New contrasts and label free imaging for life science

Optical Microscopy is an important research tool in the area of life sciences which continuously drives medical progress forward. Unfortunately, it is almost impossible to acquire meaningful microscopic images of biological specimen without significantely perturbing their biology, by e.g. exposing them to light, adding dyes or stains or having to prepare them in non-natural ways. A new technology, which would allow to extract meaningful microscopic images of biological specimen without significantly perturbing their biology could enable a better understanding of biological processes. This in turn could drive medical progress forward (such as the design of new drugs).

The development of contrast mechanisms in microscopy goes back to 1665, the year in which Robert Hooke´s masterpiece Micrographia was published. He described for the first time a stained object in microscopy: dyed wool. However, for almost 200 years, staining of biological objects in microscopy was rare and mostly an accident. In 1858 Joseph von Gerlach left a section of brain tissue in a dilute carmine solution overnight, and reported differential staining of the nucleus compared with little staining of the cytoplasm and intercellular space.

From that time onwards, more and more contrasting methods in microscopy have been developed. Techniques like phase contrast, differential interference contrast (DIC), polarization and most importantly fluorescence have revolutionized life science microscopy. Especially in fluorescence, this was always accompanied by a precise labelling of structures and molecules, e.g. with labelled antibodies or fluorescent fusion-proteins.

However, current contrasts and labels have significant limitations and drawbacks:

  • Staining of samples is time consuming and has to be planned and conducted way ahead of imaging. 
  • Introduction/expression of fluorescent labels always disturbs the integrity of a biological specimen, which makes the result less useful than it could be.
  • In addition, fluorescence excitation illumination further damages the specimen.
  • Most traditional and currently available contrasts are not selective and therefore only provide little knowledge about specific cellular or tissue structures. 

Hence, a new method is needed which overcomes the limitations of the established techniques.

The recent development of quantitative label-free contrasts (such as holotomography and quantitative phase imaging) are overcoming some of the above limitations, but specific acquisition requirements, time- and spatial resolution and dedicated instrument setups prevent their adoption for the majority of applications in the life sciences.

The goal is therefore to find a new microscopic imaging technology which would allow researchers to obtain more information from unlabeled specimen. The value of such a new method could be measured based on 4 criteria:

  • Performance in respect to spatial (2D and/or 3D) and temporal resolution
  • Performance in respect to information gain relative to traditional phase contrast
  • Degree of gentleness relative to fluorescence microscopy
  • Feasibility of sample preparation and imaging conditions for typical specimen in cell-, cancer- or neurobiology research


Fluorescence Imaging


Contrast-enhancing techniques
in transmitted light microscopy


Histological dyes in bright field microscopy



  • Specific labelling of structures and molecules of interest
  • Labeling of multiple molecules in the same sample
  • 3D imaging possible
  • Live imaging possible
  • No staining procedures necessary
  • Live imaging is possible
  • Provides information regarding structure and morphology of the sample
  • Specific structures of the sample can be visualized.


  • Label needs to be introduced into sample.
  • Label potentially perturbs the function of the labeled molecules, and as such the biology of the sample.
  • The light to excite the fluorescent labels is potentially harmful to the sample.
  • Since only the labeled molecules are visible, the context information regarding structure and morphology of the sample is missing.
  • It does not provide specific information about distinct structures and molecules.
  • 3D information is limited
  • Sample needs to undergo staining and frequently also fixation procedure.
  • Label potentially perturbs the function of the labeled molecules, and as such the biology of the sample.
  • Very limited live cell imaging compatibility
  • Limited 3D information gain.

Further reading

Introduction to Fluorescence Microscopy

Enhancing Contrast in Optical Microscopy


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