Experimental Techniques: Measuring Particles and Cells

equipment considerations

• strengths of this approach

• limitations of this approach

  • types of samples measured

SEM, TEM, and AFM have

about the same resolution

Scanning Electron Microscopy (SEM)

Imaging with a high energy beam of electrons

SEM resolution

~ 1 nm

SEM need surface of sample

to be dry and conductive, coated with gold

SEM image

3d

Transmission Electron Microscopy (TEM)

Imaging with a high energy beam of electrons

TEM runs under

high vacuum

TEM resolution

~ 1 nm

TEM sample

dry, thin, placed on a grid

TEM image

2d

Atomic Force Microscopy (AFM)

Tip can scrape a surface or oscillate over a surface

AFM change in deflection (contact) or frequency (non-contact)

of cantilever measures forces

AFM sample

first deposited to flat substrate like mica

AFM image

full 3d scan of surface topology

AFM relatively

gentle to samples

AFM resolution

~ 1 nm

AFM scanning speed

slow

Dynamic Light Scattering (DLS)

Can measure size (0.3 nm – 10 m) and zeta potential

DLS size

bulk, intensity-weighted measurement of particles in solution. Measurement is quick and easy.

DLS autocorrelation of scattered light

is used to fit for diffusion coefficient

DLS hydrodynamic radius calculated from

Stokes-Einstein

stokes-einstein equation

d = kbT / (6 pi * μ * r)

diffusion constant for protein

10^-6 cm² /s

Nanoparticle Tracking Analysis (NTA)

Can measure size (10 nm – 1 um) and absolute particle concentration in solution

NTA tracks of individual particles are followed

over time and each track is fit to stokes-einstein

NTA average

direct number-weighted

NTA capability

single particle fluorescence

coulter counter

Can size 0.4 um – 1,600 um

coulter counter counts

particles as displaced volumes

coulter counter particles suspended in a weak electrolyte solution

are drawn through a small aperture that separates two electrodes

coulter counter voltage

across aperture; is the sensing zone

What would be the best method to characterize 50 nm hydrated nanoparticles that can aggregate together?

Dynamic Light Scattering (DLS)

counting cells with microscope + hemocytometer

count squares and average; typically count left side and top side lines only

direct cell count

hemocytometer and look at Trypan Blue staining of dead cells

metabolic activity cell count

A colorimetric (absorbance) assay that uses (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or a related kit (CellTiter). More live cells = higher signal

lactate dehydrogenase count

released by dying cells. More dead cells = higher signal

apoptosis

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). DNA fragmentation caused by apoptosis

flow cytometry cell count

Fluorescent dyes such as propidium iodide can stain dead cells / DNA.

high throughput multi-label plate readers absorbance assay

concentration, cell viability

high throughput multi-label plate readers fluorescence assay

gfp expression, binding affinity

high throughput multi-label plate readers luminescence assay

luciferase expression, cell viability

high throughput multi-label plate readers advantages

Fast, Quantitative, Low sample volume, High-throughput

High-throughput Multi-label Plate Readers

average values per well, lower sensitivity