Tips for imaging cleared samples Pablo Ariel, Ph.D. Microscopy Services Laboratory Department of Pathology and Laboratory Medicine

BIG samples (>2mm diameter) DEEP imaging (>2mm deep) Assumptions: BIG samples (>2mm diameter) DEEP imaging (>2mm deep)

Workflow Sample preparation Sample mounting Sample imaging Data visualization Data management Chung et al, Nature 2013 Ertürk et al, 2012 Dobosz et al, Neoplasia 2014 Renier et al, Cell 2016 Data analysis

Before imaging During imaging

Before imaging During imaging

Trim based on your biology Typical: Studying circuits deep in brain; hemisphere as good as both sides Smaller samples clear faster, use less antibody, faster staining, easier to image, can be cut to size

Trim based on your biology Typical: Studying circuits deep in brain; hemisphere as good as both sides Smaller samples clear faster, use less antibody, faster staining, easier to image, can be cut to size

Trim based on your biology Typical: Studying circuits deep in brain; hemisphere as good as both sides Smaller samples clear faster, use less antibody, faster staining, easier to image, can be cut to size

Trim based on your biology Typical: Studying circuits deep in brain; hemisphere as good as both sides Smaller samples clear faster, use less antibody, faster staining, easier to image, can be cut to size

Trim based on your biology Smaller samples: stain faster require less antibody clear faster are easier to image Typical: Studying circuits deep in brain; hemisphere as good as both sides Smaller samples clear faster, use less antibody, faster staining, easier to image, can be cut to size

Clearing – don’t reinvent the wheel Use a clearing method that works For a question similar to yours Not just in the lab that created it With minimal equipment and fuss (avoid $$$) Good results with iDISCO+ on: brains, kidneys, liver, heart, embryos, intestine…

Labeling – (far) redder is better AlexaFluor 488 Ex 488; Em 525/50 AlexaFluor 568 Ex 561; Em 595/40 AlexaFluor 647 Ex 640; Em 680/30 1 mm Autofluorescence Scattering Absorbtion Renier et al, Cell 2014

Before imaging During imaging

What level of detail do you need? detail you want

Spacing is more important than size

Example: Labeled axons (0.2mm)

Example: Labeled axons (0.2mm) look bigger without high res optics

Problem: 2 objects close together

Problem: 2 objects close together ?

Problem: 2 objects close together ?

Spacing more important than size

Spacing more important than size

Examples of biological questions Subcellular scale Dense labelling High resolution sub-um(ish) Discerning subcellular details (spines, organelles) Counting all cells in densely packed tissue Tracing individual axons in densely labeled samples Cellular scale Sparse labelling Medium resolution um(ish) Subcellular details: counting spines, boutons; is subcellular localization of a protein different in different areas of the tissue Label all axons exiting region, trace all of them. Locate cells of interest in large pieces of tissue Trace neural circuits Trace sparsely labeled axons Analysis of vasculature

Different microscopes for different questions Subcellular scale Dense labelling High resolution sub-um(ish) Some laser scanning systems: Slow, potential for high bleaching, even resolution across field of view Cellular scale Sparse labelling Medium resolution um(ish) Some light-sheet systems: Very fast, low bleaching, uneven resolution across field of view

Different microscopes for different questions Subcellular scale Dense labelling High resolution sub-um(ish) Some laser scanning systems Need very special objectives Long working distance High resolution (high NA) Large field of view Matched to RI of clearing solutions Big (need special stands) $$$

Objectives for high resolution volumetric imaging $$$

Objectives for high resolution volumetric imaging FOV

Objectives for high resolution volumetric imaging FOV

Objectives for high resolution volumetric imaging RI DBE = 1.56

Custom sample holders: Keep sample still Minimize immersion fluid Implementation challenges Immersion fluid: $$ corrosion evaporation RI Custom sample holders: Keep sample still Minimize immersion fluid Allow space for huge objectives

Fundamental constraints of scanning techniques Low Speed (though ribbon scanning is much faster) High Bleaching For cellular and/or sparse labelling: enter light-sheet…

Features of light-sheet microscopy Objective z x Sample with fluorophores Laser sheet Detection orthogonal to sheet

Compared to scanning, light-sheet is Faster Less damaging Modified from Leica Microsystems

Ultramicroscope Objective Sample Lavision Biotec UltraMicroscope II

To optimize, know the key limitations Sheet shape Better Worse Radial aberrations Radial are zoom dependent: due to lens imperfections and 3 sheets Sheet shape affects Z dramatically. Is zoom dependent. Light propagation: also in Z, though less than in X Light propagation

Sheet shape x y z

Sheet shape Major problem in Z Bigger problem at low mag Low mag FOV 10X higher mag FOV x y z

Sheet shape Options? Major problem in Z Bigger problem at low mag Low mag FOV 10X higher mag FOV x y z

Trade Z resolution for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade Z resolution for evenness: lower sheet NA x y z

Trade Z resolution for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade Z resolution for evenness: lower sheet NA x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: crop + tile x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: crop + tile x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: crop + tile x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: crop + tile x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: crop + tile x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: crop + tile x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: crop + tile x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: Dynamic focus x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: Dynamic focus x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: Dynamic focus x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: Dynamic focus x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: Dynamic focus x y z

Trade time for evenness: Sheet shape Major problem in Z Bigger problem at low mag Trade time for evenness: Dynamic focus x y z

Sheet shape Speed Trade Z res for evenness: lower sheet NA Major problem in Z Bigger problem at low mag Speed Trade Z res for evenness: lower sheet NA Trade time for evenness: Crop + Tile Dynamic focus Quality

Radial aberrations Causes: 3 sheets, tilted in Y Objective aberrations Reason for 3 sheets tilted in Y Problem with 3 sheets tilted in Y

Radial aberrations Causes: 3 sheets, tilted in Y Objective aberrations Reason for 3 sheets tilted in Y Problem with 3 sheets tilted in Y

Radial aberrations Causes: 3 sheets, tilted in Y Objective aberrations Reason for 3 sheets tilted in Y Problem with 3 sheets tilted in Y

Radial aberrations Causes: 3 sheets, tilted in Y Objective aberrations Reason for 3 sheets tilted in Y Problem with 3 sheets tilted in Y

Radial aberrations Options? Causes: 3 sheets, tilted in Y Objective aberrations Options?

Trade shadows for evenness: Radial aberrations Causes: 3 sheets, tilted in Y Objective aberrations Options? Trade shadows for evenness: use single sheet

Radial aberrations Options? Trade shadows for evenness: Causes: 3 sheets, tilted in Y Objective aberrations Options? Trade shadows for evenness: use single sheet Trade detail for evenness: zoom out

Radial aberrations Options? Trade shadows for evenness: Causes: 3 sheets, tilted in Y Objective aberrations Options? Trade shadows for evenness: use single sheet Trade detail for evenness: zoom out Trade FOV for eveness: crop

Light propagation Options? Not a problem in good samples (well cleared, clean staining, no bubbles) Options?

Light propagation Image with 2 sheets Not a problem in good samples (well cleared, clean staining, no bubbles) Image with 2 sheets

Optimization examples

Brain vasculature - high NA, 3R sheets x y z

Brain vasculature - high NA, 3R sheets Dt = 1x x y z max Y x y z x y Sample courtesy of Nico Renier 0.25 mm x z

Brain vasculature - low NA, 3R sheets Dt = 1x x y Sample courtesy of Nico Renier 0.25 mm x z

Brain vasculature - medium NA, 3R+3L sheets Dt = 2.2x x y Sample courtesy of Nico Renier 0.25 mm x z

Brain vasculature - high NA, 3R sheets, dynamic focus Dt = 12.8x x y Sample courtesy of Nico Renier 0.25 mm x z

Brain vasculature - high NA, 1R sheet, dynamic focus Dt = 12.8x x y Sample courtesy of Nico Renier

What if we need good XYZ resolution AND a large area ? We will have to do tiling.

Best option for good tiling Radial aberrations Cropping Sheet shape Beware of edges when tiling

Gerbil cochlea – 12.6X mag, high NA, crop+tile Sample courtesy of Ken Hutson (Fitzpatrick lab, UNC)

Further reading for light-sheet https://www.ncbi.nlm.nih.gov/books/NBK536385/ https://doi.org/10.17615/C69M15

Take home Focus on what you need to answer your biological question “Quality” XYZ resolution Evenness of Z resolution Signal-to-noise Volume Imaging time Bleaching Nothing in microscopy is free

Editing and publishing Thanks for: Nico Renier (ICM Brain & Spine Institute, Paris) Ken Hutson (Fitzpatrick lab, UNC) Samples Jennifer Solomon Lynnee Argabright (UNC – University libraries) Editing and publishing $$$

Take home Focus on what you need to answer your biological question “Quality” XYZ resolution Evenness of Z resolution Signal-to-noise Volume Imaging time Bleaching Nothing in microscopy is free