MacroSPIM

(Under Construction)

ADM is setting up a new instrument  based on Lightsheet Illumination dedicated to 3D-Imaging of full organs and tumours. With a strong emphasis on studies related to oncology and neurology, this new instrument allows low to medium magnification modes on large samples (about centimetre in diameter) and challenges the traditional histological sectioning of organs, for example by allowing scientists to search single metastatic cells in a full organ or to reconstruct the 3D structure of a tumour, without dissecting it.
The technique is based on sample clearing and is not suitable to living specimen.


Instrument Details

The lightsheet illumination arm is custom built with singlet cylindrical lenses, the detection arm is a Macroscope AZ100 (Nikon) with a zoom module 1x to 8x, kind loan of Izasa SA (Spain).

Sample Mounting: We use a custom glass chamber which can be easily located and removed onto the microscope's stage. Samples are embedded in Agarose blocks and placed inside the chamber, in clearing medium.

High precision microstages displace the sample in x, y, z. Axial resolution of the illumination lightsheet is adapted to the magnification to reach almost isotropic resolution at all magnifications.

Sample Preparation

Samples are extracted, fixed in PFA and dehydrated according to the type of fluorescence.
Synthetic fluorophores and Autofluorescence are easy to image with Methanol dehydration.
Fusion proteins (GFP etc...) require a longer protocol (up to 6 days) of dehydration with Ethanol.

The last step consists in embedding the sample in a clearing solution of Benzyl-Alcohol-Benzyl-Benzoate (1:2) for some hours until the sample becomes fully transparent.

Samples conservation is generally possible over weeks or even months, however special cases involving Fusion Proteins might require imaging relatively fast after embedding in BABB.

Applications and Examples

We use this technique in two major fields of Research: Oncology and Neurology.

The ability to image samples of sizes ranging from 0.5 to 15mm brings a new alternative to histological sectioning. With a resolution close to 4microns (in Z) at intermediate magnifications (around 10x) and about 20microns at Low magnification (1x), full organ imaging can be performed in two steps: a large view taken in a single stack (sometimes 2 for large samples) and a direct zooming capability to record tiled stacks (3D mosaics) of the sample down to cellular resolution.

Autofluorescence brings insight on cellularization, tissue morphology and health.
Synthetic fluorescent markers can be used, provided labelling is properly conducted. For example blood vessels can be labeled with conjugated lectin, but staining needs to be done before fixation.
Fusion proteins work well but dehydration protocols depend on the localization of the probe, higher dehydration leads to lower intensity levels, leading to a compromise between clearing efficiency and fluorescence intensity.

- More info to come soon - contact us for more details microscopy(at)irbbarcelona.org




Mouse Lung fixed in PFA and Dehydrated in Methanol,
Autofluorescence Imaging with a Macroscope under
epifluorescence illumination @ 1x Mag.

Mouse Lung fixed in PFA, Dehydrated in Methanol,
Embedded in BABB for clearing.
Autofluorescence Imaging with a Macroscope under
epifluorescence illumination @ 1x Mag.

Mouse Lung fixed in PFA, Dehydrated in Methanol,
Embedded in BABB for clearing.
MacroSPIM imaging of Autofluorescent at 1.2x Mag,
Approx depths: 500, 800, 1200, 1500 µm.