Imaging with Microscopy

 

 

 

 

Whole slide imaging of human lung tissue showing loss of function of the cancerous tissue (left), and the normal tissue (right). This image is formed of 10,000 images that have been automatically stitched together using the Leica SCN400

 

When imaging fluorescently tagged proteins onto primary (human) tissue there is a certain amount of detected light that is from the tissue, known as autofluorescence. On excitation by a UV light source this light is derived from organelles such as mitochondria and molecules such a NADH, flavins, collagen and elastin. The spectra (light quality) can vary depending upon the tissue, for example skin and renal tissue have a high level of fluorescence with different spectral properties.  Shown above is the original data set from the microscope and the processed data when the autofluorescence has been subtracted via the Perkin Elmer Vectra system

 

 

actin labeled with Alexa 488 under gSTED

Fluorescent Histone H3 in nucleus of a HeLa cell visualised under confocal microscopy (left) and super resolution microscopy using gated stimulated emission depletion, gSTED (right). Both sides of the above image has been captured by the same methods, no image processing has been applied, the only varying factor is the method of laser illumination. As the image clearly demonstrates, there is an overall improvement in clarity and resolution.

Image: S Bagley, AIFC.

 

actin labeled with Alexa 488 under gSTED

To demonstrate the super resolution technique, actin within the cell has been labeled with Alexa488-Phalloidin and then imaged under gated stimulated emission depletion (gSTED) microscopy. Each pixel of the image is 19nm in size and the whole image took twelve seconds to capture. After image capture, deconvolution has been applied with the Huygens software resulting in an overall resolution of around 28-30nm. The spotted nature of the image is probably due to the size of the labeling complex which blocks additional binding sites. 

Image: S Bagley, AIFC.

 

Phenotype  (from the greek 'to show' and 'type')  is an array of observable features such as morphology (shape), behaviour, products of behaviour and cellular products (proteins amount, localisation and co-localisation). Phenotype is a consequence of the interaction of expressed genes (genotype) and the environment. High Content Screening (HCS) allows the automated imaging of phenotype per cell, in multiple sub-populations, across different drug treatments or environmental conditions.  HCS permits the assessment, mathematical modelling and statistical analysis across many millions of cells, in a standardised, calibrated manner for applications such as drug discovery. This technique assesses all cells and so reduces the possibility of observational bias. The image above demonstrates the power of the technique by showing a single field of view and multiple wells on a multi-well plate, with differing conditions

 

Using confocal, two-photon and high content screening techniques the assessment of spheroids and organoids has become increasingly important for the assessment of function and drug discovery. Organoids grown in the laboratory from primary tissue are miniaturised versions of an organ produced in vitro which in demonstrates realistic micro-anatomy this permitiing 3D culture assessment.  The data (above) from one of these 3D structures has been captured under confocal illumination and then 3D modelled 

 

So to accelerate translational research, High Content Screening (HCS) of 3D cultures such as spheroids and organoids are utilised for viability and morphology characterisation. By growing these cultures on 96 and 384 multi-well plates performing phenotypic assays over time becomes a routine process within the laboratory. within the facility we have automated widefield and confocal illumination systems available so to aid assessment in a GLP standardised setting.

 

MDCK cells, grown in a collagen matrix, where they form hollow spheres, called cysts. When the growth factor HGF is added to the cysts, they start to sprout, with actin-rich projections emerging from the surface of some of the cells. After one or two days, some of these cells undergo EMT, and re-orientate their axis of cell division, forming long chains of cells which can start to hollow out to form hollow tubules. This recapitulates stages in normal kidney development, and is a useful model for studying EMT and oriented cell division, and the way in which these might contribute to tumour progression. 

Image: Andrew Porter, Cell Signalling group.

 

cell division

A mitotic spindle in fixed MDcK epithelial cells, which have been stained with an anti-beta tubulin antibody (green), DAPI (blue) and an anti-centromere antibody (crest, red) which recognises the kinetochores of the chromosomes. 

Image: Cell Signalling group.

  

spb movie stills

Different structures can be labeled with fluorescent proteins or antibody/fluorescent species to allow the study of biological structures in a live cell state consequently by utilising a Sedat filter set, a variety of discriminatory illumination parameters can be set up to image, for example, the interaction of specific proteins at key stages of the cell cycle. In this example Green Fluorescent Protein, dTomato and mCherry have been utilised to image atb2, sid4 and cnp1.This data shown is from two distinct time points from forty minutes of sampling. This data was originally part of the publication in Microscopy Today (2006) 33:112-116 

Image: A.Grallert and I.M.Hagan, Cell Division group.

  

dna damage

A 355nm laser which induces DNA damage has been mounted upon a spinning disk confocal. Here images have been taken every 50 msec for two minutes and a selection of the time points are shown above. A YFP tagged component of the DNA repair process is seen to migrate to the point of laser irradiation which has induced DNA damage as the repair process initiates. After a minute the intensity of the ablated line reduces as the DNA damage has been repaired. This equipment has been designed to study mutations in the DNA repair process and study the consequences to the cell. 

Image: D Ahel, formerly of DNA Damage Response group.