Take a Revolutionary Approach to Deep Image

SCALEVIEW-S

00593_img01.png

The original recipe reported by the Miyawaki team in 2011 termed Scale was an aqueous solution based on urea that limited because the transparency process itself can damage the structures under study.
The research team spent 5 years improving the effectiveness of the original recipe to overcome this critical challenge, and the result is ScaleS, (we called SCALEVIEW-S) a new technique with many practical applications. SCALEVIEW-S creates transparent brains with minimal tissue damage, that can handle both florescent and immunohistochemical labeling techniques, and is even effective in older animals.
The new technique creates transparent brain samples that can be stored in SCALEVIEW-S solution for more than a year without damage. Internal structures maintain their original shape and brains are firm enough to permit the micron-thick slicing necessary for more detailed analyses.

Data provided by Dr. Hiroshi Hama, Tetsushi Hoshida and Dr. Atsushi Miyawaki, Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN Biotechnological Optics Research Team, Center for Advanced Photonics, RIKEN Cooperation with Olympus

Features

  • Easy-to-use
  • No special equipment required
  • Less damage to sample
  • Compatible with IF, FP and other fluorescent labels

Application1

SCALEVIEW-S Application>

Fixation Clearing Imaging
Perfusion fixation
+
post fixation
4% PFA/PBS(-)
[pH 7.6 - 7.8]
Step 1.
SCALEVIEW®
-S0
Step 2.
SCALEVIEW®
-S1
Step 3.
SCALEVIEW®
-S2
Step 4.
SCALEVIEW®
-S3
Step 5.
deScale
Solution
Step 6.
SCALEVIEW®
-S4
Step 7.
SCALEVIEW®
-SMt
Mounting
SCALEVIEW®
-SMt
Processing
Temperature
4 °C 37 °C 37 °C 37 °C 37 °C 4°C 37 °C 37 °C RT
Processing
Time
3 days 30 min 30 min 30 min 30 min 3 hrs x 2 12 - 24 hrs 1 hr

00593_img02.png

Figure 1. Transmittance images of mouse brain before and after clearing with SCALEVIEW-S Solutions.

00593_img03.png

Figure 2. Two-photon microscope imaging of YFP-H line: Mouse whole brain

Mouse Thy1-YFP-H line, 20W, ♂
Size Whole
Microscope Olympus FVMPE-RS
Objective lens XLPLN10XSVMP (NA 0.6)
Laser 960 nm (for YFP)
Image size 512 x 512, 170 tiles, Z=8000 μm, Z Step16 μm

Application2

SCALEVIEW-S Applications: IHC (AbScale)

Fixation Preprocessing
Perfusion fixation
+
post fixation
4% PFA/PBS(-)
[pH 7.6 - 7.8]
Step 1.
SCALEVIEW®
-S0
Step 2.
SCALEVIEW®
-S1
Step 3.
SCALEVIEW®
-S2
Step 4.
SCALEVIEW®
-S3
Step 5.
deScale
Solution
Processing
Temperature
4 °C 37 °C 37 °C 37 °C 37 °C 4°C
Processing
Time
3 days 4 hrs 4 hrs 12 hrs 4 hrs 6 hrs
IHC Clearing Imaging
Step 1.
Blocking
1% Blocking
Reagent (Roche)/PBS(-)
Step 2.
Staining
Antibody
stains
Step 3.
Wash
AbScale
Solution
Step 4.
Re-fixation
4% PFA/PBS(-)
[pH7.6 - 7.8]
Step 5.
Wash
deScale
Solution
SCALEVIEW®
-S4
Mounting:
SCALEVIEW®
-S4
Processing
Temperature
RT 37 °C RT 4°C 4°C 37 °C RT
Processing
Time
2 hrs > 1day 2 hrs x 1,
1 hr x 1
1 hr 6 hrs 6 - 8 hrs

*AbScale Solution:0.33 M Urea and 0.1 % (wt/vol) Triton X-100 in PBS(-) Solution

Iba1 (RF635: Green) 
Amyloid-β (Alexa Fluor 488: Red)
Tomato lectin (Texas Red: Blue)

00593_img04.png

Figure 3. 3D visualization of Aβ plaques (red), microglias (green) and blood vessels (blue) from a 17-month-old AD model mouse.

Microscope (CLSM) Olympus FV1200
Objective lens XLPLN10XSVMP (NA 0.60)

Application3

SCALEVIEW-S Applications: Fluorescent labels (ChemScale)

Fixation Preprocessing
Perfusion fixation
+
post fixation
4% PFA/PBS(-)
[pH 7.6 - 7.8]
Step 1.
SCALEVIEW®
-S0
Step 2.
SCALEVIEW®
-A2
Step 3.
8M Urea
Solution
Step 4.
SCALEVIEW®
-A2
Step 5.
deScale
Solution
Processing
Temperature
4 °C 37 °C 37 °C 37 °C 37 °C 4°C
Processing
Time
3 days 4 hrs 4 hrs 12 hrs 4 hrs 6 hrs
Fluorescent labels Clearing Imaging
Step 1.
FL labels
ex:DAPI(500nM), PI(1μg/mL)/
SCALEVIEW®-A2
Step 2.
Wash
SCALEVIEW®-A2
Step 3.
Wash
deScale Solution
SCALEVIEW®
-S4
Mounting:
SCALEVIEW®
-S4
Processing
Temperature
37 °C 37 °C 4 °C 37 °C RT
Processing
Time
6 -8 hrs 2 hrs x 1,
1 hr x 1
3 hrs 6 - 8 hrs

00593_img05.png

Figure 4. Confocal laser scanning microscope imaging of fluorescent labeled (PI) YFP-H line mouse brain slice (2 mm thick).

Mouse Thy1-YFP-H line, 42W, ♂ Microscope (CLSM) Olympus FV3000 (Inverted)
Size Coronal Slice (2 mm) Objective lens UPLSAPO10x2 (NA 0.40)
Laser 488 nm (for YFP), 561 nm (for PI )

Application4

SCALEVIEW-S Applications: Neurosphere

Fixation Preprocessing
Perfusion fixation
+
post fixation
4% PFA/PBS(-)
[pH 7.6 - 7.8]
Step 1.
SCALEVIEW®
-S0
Step 2.
SCALEVIEW®
-A2
Step 3.
8M Urea Solution
Step 4.
SCALEVIEW®
-A2
Step 5.
deScale
Solution
Processing
Temperature
RT 37 °C 37 °C 37 °C 37 °C 4°C
Processing
Time
1 hr 4 hrs 4 hrs 12 hrs 4 hrs 6 hrs
IHC Clearing Imaging
Step 1
Blocking
1% Blocking
Reagent (Roche)/PBS(-)
Step 2.
Staining
Antibody
stains
Step 3.
Wash
deScale
Solution
Step 4.
Re-fixation
4% PFA/PBS(-)
[pH7.6 - 7.8]
Step 5.
Wash
deScale
Solution
SCALEVIEW®-S4 Mounting:
SCALEVIEW®-S4
Processing
Temperature
RT 37 °C RT RT 4°C 37 °C RT
Processing
Time
2 hrs > 1day 2 hrs x 1,
1 hr x 1
1 hr 3 hrs 4 hrs

00593_img06.png

Figure5. 3D visualization of Neurosphere>/p>

Microscope (CLSM) Olympus FV1000
Objective lens

UMPLFLN10XW (NA 0.3)

References

  1. Hama,H.et al. : Nature Neuroscience 14, 1481(2011).
  2. Hama,H.et al. : Nature Neuroscience 18, 1518(2015).
  3. Hama H, et al. : Protocol Exchange (2016), doi:10.1038/protex.2016.019
  4. Molly E, Boutin, et al :Scientific Reports, 8, 11135 (2018).

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