Cellular/Molecular imaging

Imaging in biological sciences

Use of microscopy and imaging to visualise and quantify biological structures and processes in space and time

Key challenge in biological imaging

Biological systems are intrinsically complex and noisy compared to physical sciences

Reason biology has higher sample error

Biological systems cannot be simplified into closed systems easily

Advances enabling modern imaging

Developments in molecular biology biochemistry optical imaging and protocols over past 30-40 years

Resolution of the human eye

Approximately 100-200 microns

Limitation of human eye resolution

Cannot see individual cells or subcellular structures

Robert Hooke contribution

First described cells using cork tissue

Origin of term "cell"

Named because cork compartments resembled monks' cells

Anton van Leeuwenhoek contribution

First observed bacteria which he called animalcules

Walter Flemming contribution

First observed cells dividing (mitosis)

Importance of microscopy in biology

Allows visualisation of cellular building blocks and dynamic processes

Why omics data alone is insufficient

Provides little or no spatial or temporal information

Problem with proteomics and genomics samples

Samples are destroyed and analysed as static snapshots

Why imaging is essential for dynamics

Biology is inherently dynamic over time

Spatial scales in biology

Nanometres to metres

Temporal scales in biology

Milliseconds to days or longer

Imaging dynamic range requirement

Must cover wide spatial and temporal scales

Examples of whole-organism imaging

PET MRI CT

Examples of nanoscale imaging

Electron microscopy atomic force microscopy

Light microscopy resolution limit

~250 nanometres (diffraction limit)

Recent advance in light microscopy

Super-resolution techniques reaching 10-20 nanometres

Main focus of lecture

Light microscopy and fluorescence imaging

Basic components of a light microscope

Light source condenser stage objective eyepiece

Common light sources in modern microscopes

LED tungsten halogen mercury lamps

Function of condenser and field diaphragm

Focus and condition light onto specimen

Function of objective lens

Collects light from specimen and determines resolution

Eyepiece magnification

Typically 10-20x

Where cameras are mounted on microscopes

Microscope head or imaging port

Most important challenge in microscopy

Resolution and contrast not magnification

Why magnification alone is insufficient

Does not improve ability to distinguish close objects

Definition of resolution

Ability to distinguish two separate objects

Airy disk definition

Diffraction pattern produced by a point light source

Point spread function (PSF)

Three-dimensional diffraction pattern of a point object

what happnes to objects smaller than the resolution limit

objects smaller than the resolution limit are still visisble but cannot be resolved; they appear as a diffraction-limited blur (PSF) rather than their true size

Resolution comparison XY vs Z

Axial (Z) resolution is ~2x worse than lateral (XY)

Key contributors to resolution theory

Ernst Abbe and Lord Rayleigh

Rayleigh resolution equation

0.61 × wavelength ÷ numerical aperture

Variables affecting resolution

Wavelength of light and numerical aperture

Does magnification affect resolution

No

Definition of numerical aperture (NA)

Measure of light-gathering ability of an objective

Higher NA effect

Better resolution

Where NA is found on objective lens

Number after magnification (e.g. 40x/0.95)

Example wavelength used in calculations

488 nm

Why two objectives with same magnification differ in resolution

Different numerical apertures

Diffraction limit consequence

Objects smaller than limit appear same size

Why sub-diffraction particles appear identical

PSF dominates image size

Importance of knowing objective limits

Avoid misinterpreting object size

Main contrast problem in cells

Cells are mostly water and transparent

Example of low contrast imaging

Brightfield cheek cell image

Unlabelled contrast methods

DIC phase contrast polarised light microscopy

Era of contrast method development

1950s-1960s

DIC contrast mechanism

Polarised light prisms and refractive index differences

DIC advantage

No loss of resolution

DIC disadvantage

Expensive and complex optical setup

Phase contrast mechanism

Phase rings advance and retard light phase

Phase contrast advantage

Cheap and widely available

Phase contrast disadvantage

Reduced resolution

Polarised light microscopy requirement

Birefringent structures

Polarised microscopy disadvantage

Sample dependent and expensive

Use of chemical dyes in microscopy

Increase contrast by staining cellular components

Hematoxylin stains

DNA (blue)

Eosin stains

Proteins (red)

Limitation of histological stains

Low specificity and contrast

Number of proteins per cell

10,000-100,000

Number of protein complexes per cell

~6,000

Number of major organelles per cell

~13

Limitation of brightfield imaging

Cannot distinguish specific subcellular structures

Benefit of fluorescence imaging

High specificity and contrast

Examples of fluorescently labelled structures

Nucleus mitochondria actin cytoskeleton

Fluorescence excitation process

Absorption of light excites electrons

Fluorescence emission process

Return to ground state releases lower energy photon

Stokes shift definition

Difference between excitation and emission wavelengths

Typical fluorophore lifetime

0.1-10 nanoseconds

Factors affecting fluorescence lifetime

Environment pH temperature molecular proximity

Technique using lifetime information

Fluorescence lifetime imaging microscopy (FLIM)

Excitation spectrum definition

Wavelengths that excite fluorophore

Emission spectrum definition

Wavelengths emitted by fluorophore

Requirement for useful fluorophore

Good separation between excitation and emission

Example of DNA stain

DAPI

DAPI binding target

DNA helix

Why overlapping spectra are problematic

Difficult to separate excitation and emission

Purpose of excitation filter

Selects specific excitation wavelengths

Purpose of emission filter

Selects emitted fluorescence wavelengths

Function of dichroic mirror

Reflects excitation light transmits emission light

Filter cube components

Excitation filter dichroic mirror emission filter

Multi-colour fluorescence requirement

Spectrally distinct fluorophores

Number of colours possible

Typically 3 or more

Three methods to label samples

Synthetic dyes antibodies fluorescent proteins

Synthetic fluorophore example

DAPI

Chemical probe example

Phalloidin

Source of phalloidin

Death cap mushroom

Phalloidin target

Actin cytoskeleton

Why phalloidin is toxic

Disrupts actin dynamics

Benefit of conjugated probes

Choice of fluorophore colour

Resource for dyes and probes

Molecular Probes Handbook (Thermo Fisher)

Antibody specificity

Recognises specific antigens

Primary antibody definition

Antibody that binds antigen

Directly labelled primary antibody advantage

Simple and specific

Direct labelling disadvantage

No signal amplification

Secondary antibody definition

Antibody that binds primary antibody