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

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

3. 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

4. Before imaging
During imaging

5. Before imaging
During imaging

6. 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

7. 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

8. 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

9. 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

10. 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

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
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

13. Before imaging
During imaging

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

15. Spacing is 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)
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

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)
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)
$$$

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 DBE = 1.56

30. 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

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
Lavision Biotec
UltraMicroscope II

35. 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

36. Sheet shape x y z

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

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

39. 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

40. 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

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

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

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

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

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

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

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

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

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

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

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

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

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

54. 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

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

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

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

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

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

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

61. 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

62. 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

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

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

65. Optimization examples

66. Brain vasculature - high NA, 3R sheetsx y z

67. 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

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

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

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

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

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

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

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

75. Further reading for light-sheet

76. 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

77. 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
$$$

79. 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