Tips for imaging cleared samples

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

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

3. Workflow Sample preparation Sample mounting Sample imaging Data management Data visualization Chung et al, Nature 2013 Ertürk et al, 2012 Doboszet al, Neoplasia 2014 Renier et al, Cell2016 Data analysis

4. Before imaging During imaging

5. Before imaging During imaging

6. Trim based on your biology

7. Trim based on your biology

8. Trim based on your biology

9. Trim based on your biology

10. Trim based on your biology • Smaller samples: • stain faster • require less antibody • clear faster • are easier to image

11. 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…

12. Labeling – (far) redder is better AlexaFluor488 Ex 488; Em525/50 AlexaFluor568 Ex 561; Em595/40 AlexaFluor647 Ex 640; Em 680/30 1 mm Autofluorescence Scattering Absorbtion Renier et al, Cell2014

13. Before imaging During imaging

14. What level of detail do you need? detail you want

15. Spacingis more important than size

16. Example: Labeled axons (0.2mm)

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

18. Problem: 2 objects close together

19. Problem: 2 objects close together ?

20. Problem: 2 objects close together ?

21. Spacing more important than size

22. Spacing more important than size

23. 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) • Locate cells of interest in large pieces of tissue • Trace neural circuits • Trace sparsely labeled axons • Analysis of vasculature

24. 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

25. Different microscopes for different questions Subcellular scale Dense labelling High resolution sub-um(ish) Somelaser 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) • $$$

26. Objectives for high resolution volumetric imaging $$$

27. Objectives for high resolution volumetric imaging FOV

28. Objectives for high resolution volumetric imaging FOV

29. Objectives for high resolution volumetric imaging RI RI DBE = 1.56

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

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

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

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

34. Ultramicroscope Objective Sample LavisionBiotec UltraMicroscope II

35. To optimize, know the key limitations Sheet shape Better Worse Light propagation Radial aberrations

36. Sheet shape y x z

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

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

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

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

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

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

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

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

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

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

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

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

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

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