BIO265 Microscopy

What is microscopy?

visualizing small objects, usually via electromagnetic radiation (EMR)

What is histology?

the study of microscopic anatomy of tissues

What is resolution?

ability of a microscope to distinguish objects separated by small distances

Light (optical) microscope

• any microscope that uses visible light to visualize samples

• modern: compound microscope

• resolution ~200 nm, varies based on objective lens

• single light path (both eyepieces see same image)

• certain tissues block more of less light/certain types (wavelength/frequency)

What does resolution depend on?

• the light-gathering ability of the objective lens and wavelength of light source

• shorter wavelengths → greater resolution

What is numerical aperture (NA)?

measure of light collecting ability of an objective lens

What is the relationship between NA and resolving power?

higher NA → greater resolving power

Electromagnetic spectrum

• Increasing energy: long radio waves → gamma rays

• Increasing frequency: long radio waves → gamma rays

• Increasing wavelength: gamma rays →long radio waves

• visible spectrum (small range of wavelengths): increasing wavelength - 400 (violet) → 700 (red)

What do stains and dyes allow for?

enhanced contrast in light microscopy when working with thin tissue

How do stains and dyes work?

• they bind to/are absorbed by different cellular components

• provides contrast against other cellular features

• staining is primarily used with fixed tissue (can’t be done with live tissue), gives you a snapshot

What are some examples of stains and dyes?

• hematoxylin and eosin (H&E)

• DAPI

• propidium iodide (PI)

What is hematoxylin and eosin (H&E)

most common histological stain (pink cytoplasm & purple nuclei)

What is DAPI?

intercalates into the double helix of DNA, stains DNA/chromosomes blue (when excited)

What is propidium iodide (PI)?

• stains DNA red, cannot pass through membranes of viable (intact) cells (blocks PI so no access to nucleus)

• cell death marker (damage to cell membrane)

What is fixation?

• preparing tissue for imaging

• the process of using chemical methods to preserve a biological specimen

What does fixation involve/do?

• stabilizing molecular interactions (cross linking)

• denature proteolytic enzymes

• kill microorganisms

• does not work with living tissue

What are the two categories of chemical fixatives?

• dehydrating fixatives

• cross-linking fixatives

What are dehydrating fixatives & examples?

• disrupt lipids and precipitates protein molecules

• methanol, ethanol, acetone

What are cross-linking fixatives & examples?

• create covalent chemical bonds between proteins in tissue

• non covalent interactions (not as stable, degraded with time) → covalent bonds that prevent breakdown

• formaldehyde, paraformaldehyde, and glutaraldehyde

immersion fixation

• just drop tissue into fix

• great for small tissues like fly brains (use 5 to 10 times the volume of tissue being fixed)

perfusion fixation

delivering a fixative through an animal’s cardiovascular system

How do you choose a fixative?

choice of fixative depends on downstream applications

Stereomicroscopes (Dissecting Microscopes)

• two separate lens systems (light passes through each lens independently) rather than one → gives sample depth, three-dimensional appearance, aids in fine manipulation

• used for examining specimens/aiding in dissections and preparation of small tissues

• lower magnification than compound microscopes → lower resolution (but can act in real time)

• not for generating images

What are some advantages of fluorescence microscopy?

• can be used to detect specifically labeled fluorescent molecules

• multiple fluorophores can be imaged in same sample

• high signal to noise ratio → sensitive

• can image live cells without the need for fixation and staining (can catch dynamics of structure changing over time)

• enhanced contrast, high quality images, can make out different structures

What is fluorescence?

when EMR (light) is emitted by a molecule after being excited by EMR of a shorter wavelength

What is a fluorophore?

• a fluorescent chemical compound that can re-emit light upon excitation by light

• the new wavelength will always have lower energy and longer wavelength → also means a different color

• can use pseudocoloring to enhance contrast & assign colors to wavelengths

tetramethylrhodamine (TRITC)

• “trit-see”

• orange-red (544nm excitation, 570nm emission)

fluorescein isothiocyanate (FITC)

• “fit-see”

• green (488nm excitation, 516nm emission)

• excitation-emission spectrum is not all or nothing

Immunofluorescence

• use antibodies to visualize the presence of proteins and other molecules in cells and tissue, usually through a fluorescent molecule (immunofluorescence) or an enzymatic reporter bound to an antibody

• can be used in conjunction with fluorescent stains like DAPI (stains DNA)

• amt of fluorescence corresponds to amt of protein present

• requires fixed tissue

Fluorescent in situ hybridization (FISH)

• design nucleic acid “probe” with complementary sequence to target

• fix + permeable tissue

• incubate tissue with probe

• visualize fluorescence in sample (indicating locations of gene expression)

Fluorescent fusion proteins

• allow you to visualize the location and amount of a particular protein in a cell

• coding sequence for fluorescent protein is genetically “fused”to coding sequence for gene of interest

• can be used with fixed or living cells

Example of live cell fluorescence microscopy

neuron activity reporter (can test with various neurotransmitters)

Epifluorescence (wide-field) microscopy

specimen is illuminated by excitation wavelengths and emits light from excited fluorophores throughout the entire thickness

What are some advantages of epifluorescence (wide-field) microscopy (over other forms)?

• simpler and faster image collection methods than other forms of fluorescent microscopy (confocal, two-photon)

• cheaper

What are some disadvantages to epifluorescence (wide-field) microscopy?

out of focus fluorescence can cause blurry pictures, making structures difficult to resolve

Confocal microscopy

• confocal microscopes produce clear images within relatively thick tissues/specimens (blocks out of focus light)

• most common - confocal laser scanning microscopy

• excitation light passes through pinhole - limits illumination to a single plane

• out of focus illumination is blocked by the use of pinhole apertures, so only in focus emitted light is collected

• optical sectioning (images at different Z-axis positions) creates sharp, in-focus images

• 3D “stacks” can be generated using images from multiple planes

• cost more, image acquisition is slower

• still a limit of tissue depth

Two-photon (multiphoton) microscopy

• fluorophores in a thin focal plane are selectively excited by absorbing the combined energy of two photons simultaneously that cannot excite fluorophores on their own

• each photon has wavelength 2x usual excitation wavelength (1/2 the energy)

• longer wavelengths of laser illumination allow deeper penetration of fluorescence excitation

• clearer images than traditional confocal microscopes, can image thicker tissue samples

• expensive equipment

• other advantages: in vivo imaging of intact organisms, long term fluorescence imaging

• limiting to particular point in focal plane

Electron microscopy

• all light microscopy techniques are limited by the wavelength of visible light (resolution)

• electrons have much shorter wavelengths than photons (1000 fold increase in resolving power)

• ideal for imaging specific cellular structures/components (e.g., synapse, vesicles, proteins)

• expensive and requires sample prep

Scanning EM (SEM)

• detect secondary electrons scattered off the surface of the sample (bombarding surface)

• appears 3D - 3D images can be obtained using computer rendering software and multiple images

• specimen must be coated with thin film of gold or platinum (can be expensive)

• cannot be used on live cells, harsh processing conditions can cause artifacts, requires specialized equipment (rendering software)

• cannot use live cells

Transmission EM

• electron beams transmitted through ultrathin sections

• nanometer resolution

• cannot be used on live cells, harsh processing conditions can cause artifacts, requires specialized equipment

• ultrastructure of cells, synapse structure

Cryo-ET/Electron tomography

• rotate specimen to take TEM images from multiple perspectives and create 3D reconstruction

• provides 3D organization information

• cannot be used on live cells, requires intensive computation to reconstruct TEM views

• 3D cellular ultrastructure and organization, protein structure

• Cryo-ET - use cryogenic temperatures rather than fixative to preserve structure