Microscopy

Question 1

Light Microscopy

Answer 1

• Uses visible light and system of lenses to magnify images
• Commonly used to examine tissues

Question 2

Super-Resolution Light Microscopy

Answer 2

• Encompasses fluorescence techniques with capabilities to resolve objects below the classical diffraction limit of optical resolution

Question 3

Electron Microscopy

Answer 3

• Uses a beam of concentrated electron against the sample at a greater resolution
• Does not work on living cells because it will fry them

Question 4

Atomic Force Microscopy

Answer 4

• Scanning probe microscope which primary roles include measuring properties such as magnetism, height, and friction

Question 5

Fluorescent Microscopy

Answer 5

• Revolutionized the field of cell biology
• Provides an enormous contrast (black, white, etc.)
• Linked to specific probes, such as antibodies or small molecules to localize discrete cell types or cellular compartments
• Possible by virtue of the principle of fluorescence

Question 6

Principle of Fluorescence

Answer 6

Increased absorption of photons -→ reaches an excitatory state

When released from the excitatory state, emission of photons at a longer wavelength

Question 7

First Barrier Filter

Answer 7

Lets through only blue light with a wavelength between 450 and 490nm

Restrict; Allows light that excites

Question 8

Beam Splitting Mirror

Answer 8

Reflects light below 510nm and transmits light above 510nm

Question 9

Second Barrier Filter

Answer 9

Cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560nm

Restricts; Allows you to view the range you’re expecting

Question 10

Phase Contrast Microscopy

Answer 10

• If staining is not possible
• Takes advantage of the change in phase caused by regions of the sample have differential refractive index to introduce context into the image
• Look at occlusion within cells

Question 11

Refractive Index

Answer 11

Measurement of a bending of light ray when passing through one medium and into another

Question 12

Differential Interference Contrast Microscopy

Answer 12

• If staining is not possible
• Another method of generating contrast is based on the interference pattern generated by light passing through adjacent regions of a sample

Question 13

Fluorescent Protein Technologies and in vivo Imaging

Answer 13

• The creation of fusion proteins between a protein like GFP and a cellular gene in yeast or any other organism permits us to light that cellular product in a living organism
• The presence of GFP does not interfere with the function of the cellular protein
• No antibody or fixation required

Question 14

Jellyfish Aequorea Victoria - Green Fluorescent Protein (GFP)

Answer 14

• System to TURN ON GFP: Aequorin + Coelenterazine  (Ca²⁺)  Aequorin (Blue emission energy transfer to GFP)  GFP
• Light around 480nm will image a longer wavelength

Question 15

GFP and DsRed

Answer 15

• Two families of fluorescent proteins
• Both tinkered and engineered to emit most of the visible spectrum
• DsRed and GFP functions are similar but DsRed can emit different wavelengths

Question 16

Photoactivable Fluorescent Proteins

Answer 16

• Useful for looking at the dynamics of specific molecules
• Can be turned on with a laser of specific wavelength and turned off by an intense laser, photobleached (bright light)

Question 17

PhotoActivable Localization Microscopy (PALM)

Answer 17

• Only activates specific molecules at a time
• Current iterations of PALM technology enable 20nm resolution

Question 18

Eukaryotic Cell

Answer 18

The size of an organelle is just at the limit of resolution for a conventional microscope

Question 19

Factors that affect the Quality of Data

Answer 19

1. Contrast
2. Microscope Design
3. Resolution

Question 20

Contrast

Answer 20

• The use of stain to selectively mark cellular components
• To observe cellular structure stains or dyes were necessarily introduced into samples to increase contrast between different cellular constituent

Question 21

Stains

Answer 21

Stains impart differential absorption of light as it passes thru samples, thus providing contrast to an otherwise uniformly translucent sample

• Different stains will bind to different regions
• “Differential absorption of light”
• Common stain: Hematoxylin/Eosin (H&E)

Question 22

Hematoxylin (BLUE)

Answer 22

Binds arginine and lysine AA side chains enriched in nuclei (Basophillic)

Question 23

Eosin (PINK)

Answer 23

• Stains protein the cytoplasm
• Staining is done on fixed tissue (dead)

Question 24

Benefits of Fixed Tissues

Answer 24

• Fixation procedures allow for stains to enter the sample (more stable)
• Helps in catalog and archiving samples

Question 25

Antibody Probes

Answer 25

Antibodies can be linked to fluorescent molecules and become stains themselves * Antibodies to many cellular antigens exists to recognize many cellular constituents

Question 26

Covalently Coupled Markers

Answer 26

Enhance contrast in the cell or sub-cellular region in which the antigen was contained

Question 27

Cell Impermanent

Answer 27

Opens hole using alcohol to allow the stain to enter

Question 28

Digitization

Answer 28

* Of image data using digital cameras extend the ability to accurately detect very weak signals and increase contrast * Digitization permits processing to enhance image quality or presentation

Question 29

Digitization Examples

Answer 29

* In Calcium imaging, where the sperm enters. Color coding identifies different concentrations of calcium * 3D structures of organelles can be determined through digitization and processing

Question 30

Microscope Design - 3D Reconstruction

Answer 30

* Removes out of focus light * Amending the microscope design

Question 31

Standard Fluorescence Microscope

Answer 31

* Out of focus light * As you image a particular focal plane (optical section) light from above and below that plane is illuminated transmitted to the detector * Fuzzy image that is a composite of the plane of focus and the out of plane light

Question 32

Cortical Microscope

Answer 32

Producers an image of all the light coming off the sample both above and below the point at which you are focused * “ Just look where you are focused”

Question 33

Deconvolution

Answer 33

Algorithm is used to remove out of focus based on the behavior of light in the system

Question 34

Laser Scanning Confocal Microscope

Answer 34

* Removal of out of focus light through fine engineering * Confocal pinholes are controlled precisely by and relative to one another where light illuminates to the point of focus and what emitted light makes it to the detector * Only fluorescence emitted from the point of focus makes it through the second pinhole to the detector * Coupled with digitization of data permits us to view the cell in 3D

Question 35

Resolution

Answer 35

Development in super-resolution technology

Question 36

Diffraction Limited Resolution

Answer 36

The Raleigh criterion defines the lower limit of resolution

Question 37

The Raleigh Criterion

Answer 37

two images are just resolvable when the center of the diffraction pattern of one is directly over the first minimum of the diffraction pattern of the other * The size of the disc, its interference rings, and the overall resolution of the optical system is a function of the following: (Resolution eq: wavelength/numerical aperture) * Lower value for resolution is desirable: Decreased Resolution = Increased ­ Light