Cell bio Ch.9 - Visualizing cells

bum ahh exam

What makes it difficult for cell biologists to visualize individual cells?

Small size and transparency

Resolution

The ability to distinguish 2 objects that are close to each other

what is light microscopy limited in

resolution

True or False: Magnification is a limitation of light microscopes

FALSE

Define fixation

the process of killing and preserving cells in order to reveal details of cells

how do chemical dyes produce color

by absorbing specific wavelengths of light

Fluorescent molecules _____ light at one wavelength and _____ light of a longer wavelength

absorb, emit

Fluorescent Microscopy Filters

• first barrier filter

• beam-splitting mirror

• second barrier filter

First barrier filter

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

beam-splitting mirror

• reflects light below 510nm but transmits light above 510nm

• Reflects high energy but transmits lower energy

Second barrier filter

cuts our unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560nm

A resolution with a smaller number is a higher/lower resolution

higher

Fluorescence microscopy limitation

fluorescence comes from out-of-focus parts of cells

Confocal microscopy

uses a scanning laser and pinhole apertures to limit detection to the focal place

Optical section

image at each point, slice of the cell

Electron Microscopy

a focused beam of electrons replaces light

What is required for electron microscopy

• vacuum

• cells must be fixed

• cells are often desiccated and sliced into this sections

______ materials are used to stain cells for electron microscopy

electron-dense

Scanning electron microscopy (SEM)

images the outside surface, staining but no sectioning

Transmission Electron microscopy (TEM)

Images internal structures and slightly better resolution that SEM

Why does an electron beam give more resolution than light

The wavelength of an electron is much smaller than the wavelength of a photon

Shorter/Longer wavelength means better resolution

Shorter

What is Flow cytometry

Live cells are analyzed in real-time in an aqueous stream as they pass through a laser

Flow cytometry collects…

• fluorescence data (color, intensity) and laser scatter (reflecting size, shape, and internal architecture)

• Quantitative data

What does a cell sorter do?

allows cells to be separated based on fluorescence and scatter properties

Limits to light microscopy

Resolution

Ways to provide contrast and sensitivity for light microscopy

• chemical stains

• fluorescent molecules

Flow cytometry uses fluorescent labels to…

measure levels of specific biomolecules and ions, and to sort cells based on expression levels