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    Clearing biological samples: issues, methods and applications

    3D imaging of complex organs or tissues is a major issue in photonic microscopy applied to biology.

    However, the phenomena of light diffusion and absorption within such environments are an intrinsic obstacle to the deep exploration of thick samples, despite the advent of imaging techniques such as multiphoton microscopy or light sheet Single Plane Illumination Microscopy (SPIM). To meet the needs of 3D imaging of large biological samples, a considerable number of sample transparency techniques have been developed, some based on organic solvents, others based on aqueous solutions, with different properties and various applications. The community of biologists is very interested in this process of clearing but often overwhelmed by the multitude of techniques described in the literature since more than 5 years. We propose you to see more clearly by coming back to the origins of the method and then describing its principle and some direct applications.


    Why do we need to clear biological samples?

    In biological samples, light scattering and absorption make imaging very difficult. Indeed, since the different molecules forming the biological material all have a different refractive index, the light is highly diffused, from the first micrometers reached. As a result, from few years ago to now, the thick optically dense biological samples were cut into thin slices to allow volume imaging. Over the past decade, fantastic progress has been made to make specimens transparent to visible light, enabling microscopic visualization of entire organs.

    Legend: cleared mouse brain with Clarity technique.
    Image from Kwanghun Chung and Karl Deisseroth, Howard Hughes Medical Institute/Stanford University


    Clearing techniques

    Sample clearing processing has 3 objectives:

    • Remove (or reduce) cell opacity due to membrane lipids without damaging cells
    • Discolour pigments
    • Homogenize refractive indices to limit refraction and diffusion phenomena

    Each method will have advantages and disadvantages in the preservation of tissue structures, volume, endogenous fluorescence, etc. These methods of transparency can be classified into two broad categories:

    Organic solvent based techniques:
    The biological sample is first dehydrated, delipidated and then discolored, as if we use bleach. Then the refractive index of the sample is homogenized with organic solvents. The most common techniques are BABB [Spalteholz, 1914], 3DISCO [Ertürk et al., 2012], iDISCO+ [Renier et al., 2016] and uDICO [Ertürk et al., 2016]. These techniques are reliable, reproducible and perfectly adapted to clearing large volumes. However, they are highly toxic due to the use of organic solvents, and can induce artifacts such as shrinkage or fluorescence quenching of endogenous proteins such as GFP for example.

    Techniques based on hydrophilic agents:
    This category can be divided into three subgroups:

    1. Simple immersion.
      Here, the refractive index is adapted to the sample by dipping it simply in a suitable medium (TDE, Fructose - SeeDB method [Ke et al., 2013; 2016], Glycerol). This technique is simple to perform, works perfectly for small samples, does not induce shrinkage, is compatible with immunofluorescent staining and keep endogenous fluorescence. On the other hand, transparency is rarely complete, is very slow, and does not work on large volumes.
    2. Hyper-hydration.
      The lipids are removed using detergents and then, sucrose or urea allow the hydration and the transparency of the samples. The usual techniques are: CUBIC [Susaki et al., 2014; 2015], Scale [Hama et al., 2011; 2015], Fruit [Hou et al., 2015]. These methods are effective, do not quench the endogenous fluorescence but they are slow and induce a slight expansion of the sample.
    3. Hydrogel inclusion.
      The sample is embedded in hydrogel and the lipids are removed with detergent or electrophoresis. Homogenization of the refractive index is achieved using media such as TDE, FocusClear, RIMS, or Glycerol. The most used techniques are CLARITY [Chung et al., 2013; Tomer et al., 2014], PACT / PARS [Yang et al., 2014; Treweek et al., 2015], or SWITCH [Murray et al., 2015]. These methods are suitable for the clearing of small whole animals, do not induce quenching of endogenous fluorescence but are rather slow and sometimes require special equipment.


    Imaging of clearing samples

    Imaging of biological samples in volumes requires microscopy techniques with the following particularities:

    • Optical sectioning in z (not a wide field microscope)
    • Large field of view (exit too high magnification lens)
    • A localized excitation at the focus of the objective would be a plus not to degrade the sample too quickly.

    Then, to be able to correctly image the transparent tissues, it is necessary to keep them dipped in their clearing medium allowing the homogenization of the refractive index. Indeed, out of this environments, these samples become opaque again very quickly. Taking into account this constraint, many tips to allow the preparation of samples for imaging purposes have been conceived (chambers printed in 3D for example). Two optical microscopy techniques are particularly suitable for in toto imaging of thick tissues: confocal / multiphoton modalities and light sheet microscopy.


    Legend: Whole mouse kidney cleared with BABB method ; red : blood vessels and podocytes in nephrons/griffonia simplicifolia agglitinin ; green : tubular cells/peanut agglutinin (lectins)
    Multiphotonic imaging technique; Marco Pontoglio / Thomas Guilbert / IMAG’IC, Institut Cochin

    Confocal microscopes are widely used in research laboratories. Point-scanner or spinning-disk, the image acquisition speed is fast enough, the available range of lenses is such that it is possible to combine large field of view and good resolution, and optical sectioning is performed at the detection. It should be noted that there are now on the market immersive lenses that can be directly dipped in certain mediums of transparency. However, if the transparency of the sample is not perfect, the phenomenon of photon scattering in biological material will lead to a deterioration of the quality of the images as the z progresses. Bulky sample imaging will also be problematic since the excitation of fluorescence occur in volume, increasing the risk of photo-bleaching. Finally, still for a large sample, the number of images needed to cover the entire sample can become considerable, as can the weight of these (several hundred GB) thus making it difficult to reconstruct 3D volume, nor the resulting image processing.


    Multiphoton microscopy has grown considerably since the beginning of the 2000s. Added to the advantages of confocal microscopy, intrinsic optical sectioning linked to the technique itself, as well as the excitation in the infra-red, which gives it a lead in the imaging of thick samples field. However, issues related to the number and the volume of images remain the same as with confocal microscopy.

    Selective Plane Illumination Microscopy is a technique that has undergone many improvements in recent years, although its creation back to the 1900s (developed by Siedentopf and Zsigmondy). With this technique, an optical sectioning is obtained no longer as a point as in multiphotonic excitation, but over an entire surface by guiding the exciter laser beam through a cylindrical lens. A second objective, to capture images, makes it possible to acquire the fluorescence emitted by the sample on a surface corresponding to its field of view, with a resolution in z - the thickness of the light sheet - of a few μm. Apart from the systems developed in the laboratory, the most widespread system is a macro-SPIM (12x max magnification), and if we lose in resolution compared to the techniques described above, we gain considerably in acquisition time.


    As we have seen, clearing biological samples is in full swing, new methods appearing every year. We must kept in mind that the biological question remains fundamental when we try to use this type of approach, because depending on the sample to be observed and the imaging system available in labs, there will necessarily be a more appropriate technique than the others, but which will, in no case, be "magical".



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