Category Archives: techniques

IN-FOCUS: Cutting through the fog: reducing background autofluorescence in microscopy.

Autofluorescent bone sample

Above: Autofluorescence from mixed connective tissues imaged by confocal microscopy (left). The autofluorescent emissions can be spectrally-resolved through wavelength scanning (right). Excitation at 488nm.

Whilst autofluorescence from endogenous fluorophores can reveal much about the biochemical composition of a sample, it can also hamper the microscopic detection of targeted fluorochromes if they emit light at the same wavelengths as endogenous fluors. Indeed, without proper controls, complex background autofluorescence can lead to misinterpretation of image data and generation of false positive results.

Autofluorescence derives from multiple sources within the sample – the main culprits are  NADH and NADPH, lipofuscins, flavins, elastin and collagen (and lignin and chlorophyll in plants). The excitation and emission ranges of the worst offenders have been shown below. It follows that tissues with high collagen and elastin contents, e.g. skin, tendon and cartilage, autofluoresce very brightly; as do tissues that are rich in metabolic breakdown products such as lipofuscin, e.g. liver, spleen etc.

Autofluorescent data

Adding to the problem is the effect of chemical fixatives (e.g. formalin, glutaraldehyde etc) and solvents used to preserve tissue architecture for microscopy: the cross-linkages generated by these chemicals increase autofluorescence, which can be worsened further by long-term storage of the fixed processed tissues.

So, dear reader, here’s some simple advice on steps that you can take to address this common problem:

1. Include an unlabelled control to evaluate the level of autofluorescence within your sample.

  • Observation of unlabelled samples through RGB fluorescent filters (note their transmission characteristics) will help identify where in the visible spectrum the autofluorescent signal is brightest.
  • Spectral (lambda, wavelength) scanning will allow you to precisely identify the fluorescent emission spectra from endogenous fluorochromes and can help separate their emissions from those of your fluorochrome (see above figure).

2. Select fluorochromes that are outside the range of the autofluorescence.

  • If the autofluorescence signal is high in the blue, then move into the green; if it’s high in the green, move into the red – or better still, the far red (if your system can detect in this range).
  • Use modern fluorescent probes (e.g. Alexa Fluor, Dylight, or Atto range) instead of first generation fluorochromes.  They are brighter, more photo-stable and have narrower excitation and emission bands. They are also available in variants that span the near UV, visible and far red range of the spectrum, affording you plenty of choice.

3. Use a microscope with filters optimised for your choice of fluorochromes.

  • Band-pass filters which collect emissions within a specific range may be more useful than long-pass filter sets which collect all emissions past a certain wavelength. The narrower the range of the band-pass filter, then the better it can separate fluorophores with close emission spectra.

4. If the autofluorescence is unevenly distributed within your sample, use targeted microscopy to avoid it.

5. If you can’t avoid the autofluorescence, then take measures to remove or reduce it.

  • Analyse the pixel intensity distribution within your image and try thresholding out the lower intensity autofluorescence signal.
  • Pre-bleach your samples in a light box using a high intensity illumination source prior to fluorescent labelling (see below reference)
  • Treat samples with a chemical reagent (e.g. sodium borohydride, Sudan black B, ammonium ethanol etc) to reduce background autofluorescence (see below reference)

6. If all else fails, consider the following:

  • use cryoprocessed material as an alternative to chemical fixation and paraffin wax processing.
  • avoid long term storage of material/archival tissue samples.
  • try a different detection modality (e.g. immunoperoxidase instead of immunofluorescence)

AJH

Further reading

Wright Cell Imaging Facility. Autofluorescence: Causes and Cures

 

IN-FOCUS: Better to burn bright than to fade away: reducing photo-bleaching in fluorescence microscopy.

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Above: Photo-bleaching (fading) occurs when a fluorochrome permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification. 


Hands up if you’ve spent hours preparing a sample for fluorescence microscopy only to see the signal disappear before your eyes upon excitation? Frustrating eh (unless, of course, FRAP is your objective)? Well here’s some simple and sound advice on how you can minimise photo-bleaching and get the best out of your samples under the fluorescence microscope.

1. Visualise your samples immediately after fluorescent labelling – this is when they are at their brightest.

  • If this is not possible then loosely wrap your samples in aluminium foil and keep them in the dark at 4oC until you get the opportunity to image them.

2. Minimise their exposure to light in order to reduce photo-bleaching.

  • visualise your samples under low light conditions.
  • use transmitted light to find a region of interest (ROI) and then switch to epifluorescence observation – avoid dwelling too long on the ROI.
  • step down the intensity level of excitation light or insert a neutral density filter into the light path.
  • set up imaging parameters on a neighbouring region and then return to the ROI for image capture.
  • use image binning to reduce exposure time.
  • use the microscope shutter to switch off the light source between images.
  • create a photo-bleach curve from a timed series of images. This can be used to normalise for loss of fluorescence intensity.

3. Switch to a mounting medium with anti-fade protection e.g. Vectashield, Prolong Gold/Diamond, SlowFade Gold/Diamond. These work by reducing the oxygen available for photo-oxidation reactions, thus reducing photo-bleaching. N.B. Many of these are available with a nuclear counterstain (e.g. Dapi) included in the formulation. Alternatively, make your own anti-fade reagent (instructions below).

4. Switch to brighter, more photo-stable fluorochromes. First generation fluorochromes such as FITC and TRITC photo-bleach readily (and are pH sensitive) thus should be replaced with modern dyes such as the Alexa Fluor, Dylight, or Atto  range of fluorochromes, which are much brighter and far more photo-stable.

Good luck!

AJH

 

Further reading

IN-FOCUS: Development of a 3D printed pollen reference collection.

pollen montage 1
pollen montage 2

 

 

 

 

 

 

 

 

 

 

 

 

 

Above: surface-rendered confocal reconstructions of pollen samples (left) and their corresponding 3D printed models (right).

Isn’t the World Wide Web a wonderful thing? Not so long ago I wrote a short blog explaining how we had developed methodology to convert volume datasets from the confocal microscope into 3D printed models – perfect solid scale replicas of samples the size of a pollen grain etc. Well, shortly afterwards I received an email from someone who had not only read the blog but, serendipitously, wanted to do this very thing! What is more, she was located not a million miles away: in fact, little more than 400 yards down the road from us, working as a researcher within Cardiff University’s School of History, Archeology & Religion. Please excuse the pun, but it really is a small world!

Rhiannon Philp is an archaeologist – or palynologist to be precise – someone who studies ancient pollen grains and spores found at archaeological sites. Pollen extracted from archeological digs can be used for radiocarbon dating and for studying past climates and environments by identifying plants growing at the time. Rhiannon is using this information to develop an understanding of prehistoric sea level changes in South Wales as part of the Changing Tides Project.

Rhiannon asked if we could generate a reference collection of 3D pollen prints that could be used for teaching and outreach activities as part of a new Archaeology engagement project called Footprints In Time. Indeed, some of her pollen samples were from sites containing both human and animal footprints made over 5000 years ago!

You can see some of our results above: on the left are the surface-rendered confocal volume reconstructions and, on the right, their corresponding 3D printed facsimiles – courtesy of the BIOSI 3D printing facility.

If you’re at the National Eisteddfod in Abergavenny this week (29th July – 6th August), then please pop by to see Rhiannon’s stall within the Cardiff University tent – all of the models will be on display there, together with a lot more.  Any further interest, then please get in touch.

AJH

 Further reading:

IN-FOCUS: Bigging it up: 3D printing to change the shape of microscopy.

3d pollen

Virtual to reality: a surface-rendered digital image of a single pollen grain generated by confocal microscopy (left) is 3D printed into a 2000x scale replica model (centre & right).

Imagine being able to generate a highly accurate, solid scale replica of the sample that you are visualising down the microscope; a perfectly-rendered pollen grain, or blood cell, or microscopic organism, but big enough to hold and examine in your hand.  It would allow much better 3D conceptualisation of the sample, particularly for blind or visually-impaired individuals, and would have enormous utility in teaching and in engagement activities, and what researcher wouldn’t want a tangible, physical embodiment of their research to help explain their work (and impress their colleagues) at scientific meetings? Sounds like the stuff of science fiction doesn’t it? Well, not any more. Thanks to 3D printing technology (and the help of Dr Simon Scofield‘s lab) we have started taking volume datasets from the confocal microscope out of the virtual world and making them a reality. If you would be interested in generating a highly accurate scale model of your favourite biological sample (or would simply like to handle a giant pollen grain!) then please feel free to get in touch.

AJH

 Further reading: