Lecture 1: Light microscopy

Why may something not be visible

Not enough photons

Wrong kind of photons (outside visible spectrum)

Objects smaller than visible wavelengths of light

Objects do not interact with or emit visible light

Object interacts with light the same as surrounding medium

Materials bend light to cloak object

How does a convex lense work

Refracts light in a way which increases the angle between the top and bottom of the object, spreading out the light hitting the detector and making the object appear larger

What type of lenses do microscopes use

Compound lenses

Light from the specimen hits the

Detector

The eyepiece lens does what

Magnifies image from objective lens

What are the 3 lenses in a compound microscope

Objective

Eyepiece

Condenser

What does the condenser lens do

Focuses light onto the specimen, maximising the number of protons hitting the specimen

How can you overcome there not being enough photons to make an image visible

Use condenser lens to focus light on sample

What if its the wrong kind of photons

Use detectors sensitive to other wavelengths

Objects to small for array?

Use compound lenses to magnify sample

Resolution definition

Smallest distance at which 2 objects can be seen as separate

What determines resolution

How much light is lost between specimen and lens

How much light is gathered by the lenses

Wavelength used fro illumination

Matching the refractive index of two mediums will

Give a higher resolution

What substance can be used to prevent loss from refraction on a microscope slide

Special oil with the same refractive index as glass

What is the angular aperture

The half angle of a cone of light netting the objective lens, dictates how much of the light is collected by the objective lens

Higher the angle, more ight gathered

What is the numerical aperture

Number that describes the light-gathering ability and resolving power of an objective lens

Hig number = more light lans gathers

What is the resolution of a light microscope

200nm

What can you do if objects interact with light the same as surrounding medium

Use optics that boost contrast between components with different properties (Phase contrast or DIC)

What is phase contrast

Boosts contrast resulting from differences in refractivity between cells and cellular structures

What is DIC

Uses polarised light to boost contrast from diffraction of light in the sample

What can you do if an object doesn’t emit visible light

Use stains or radioactive labels detected by eyes or cameras

What is a fluorophore

Molecule that emits energy in the form of photons with a longer wavelength when hit with shorter wavelength protons (a type of transformer)

Can be attached to molecules to label cellular structures

How does fluorophore work

Photons excite electrons in fluorophore and energy is released when they fall back to ground state (less is released than light absorbed due to some lost as heat, so emitted photons have longer wavelengths)

How does fluorescence microscopy work, what filters are passed through and what is a dichroic mirror

Light passes through objective lens to and from the sample through excitation and emission filters

Dichroic mirror reflects shorter wavelengths onto sample and longer pass to detector

How to get better resolution in fluorescence microscopy

Reduce out of focus fluorophore excitation

Reduce out of focus emitted light collection

How can you do the latter

Use a two photon confocal microscope

What else can be used to label cellular components

Antibodies - called immunolabelling

What is direct imuno-labelling

Where the primary antibody binds to the epitopes (part of antigen that antibody recognises)

Indirect immuno-labelling?

A secondary antibody binds to the primary antibody and the antigen for the secondary antibody is the immunoglobin of the primary antibody

What does multiplexed fluorescent labelling mean

Combination of dyes and antibodies to highlight different parts of the cell