light through, etc. - INTERFERES W/ POLARITY NOT PHASE this give another definition of the cell – by recombining the lights you see shadows as well as topographical features of cells - It also defines the outline of large organalles within the cell - Phase contrast sometimes results in brightness in certain areas which does not occur w/ DIC – Results in HALO effect - SUMMARY 3 different types? Fluorescence Microscopy - Uses a property of certain molecules to fluorence, i.e. to emit visible light when they absorb light at a specific wavelength (e.g. invisible UV light) - Location of fluorescent dyes or fluorescent protein molecules can be imaged. - Can visualize more than one protein or cell structure  Green – Tubulin  Red – Mitochondria  Blue- Nuclei - Specific details can be seen with this - Chemicals used that will emit light – visible light (specific colours) - Adds dyes that attach to certain structures w/ in cells that emit colours - dyes or “FLUOROPHORES” absorb energy kicking electrons (e ) into a higher orbital (unstable) - instability causes e to drop into its normal orbital releasing energy as visible light “FLUORESCENCE” - Chemicals w/ unique ppt i.e. can be excited by certain wavelength of light - - the e in outer orbit jump up to exited state and drop back down – emits light of different wavelength (i.e. excited by blue light, drops back down and emits green light) - You have excitation wavelength and optimal wavelength and emitation wavelength - Optimal wavelength of light excites particular phorphores at its maximum and emits a wavelength of light at a maximum. - Difference b/w these two maximum is called a strokes shift How are fluorescence images obtained? - The microscopes do not have to be two different types (one can be used to conduct light or fluorescent microscopy etc.) - In fluorescence microscopy:  Different light sources and different light paths are used (IMPORTANT TO KNOW DIFFERENCE IN PATHS) - Light (on left) from source(incandescent) passes through specimen and magnified and see through the eye piece - Fluoroscent microscope – differenet light source – broader spectrum covered bright light - Passed through filter that only allows through a specific wavelength of light, in this case 540 (green) - The green light goes through filter and its dichronic mirror that reflects down through objective, hits specimen – the flourophore is excited and then emits a red light (630) - The dichroic mirror reflects low wavelengths down and high wavelengths up - The red light goes back through and is reflected up – the emission filter only allows the purest red light through - LIGHT DOES NOT START FROM BELOW THE SPECIMEN (I.E. NEVER GOES THROUGH)  Understand difference b/w 2 types of microscopy Many Colours of Fluorophores - Signals are bright on a black background. - A variety of fluorophores exist with different excitation and emission wavelengths that allow labeling of more than one protein or organelle at the same time - Fluorescent dyes are available to stain cell structures and organelles (e.g. DAPI to stain nuclei blue, Mitotracker Red to stain mitochondria red). - A dye can be conjugated with antibodies to localize any molecules of your interest in cells (immunofluorescent staining). - Fluorescent images: Black background - Where ever the flueroflor binds lights up??  DAPI binds to DNA very well – mitotracker red as it only binds inside mitochondria??  Mitotracker red only fluoresces when taken up in mitochondria  Interested in protein – we have 10000000000 different proteins  This is by using chemicals or dyes that recognize specific structures  But how to locate proteins?? By using antibodies Monoclonal Antibodies - HAT medium (a selection medium) is toxic for myeloma cells, which have mutation of specific gene. - Hybrid cells survive in HAT medium because they obtain a missing gene product from spleen cells. - Hybrid cells are immortal like myeloma cells and produce antibody. - B cells produce antibodies to target foreign invaders, bind, and trigger the influx of killer cells to destroy pathogen - Take advantage of this immune response by using very specific antibodies, monoclonal antibodies that are found in mice - Proteins synthetically produced, injected into mouse, mouse senses it’s a foreign invader, mouse b cells produce antibodies which tend to accumulate in the spleen - Nodules in the spleen are clusters of b-cells that have proliferated and are all making antibodies (primary cells – will only grow and produce antibodies for a limited amount of time) - A hybrid cell line containing a mutant mouse cancer cell that is fused to the b-cells creates an immortal cell line with 2 properties: 1. If fuse successfully, grow indefinitely 2. Because spleen cells has effectively fused with the cancer cell and resistant to the hat media, the fused cells will now grow (plain cancer cell does not gorw) - Grown to create clonal populations of pure hybrid cell line to create antibodies indefinitely - HYBRIBOMA cells pure – colonal population Immunofluorescence Microscopy - Antibodies are made to specific proteins (i.e. microtubules, actin) - Antibodies are added to cells fixed on a slide which bind the specific protein they were designed to recognize - Secondary antibodies with attached fluorophores are added and bind the primary antibody - Each fluorophore has a unique excitation and emission wavelength that can be detected with appropriate filters in the microscope - The pure antibody obtained that recognizes protein of interest is now made - Cell grown on glass – antibody solution aggeg to cells causing the cell to undergo fixation process that kills the cell and
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