Observing Microscopes through a microscope Flashcards

Question

What is Darkfield microscopy and does it work? What is it commonly used for?

Answer 1

Darkfield microscopy: Light objects are visible against a dark background -Light reflected off the specimen ENTERS the objective lens -Useful for LIVE organism Not visible with a light microscope, cannot be stained or distorted by staining -Commonly used to visualize Thin Spirochetes from syphilitic chancres (sores; used to diagnose syphyllis) ex: treponema pallidum

Answer 2

Light path for Dark microscope unique opaque disc between condenser lens and light source. The disc will block any light that will directly traverse to and objective lens no specimens on stage- field dark If there is a specimen on stage, and light hits it at an angle, the specimen will deflect some of light into objective lens and you will be able to see organism.

Answer 3

Brightfield microscopy is Inadequate for Visualizing TRANSPARENT and COLORLESS specimens (most bacteria are colorless; unless photosynthetic)

Answer 4

Two ways to increase contrast in light microscopy; 1) Use a stain; you will have chromofore that will absorb particular wavelengths of light from white light and you will see that color to get good contrast (amplitude of light decreased when light pass though stained cell) 2)observing Phase shift in unstained cell; that occurs when light travels through specimens and parts of specimen have different refractive index than other parts. Light passes through sample, amplitude does not decrease, but phase of light shifted. (initially before light passes bacterial specimens, trough and peaks of light are the same, but after light passes specimens, trough light has dropped, phase shift)

Answer 5

Two waves in phase: BRIGHT Two waves OUT of phase; DIM when two light waves combine in phase, the resultant wave is larger/brighter. When they combine out of phase, the wave is smaller/dimmer.

Answer 6

These 2 types of microscopy exploit light wave Interference effects to obtain greater contrast in light microscope

Answer 7

Phase contrast Microscopy produces much better CONTRAST than brightfield. The EDGES- have largest phase shift in phase contrast image.

Answer 8

Phase contrast; has HALOS in the image, and you can see the internal structure of the cell -you also cannot use phase contrast on bacteriophage (too small in terms of resolution) DIC (aka Normarski, differential interference contrast); gives you more 3D image, and is a light gray color. Advantages: - DIC has INCREASED Depth of Field -Phase contrast better visualize INTERNAL structures

Answer 9

DIC (aka Differential interference contrast, or Normarksi): -Objects typically appear black to white on a grey background and appears nearly 3-dimensional. (side note: the colored image from textbook requires insertion of birefringent compensator plates, Intel optical pathway of microscope; uncommon)

Answer 10

Both phase contrast and DIC Microscopy; can be used with LIVING cells -DON'T require cell fixation/attachment slides -DON'T require cell staining

Answer 11

Flourescent Microscopy: takes advantage of fluorescent dyes (fluorochromes) that absorbs short wavelengths of light (UV or near UV) and emit at another, longer wavelength(visible) pathway for Flourescent Microscope: the specimen will have green fluorescent protein: it absorbs blue light. 1. set first barrier filter: to let through ONLY blue light (with wavelength between 450 and 490 nm) 2. it hits Beam-splitting mirror: reflects light below 510 nm but transmits light above 510 nm (*specimen absorbs blue light, and emits green light, which will go through second beam-splitter barrier, which only allows green light through ; up to eyepiece* ) 3. Second barrier filter: cuts out unwanted fluorescent signals, passing the specific green fluorescing emission between 520 and 560 nm

Answer 12

If you absorb red light, or any other color, you must change barrier filter to allow red light to be deflected and another color of light go back up. *There are different filters and mirrors you have to change for each fluorophore used. ***Fluorescent Microscope ONLY used If specimen itself Fluoresces or if specimen is bound to something that fluoresces. since it requires fluorochromes.

Answer 13

Fluorescent compounds -CFP, GFP (green flourescent protein from jelly fish), YFP and RFP are proteins, while the remaining are small molecules (fluorochromes) ex; DAPI, FITC, rhodamine b, Cy3, Alexa 568 Cy5 DAPI has affinity for DNA (DAPI small molecule that binds to DNA and allow it to fluoresce) some fluorochromes have affinity for different microbes -aurimine O, Myobacterium tuberculosis (binds to mycolic acid) -FITC, Bacillus anthraces -The remaining molecules are typically conjugated to antibodies *8GFP has modified this protein to produce other proteins that are fluorophores.

Answer 14

*immunofluorescent can be an important DIAGNOSTIC tool -(used to detect bacteria, and confirm antibody you're looking for) -*Antibodies conjugated to fluorochromes can be used to quickly detect bacterial cells. if antibody is specific to bacteria, it will bind to bacteria (with flourochrome attached) allowing bacteria to fluoresce. ex: Treponema palladium cells visualized with antibodies conjugated to fluorochrome (bacteria glows) used antibody recognized protein surface, antibody is conjugated with flurophore)

Answer 15

Fluorescent compounds each Absorbs at SHORT wavelength (high energy) and Emit at LONGER wavelength (lower energy) SHORTER wavelength- more energy absorb- lose energy

Answer 16

Multiple optical systems can be combined into ONE microscope Light microscope with bright field, phase contrast and DIC optics (each has its own objective and ocular; different white source (white, UV light) -insert different filters or prisms, you can change the type of microscope.

Answer 17

Standard fluorescente: ex: image of microtubules tagged with fluorochrome: blurry image (fluroescence focuses light on one particular plane; The sum of photons of light activate things below and above plane of focus, making image is out of focus. Confocal Microcopy: Eliminates OUT OF FOCUS fluorescence (due to very narrow depth of field)

Answer 18

Confocal Microscopy: technique of eliminating out of focus fluorescence; create a clear or sharper image. Cells must be stained with fluorochromes -the appropriate wavelength of light is used to excite the dyes -The light ILLUMINATES each plane in a specimen to produce a three-dimensional image -up to 100 um deep **the focal point of the formed image is at the pinhole "confocal". The microscope can filter out the out-of-focus light form above and blew the point of focus in the object.

Answer 19

A fluorescence confocal microscope is similar to fluoresce, but instated of illuminating the whole field, only ONE plane of a small region is illuminated -at any given instant, only one point of sample is observed. Laser is necessary because you need a Strong light to get through the small hole and illuminate specimen pinhole used to get very tight focus (on one small set)

Answer 20

By having a confocal pinhole, the microscope is very efficient at rejecting out of focus "haze" fluorescent light. By scanning the beam of light across the specimen, a very SHARP two-dimensional image of the exact plane is built-up that is not significantly degraded from other regions of the specimen.

Answer 21

emitted flourescent light from in-focus poin: is focused at pinhole and reaches detector Emitted light from Out-of Focus point is out of focus at pinhole and is largely EXCLUDED from detector

Answer 22

2 pinholes; 1 where the laser goes through and another one in front of the detector things out of focus will not be able to go into detector. confocal scans image ( scan different layers of image that are in focus of plane) multiple times and combines them together to get a clear image

Answer 23

Two-Photon Microscopy: Takes advantage of the fact that flurophores can be excited by near simultaneous absorption of 2 lower-energy photons This microscope uses two photons (lower energy) to excite fluorochrome (side note: Advantage; -No out of focus excitation -red/near infrared light penetrates deeper and is less damaging

Answer 24

Two-photon microscopy: (more 3D image) Alternative to confocal -Cells all stained with flurochromes (like confocal) and uses focused beams scanners in Raster pattern to generate images **Two photons of Long-wavelength (lower energy) are NEEDED to excite then dyes hence lower energy, less damage to cells -NO pinholes required, the two-photon hit the requirement ensures no fluorescence outside of focal plane -Fluorochrome Photobleaching and cell photodamage (single oxygen) outside focal plane almost non-existent (not usually occur with this microscopy) -Used to study cells within live, deep tissues (up to 1 mm deep vs 100 um for confocal)

Answer 25

Photobleaching: When Flurochrome loses ability to fluoresce, absorb and emit light, due to photon-induced damage. Covalent bond cleavage or non-specific reactions between the fluorochrome and surrounding molecules occur Photobleaching can be a problem when you are looking at something in cell with microscope and tracking it over time; you want to fluoresce the cell, but signal gets DIM

Answer 26

Super-Resoltuon Flourescence Techniques: -3 approaches are available to get around resolution constraints due to the classic diffraction of light -some can increase resolution from 200 nm to 20 nm. -Stimulated Emission Depletion (STED) microscopy (won the Nobel prize in 2014)

Answer 27

B/c a single source of light appears as a blurred disc and two points merge into one (sensitivity/Detection vs Resolution) COME BACK TO THIS (slide 46)

Answer 28

Since Fluorescent excitation spots are blurred, fluorescent objects closer than 200 nm to each other will appear as one blurred spot. One method breaches the light diffraction limit by REDUCING the size of 3-D blurred image formed when a single point source of light is brought to focus with a lens - "Structures smaller than 200 nm are lost in a blur"

Answer 29

Stimulated Emission Depletion (STED) microscopy: -method increases resolution by adding a second bright laser beam that wraps around the excitation like a torus. This beam switches off fluorescent molecules at the periphery of the blurry excitation spot. As the specimen is scanned, fluorescent molecules are turned ON and OFF and the effective fluorscent spot is recorded -This method requires special photoswitchable fluorescent probes

Answer 30

Confocal vs Super-Resolution microscopy -Fluorescent labels synaptic vesicles in live neurons -Fluorescent labeled replication factories in the nucleus of a cultured cell. COME BACK TO THIS

Answer 31

Electron Microscopy -Uses ELECTRONS instead of light -The SHORTER wavelength of Electrons, gives GREATER Resolution -In an electron microscope (with an accelerating voltage of 100,000 V), the wavelength of an electron is .004 nm -This predicts a Resolution of 0.002 nm.

Answer 32

The practical resolving power (quality, sharpness of image) of most modern Electron microscopes is much less resolving power (ability to distinguish two adjacent points in an object separately) -For biological samples, problems of specimen preparation, contrast and radiation damage, generally LIMIT RESOLUTION to 2.5 nm (25 angstroms)

Answer 33

- They cannot analyze live specimens -They only appear in black and white images

Answer 34

The Overall design of Transmission Electron microscope (TEM) is similar to light microscope, although it is LARGER and Upside Down. TEM uses an electron gun, whereas light micscorpe uses a light source. Similarities: Both TEM and light microscope use condenser lens, specimen, objective lens. However, light microscope has an eyepiece lens, and image is viewed directly. ,meanwhile. TEM has a Projector lens, and will have viewing screen or photographic film Differences: -TEM is 2 meters in height (taller, and bigger than light microscope) -Electrons are scattered by stained structures in the specimen -Magneitc coils in a vacuum instead of glass lenses in air (light) -Electrons that pass through the lens are detected to form an image.

Answer 35

TEM requires special sample preparation (No possibility for viewing living cells) 1) Glutaraldehyde fixes proteins while Osmium tetroxide binds and stabilizes proteins and lipids 2) Dehydrate specimen and permeate it with monomeric resin to form a block of plastic 3) Cut fixed tissues to be cut into ultra-thin sections (50-100 nm thick) it has to be 1/200 thickness of mammalian cell because electrons cannot pass through with thick membranes. 4) Place thin sections on a circular metal grid for microscope viewing

Answer 36

Contrast in the electron microscope depends on the ATOMIC NUMBER of the atoms in the sample -Elements with HIGHER atomic numbers scatter electrons better and produce GREATER contrast -Biological tissues are composed of atoms of very LOW atomic number (C, H, N, O) -Contrast is INCREASED by impregnation with the salts of heavy metals (Ur [92], Pb [82] Os [76} ) 'elements with high atomic number -Different cellular components are revealed to various degrees depending on their impregnation with these salts. DENSE regions of the specimen absorb/reflect more electrons and therefore look dark.

Answer 37

INTERNAL STRUCTURES in the slice of an organism are visible with TEM -TEM's are always black and white images (no color)

Answer 38

Positive staining: you stain the SPECIMEN Negative staining: you stain the BACKGROUND Negative staining is preferred when looking at intact microbrial structures (that do not change its morphology) ex: Negative TEM staining seen in intestinal bacterium and bacteriophages. (negative staining allows you to observe shape, size and arrangement of bacterial cells)

Answer 39

Thin sections do NOT convey 3-Dimensional shapes in TEM and can be misleading -although tedious, this third dimension can be reconstructed form serial sections. TEM only provides 2D images REVIEW -TEM's can also be misleading because even thin sections have significant depth compared to resolution of the electron microscope -Thus all parts of the specimen are in focus and objects that lie at different depths can be superimposed.

Answer 40

Scanning Electron Microscopy (SEM): -An electron gun produces a beam of electrons that Scans the SURFACE of a WHOLE SPECIMEN -Secondary electrons emitted (Knocked out) from the surface of the specimen produce the image. The specimen is coated with heavy metal that is deposited uniformly on the surface. (SEM also has amplifier and electron collector)

Answer 41

SEM (Scanning Electron Microscopy): Advantages: Sectioning is Not required. However, cells must still be FIXED (or FROZEN) and dehydrated and coated with a heavy metal -provides great depth of field -Image has highlights and shadows that give it a 3-dimensional appearance -SEM is smaller, Simpler and Cheaper than TEM Limitations: -Only SURFACE features can be examined -resolution is LESS than TEM (about 10nm; 1000-10,000X (magnification) )

Answer 42

In SEM, the specimen is scanned with a narrow beam of primary electrons that scan the specimen. These electrons are measured by a detector that controls the intensity of a image built on a video screen. -TEM, use transmitted electrons (that pass through a specimen to create an image. TEM has more lenses (condensor, objective, projector) and eyepiece; more equipment SEM- is simpler, less parts of microscope (TEM produces more MAGNIFIED, 2D images, with Better resolution for internal details of organism ; while SEM is less magnified and makes 3D images; used for surface details)

Answer 43

Scanned-Probe Microscopy: a general term for several related techniques that use a sharp Probe to Scan a surface -Resolution is NOT constrained by the wavelength of light or electrons, thus can Resolve at the Atomic level (better resolution than EM) -Special specimen preparation is NOT required -Only SURFACE features are observed. -

Answer 44

Scanning Tunneling Microscopy (STM): -Uses a sharp metal probe to scan the surface of the sample -The flow of electrons form the surface to probe tip (called Tunneling) is dictated by their distance -The stylus is raised and lowered in order to maintain a constant current/fixed distance (diameter of one atom) -Thus the topography of the sample is created. STM has BETTER resolution than TEM (100 pm for TEM vs 2.5 nm for STM)

Answer 45

Atomic Force Microscopy (AFM); -The AFM was developed to overcome a basic drawback with STM (scanning tunneling)- it can only image Conducting or semiconducting surfaces Process: -A probe is gently forced down onto a specimen -As the probe moves along the surface, its movements are recorded. (use laser to test up and down and record movement of probe) -As with STM, AFM does NOT require special specimen preparation Resolution is similar to STM (AFM : 1-5 nm)

Answer 46

Live or Unstained cells have LITTLE contrast with the surrounding medium. Staying provides contrast for light microscopy -Prior to staining, cells must be FIXED to microscopic slide -Heat fixation kills themicorbes and adheres them to slide -Smear: a thin film of solution of microbes on a slide. (Steps; Smear, fix, stain)

Answer 47

Stains are chromophore salts composed of a positive and negative ions -in a BASIC dye, the chromophore is a cation (positive) - Violet: typicaly neutralized by a Cl- ion. (basic dyes have chromophore and auxochrome groups) -In an ACIDIC dye, the chormopore is an anion (negative charge) -Eosin: typically neutralized by Na+ ion

Answer 48

Staining bacteria with dye -Bacteria are slightly Negatively charged at pH 7 -The colored positive ion of the BASIC dye is ATTRACTED to the bacterial cell -ACIDIC dyes are REPELLED by the bacterial cell and thus Stain the Background instead.

Answer 49

Four kinds of Staining Techniques: -Negative -Simple -Differential -Special

Answer 50

Negative staining: A method where the background is stained instead of the cell Advantages: 1) Useful for determining the overall cell morphology and size because they are highly visible against a contrasting background; and cell distortions are reduced because cells don't pick up stain and Heat Fixation is NOT necessary 2) Difficult to Stain cells (ex; Mycobacterium, and Spirochetes are easily observed) **ACIDIC dye is used. Cells are added directly to stain and allowed to dry (ex: B. subtitles, Nigrosin)

Answer 51

Simple Staining: Method of staining microorganisms with a Single BASIC Dye (ex: methylene blue, carbolfusion, crystal violet, safranin) The Goal is to HIGHLIGHT the entire microorganism to visualize cellular shapes and basic structures

Answer 52

Differential Staining- method that uses More than One chemical stain to differentiate between different organisms -Gram Stain (Danish bacteriologist Hans Christian Gram) -Acid-fast stain

Answer 53

Gram Positive - thick mesh layer of peptidoglycan; purple color Gram Negative: thin Peptidogylcan layer; pink color

Answer 54

Gram Stain process: Fixation--> Crystal Violet--> (water wash prior) to Iodine treatment--> (water wash prior) to Decolorization (alcohol) --> Counter stain Safranin Gram stain mechanism; -Crystal violet-iodine crystals form in the cell *Gram-Positive: -alcohol dehydrates thick peptidoglycan layer -CV-I (crystal violet-Iodine) chrysalis Do NOT leave (hence purple color) Gram Negative: -Alcohol dissolves outer membrane leaving a very THIN peptidoglycan layer -CV-I washes out -Safranin is added (the stain turns PINK color)

Answer 55

b/c the color of Safranin is Masked by crystal violet. (purple is a stronger color)

Answer 56

Mordant: A substance added to a staining solution to hold the stain, or coat the specimen to make it thicker and easier to see after staining is employed when gram-staining The importance of each step of gram-stain is well described in textbook (page 66)

Answer 57

Gram reaction indicates cell wall structural differences -Gram-positive bacteria have THICKER peptidoglycan cell wall while MOST Gram-Negative bacteria have a LPS layer and thinner peptidoglycan cell wall -Gram reaction provides Disease Treatment information (gram negative bacteria: Vibrio; gram-positive are Rod and Coccus)

Answer 58

Gram-NEGATIVE bacteria are generally more RESISTANT to antibiotics because of the LPS layer that the antibody must get through

Answer 59

Gram Positive bacteria tend to be killed by Penicillin and cephalosporins because both Antibiotics INHIBIT it during the synthesis of forming of peptidoglycan (penicillin is modulated to get through LPS layer)

Answer 60

ACID-FAST Stain (2nd differential stain used to distinguish between acid fast stain and Non-acid fast stain ) -Only binds strongly to bacteria that have WAXY material in their cell walls -Mycobacterium and Nocardia are Acid-fast -Two important pathogens are M. Turbuculosis and M. Leprae

Answer 61

Acid Fast-Staining process: Fixation--> Carbol Fuchsin steam for 5 mins--> Cool, water wash--> acid alcohol wash--> Counterstain with Methylene blue Acid Fast stain- can resist decolorizing after accepting a stain, hence its RED color (many acid-fast have high mycolic content; ex: mycobacterium, E. coli, M. leprae) Non-acid fast stain: are readily decolonized by acid after staining hence its BLUE color

Answer 62

Special Stains: used to distinguish specific parts of cells -Capsule stain -Endospore stain -Flagella stain

Answer 63

Negative staining REVEALS the Presence of capsules 1) Mix cells in negative stain for contrasting background (allow to Dry) 2) Add appropriate simple basic stain (ex: Safranin) and GENTLY wash ( or just told slide to remove excess) Capsules are water-soluble and easily dislodges or removed by washing. Cells are NOT heat-fixed (or fixed period) Capsules contain over 95% water, heat will cause distortion and shrinkage of capsule. capsules don't accept most stains ex: Klebsiella pneumoniae has capsule

Answer 64

because microbes with capsules tend to be more Virulent (more pathogenic)

Answer 65

Endospore Staining (Schaeffer-Fulton Stain) -Primary stain: Malachite green, steamed 5 mins -Remove stain from all the cells structures except endospore; water/30 secs -Counterstain: Safranin (stained red color, allow you to see cells)

Answer 66

YES, spores can be detached in a light microscope without staining because they are very DENSE and give GOOD CONTRAST

Answer 67

Flagella Staining -MORDANT- used to Coat Flagella with stain until Thick enough to be seen -Carbolfuschin simple stain used to give CONTRAST