BIOS 3450: Lecture 12: Chapter 9: Visualizing Cells and Their Molecules

resolution

ability to distinguish two nearby objects as separate, relevant as cellular structures are extremely small

light microscope limit

can only resolve 200 nm (0.2 um)

electron microscope limit

can resolve structures down to <1 nm, allowing macromolecules and viruses to be seen

size of a cell

10-100 micrometers

size of a nucleus in a cell

5-10 micrometers

size of mitochondrion in a cell

1-2 micrometers

size of bacterium

1 micrometer

size of virus

20-300 nm

size of protein

2-10 nm

long wavelength energy and resolution

lower energy, lower resolution

short wavelength energy and resolution

higher energy and resolution

traditional versus electron microscopes

visible light is used in traditional microscopes, electron microscopes use electron beams with short wavelengths and have higher resolution

light microscopy

uses visible light to illuminate specimens, can only be used on living cells, is inexpensive, with easy preparation, but has limited resolution, 4 types

bright-field microscopy

basic microscope, light passes through specimen

characteristics: dark specimen, bright background

problem: stain often required

uses: histology, tissue sections, general cell observations

phase-contrast microscopy

observe living unstained cells

principles: different parts of cells slow light by different amounts, microscope converts into contrast

advantages: no staining, living cells

good for: cell shape, movement, and division

Differential Interference Contrast (DIC)

produces: sharp images, 3D like appearance

advantages: visualization of organelles, cell boundaries, and living cells

Dark-Field Microscopy

principle: only scattered light enters objective

appearance: bright specimen, dark background

useful for thin specimens

microtome

instrument that cuts extremely thin slices, 5-10 micrometers

staining

prepares samples for light microscopy

hematoxylin: stains DNA, nuclei, blue/purple

eosin: stains cytoplasm and proteins, pink

H&E stain: common

Cryostat

rapidly freezes tissue before sectioning

advantages: faster, preserves proteins, immunostaining

electron microscopy (EM)

uses electron beams instead of light

adv: high resolution, reveals ultrastructure

disadv: must be fixed, expensive, complex preparation

Transmission electron Microscopy (TEM)

electrons pass through specimen (thin)

produces: 2D images of internal structures

best for viewing: membranes, ribosomes, mitochondria, nuclear pores, ER

Scanning Electron Microscopy (SEM)

electron beam scans specimen surface

produces: 3D like surface image

best for viewing: cell surfaces, microvilli, cilia, tissue topography

Cryo-Electron Microscopy (Cryo-EM)

revolutionized structural biology

Steps: 1) sample in water, 2) rapid freezing, 3) ice crystals do not form, 4) molecules in native state

adv: proteins appear exactly as they do in living cells

applications: determine structures of ribosomes, ion channels, viruses, and protein complexes

immunofluorescence

locates specific proteins inside cells

principle: antibodies recognize specific proteins; fluorescent dyes attached to antibodies glow under specific wavelengths

process: 1) antibody binds target protein, 2) fluorescent tag attached, 3) microscope detects emitted light

result: scientists can see exactly where a protein is

DAPI

fluorescent dye that binds DNA, visualizes nucleus, appears blue, allows for comparison of protein location relative to nucleus

BmLz

in nucleus, transcription factor

fluorescent molecules

absorb one wavelength and emit another, fluorophores, GFP absorbs blue light and emits green light

reporter gene

shows when gene is active, can be inserted into cells, if gene is turned on: GFP is produced and cell glows green

uses: measures gene expression, promoter activity, and protein localization

fusion proteins

scientists attach GFP to another protein and the protein glows green (or color of GFP), allowing scientists to watch movement, localization, and transport in living cells

GFP

allows for observation of living cells, real-time protein movement, developmental processes, and signaling pathways without killing cells

FRET (Fluorescence Resonance Energy Transfer)

purpose: measures whether two molecules interact

concept: 2 fluorescent proteins, donor → acceptor, when proteins are extremely close (-1-10 nm), energy transfers from donor to acceptor

interpretation: FRET present = interaction, no FRET = no interaction

allows scientists to observe protein interactions, cell signaling, activation of pathways in real time inside living cells

flow cytometry

analyzes thousands of cells per second

How: cells are labeled with fluorescent antibodies, cells pass single file through laser, laser excites fluorophores, detectors record signals

measures: cell size and complexity, protein expression, cell type

forward scatter and side scatter

Forward Scatter (FSC)

measures cell size, larger the cell = more FSC

Side Scatter (SSC)

measures internal complexity, more organelles = higher SSC

FACS (Fluorescence Activated Cell Sorting)

specialized form of flow cytometry, measures and separates cells

process: cells with desired fluorescence are given electric charge and deflected into collection tube

uses: isolates stem cells, sorts immune cells, purifies GFP-expressing cells