light microscopy

history of cell biology

• 1655 robert hoke 

• called pores celluae – little rooms - (they were not cells but empty cell walls of dead plant tissue) 

• Observed plant cells 

• Anton van leeuwenhoekhoek 1674 observed bacteria (animalcules) 

cell theory

• Scheiden and schwann (1830s)  

• All organisms are composed of 1 or more cells 

• Cell is the strucutural unit of life 

• 1855 cells can arise only from division of pre exisiting cell 

functions of cells

• Cells are complex and organised 

• Possess a genetic program 

• Capable of producing more of themself 

• Aquire and utilise energy 

• Carry out chemical reactions (metabolism) 

• Engage in mechanical activities 

• Able to respond to stimuli 

how does light interact with matter

• Transmission 

• Reflection 

• Refraction 

• Diffraction 

• Scattering 

• Absorption 

why are things not visible

• Not enough photons 

Use condenser lens to focus light on sample 

• Wrong kind of photons (outside the visible spectrum) 

Use detectors that detects other wavelengths 

• Objects too small for array of photoreceptors to resolve 

Use compound lenses to magnify the sample 

• Objects do not interact with (or emit) visible light 

Use stains or labels that can be detected by eyes or cameras 

• Objects interact with light the same as the surrounding medium 

Use optics that boost contrast between components with different material properties 

• Materials or lenses bend light to cloak the object 

compound microscopes

• Use compound lenses  

• Objective lens magnifies light from the specimen to it (also compound lens) 

• Eyepiece (ocular lens) magnifies the image from the objective lens 

• Condenser lens focuses incoming light onto the specimen (more photons focuses on object) 

• By matching refractive index, higher resolution can be achieved 

• When there is refraction at the interface, less light enters the objective lens 

• By using a special oil with a refractive index, this can be remedied 

what determines resolution

• How much light is lost between specimen and lenses (more = lower resolution) 

• How much light is gathered by lenses (more = higher resolution) 

• Wavelength used for illumination (shorter = higher resolution) 

 

• Limited refraction between specimen and lens 

• Higher numerical aperture to gather more light 

• Shorter wavelength of illumination (shorter wavelength photons carry more energy) 

limit of resolution

• 200 nm for light microscopes 

• 2nm for electron microscopes 

identifying structures

• Can see some organelles without a stain due to them having different diffractions or refractions 

• Phase contrast 

• DIC (uses polarised light) 

• Can attach fluorescent molecules to molecules or proteins that label cellular structures 

• Photons of a particular wavelength carrying a particular amount of energy excite electrons in the fluorophore 

• As electrons go back to ground state, energy is released as emitted photon 

• Less energy is released as light than was absorbed (finish later 

• Use antibodies to label cellular components in immuno-labelling (bind to an antigen by recognising the epitope) 

• Monoclonal (antigens that bind to one epitope) 

• Polyclonal (mixture of antibodies that recognise multiple epitopes on the same antigen) 

fluorescent labels

• DAPI and Hoechst (small molecules that bind to DNA) 

• Fluorophore-conjugated molecules 

• Fluorescent molecules that localise to cells or organelles 

• GFP (green fluorescent protein) (can be sequenced at the beginning or end of a protein to make a hybrid tagged protein) 

how to improve resolution in fluorescence microscopy

• Improve resolution by removing out of focus light (use confocal microscope) 

• Reduce out of focus emitted light collection 

• Digital deconvolution (mathematically improve images using information about the diffraction of light)