MED 204: Visualization of Cells and Tissues

what is it: Contrast

diff in light intensity between specimen and background 

Generate through stains, dyes, fluorescent dyes  

what is Magnification?

microscope make image larger than actual 

• Useful ONLY when see details that u can't with your unaided eye 

• objective lens ENLARGE image  

What is Resolution?

distinguish detail  

• min distance where 2 points of specimen can be seen  

• Objective lens, imaging media, wavelength of light determine fine detail 

X-ray

x-ray radiation to detect tissue

what is CT?

computed tomography)- compilation of x rays (greater resolution than X-ray) 

What is MRI?

- use radio waves and magnets to detect tissue

What is PET scan?

positron emission tomography)- radioactive tracers to track blood flow, O2 intake, metabolic activity (ex: detect change like cancer)  

Microscopes use (blank) or (blank) to make image

Microscopes use light or electron to make image !  

Light Microscope

light pass through specimen is altered to see contrast 

Transmission Electron Microscopy

What is it?

accelerated electron beam through specimen to phosphor screen  

(dense obj= scatter electrons = dark) 

Scanning Electron Microscopy

What is it?

focused electron beam. Electron interation with atoms on sample makes scatter that generates image  

Formula: Total Magnification

multiple magnification of each component  

Ex: (10x eyepiece) x (40x objective) = 400x 

Visible light ?
What is relationship between wavelength and resolution?

type of electromagnetic radiation (no mass only photons)  

Shorter wavelength= higher resolution = more damage to tissue  

Refractive Index (RI)

ratio of speed of light in Air to speed of light in Material 

2 types of specimens

Phase specimen and Amplitude specimen

Phase specimen

colorless, retarded slows down, amplitude don’t change  

• Microscope components

Amplitude specimen

stained visible dye, amplitude changes, looks darker , light is being absorbed 

• Stains and dyes 

• Brightfield microscopy 

Diffraction

Tendency of waves to bend around edges  

Magnification

make image bigger with objective lenses (4x, 10x, 40x, 100x)  

Smaller angle, fewer diffraction  

Resolution

shortest distance between 2 points on a specimen that can still be distinguished by observer or camera as separate entities – u need space between them to resolve the image  

RESOLUTION IS INVESERLY PROPORTIONAL TO WAVELENGTH  

Abbe diffraction limit

resolution in light microscopy is Limited by light's physical properties 

Rayleigh Criterion

2 objects can be resolved if space between (valley) between overlapping brightness is reduced by 20% between two maxima to show 2 diff objects 

Point spread function

3d representations of diffraction pattern 

Prepping Samples :
Light microscopy?

Electron microscopy?

Light microscopy-  LIVING or FIXED  

Electron microscopy- MUST BE FIXED 

Contrast

stains and dyes- which specimen?

Microscope components- which specimen?

made by stains, dyes, enzymatic color change

• Stains and dyes- amplitude specimens  

• Microscope components: Phase specimens  

  Phase optics, DIC optics,  

Brightfield microscopy

What is it and which specimen?

light DAMPENED when passed through cell that have been stained with dyes  

=Amplitude specimens- amplitude of light waves change when passed through samples

Generate contrast with dyes and stains

Fixed or live samples?

must be first FIXED  

Generate contrast in light microscopy with optical components

Fixed or live samples?

  live or fixed  

Phase contrast microscopy

physical impediments to shift light waves as they pass through specimen= destructive interference  

• Study internal cell structure without need to strain and kill cells 

primarily used on live samples, unstained

Differential Interference Contrast Microscopy (DIC)

use polarized light to generate contrast/details as light pass through specimen 

(google says both live and fixed)

Generate contrast with Fluorophores

Fixed or live samples?

cells living or fixed  

Fluorescent moles must be excited by light at 1 wavelength to emit light at a second wavelength  

Advantages of Flourophores?

Advantages: sensitivity, resolution 

• Specificity 

• Sensitivity (50 m/mm3) 

• Fluorophores Sensitive to physical-chemical environment 

• Temporal resolution (10^-9 sec) 

• Spatial resolution (can detect single molecules) 

• Greater resolving power than staining with visible dyes or color enzyme assay 

Generating contrast in light microscopy with immunological reagents

Fixed or live samples?

- usually FIXED  

Immunolabeling to visualize specific compenents of cells –

Fixed or live samples?

usually FIXED  

Direct labeling of a molecule of interest to see its cell components

What is it? Fixed or live samples?

– LIVING or FIXED cells = molcularly manipulating gene of interest to add a detectable tag 

• Introduce Plasmids--> tagged protein 

• Fluorescent protein tag (GFP), visualized by shining wavelength of light on sample 

• Non-Flourescent epitope tage (myc, His, GSt, FLAG, HA), visualized by immunostaining & indirect flourscence 

• Use molecular cloning to insert gene of interest into expression vector

Electron Microscopy? has HIGH ___ & ______

Electron Microscopy: HIGH MAGNIFiCATION + RESOLUTION 

Transmission EM (TEM)

2D, fine detail since sample sliced thin  

More Dense objects= scatter electrons= dark in TEM  

Less dense = don’t scatter electrons= brighter  

Super fine sample, high resolution  because super thin! So your only looking at a piece of the cell  

Scanning EM (SEM)

3D, surface features  

Freeze fracture/etch with scanning EM

Freeze fracture/etch with scanning EM: 3D view of inside cell 

Numerical Aperture

Numerical Aperture- angular acceptance for incoming light