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BioScope Resolve BioAFM The Most Advanced AFM for Life Science Research
Masami Saito Application Engineer, Bruker Nano surface Devision
Bruker Nano Surfaces Division
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How Does AFM Work?
9/3/2015 3 Bruker Nano Surfaces Division
(Figures and video from www.freesbi.ch)
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Bruker Series of AFMs for Life Science
9/3/2015 4
MultiMode® 8
Highest resolution AFM for molecular imaging
BioScope Resolve™
True functional integration of AFM and
optical microscopy
Dimension FastScan Bio™
High-speed imaging of biomolecular and cellular
dynamics.
• Multiple Bruker AFM Platforms being used in leading biological research and biotechnology industry.
Bruker Nano Surfaces Division
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FastScan Bio AFM 高速・高分解能測定
0.5fps
2.0fps
1.0fps
3.0fps
Lamba Digest DNA in buffer.
(500nm x 500nm x 2nm)
Bruker Nano Surfaces Division 9/3/2015 5
高速測定でも:
イメージ分解能の劣化を最小に抑え
DNAを破壊することなく測定
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Mouse embryonic stem cells. Obtained over 12hrs with a 40x phase contrast objective.
© The Exploratorium, www.exploratorium.edu
50 mm
50 mm
Obtained over ~1hr at 40sec/frame at 512 x 128 pixel resolution. Phase images shown. (~10 mm scan size)
FastScan Bio AFM of Cell Structure Dynamics 哺乳類の細胞移動
Bruker Nano Surfaces Division
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Bruker BioScope ResolveTM BioAFM 比類のないイメージ分解能をもち、機械生物特性が計測できる PeakForce Tapping®を搭載
9/3/2015 8
• 細胞と分子メカニクスのために開発した最も包括的なパッケージ
• 新 PeakForce QNM® は、生細胞上で最高速、最高分解能の
ナノ機械特性が得られる
• 新 FASTForce VolumeTM は新たな特性マッピングを搭載し、
さまざまな速度と荷重感度でデータ取得できる
• BioAFMの中で、実質的に最高の感度をもつ
• ルーチン測定で最高分解能の分子構造測定が可能
• 生細胞の全体をアーティファクトなくScanAsyst自動イメージング
• AFMと光学顕微鏡が完全に融合し、高度な相関が取れる
• 完全な融合により、AFMイメージと様々な光学測定技術をシンクロできる
• 自動測定機能により、ハイスループットでデータを取得
• 生物試料のハンドリングに優れ、生細胞研究向けの高度な環境制御オプション
Bruker Nano Surfaces Division
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BioScope Resolve バイオ実験のための完璧なアクセサリー
• ディッシュ、スライドガラス、カバーガラスいずれの サンプルにも、容易にアクセスできる
• EasyAlignプローブ取付ステーション
• マイクロボリュームセル (60µL)
• サンプルヒーター
• 還流ステージインキュベーター (PSI)
• 直上観察用光学顕微鏡
9/3/2015 9
Unrivalled open design for easy sample access
New EasyAlign platform for Easy Probe Loading
Bruker Nano Surfaces Division
New Cantilever Holder & Micro Volume cell (60µL)
Top View Optics with stand
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New BioScope Resolve User Interface 究極の Ease-of-Use データ取得
9/3/2015 11 Bruker Nano Surfaces Division
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MIROView Optical Synchronization 光学顕微鏡とAFMのシームレスな融合
• CCDカメラや共焦点顕微鏡で高機能な蛍光像や明視野像を取得し、自動的にAFMソフトに インポート
• 光学像が取得できるタイミングは:
• AFMスキャンラインごと
• AFMイメージごと
• フォースカーブごと (アプローチやリトラクト)
• AFM と光学像の位置合わせは、後処理もできる。光学データはナノスコープ上のムービーメーカーで再生可能
12 9/3/2015
Synchronization of AFM and Confocal Imaging of MDCK cells. Actin fibers are fluorescently labeled with Alexa Fluor® 546 Phalloidin.
Confocal images showing deliberate photobleaching of the dye are captured every 25s during continuous AFM imaging of the cell structure.
Bruker Nano Surfaces Division
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Approach Retract
z Δz
Zajac & Discher (2008) Curr Opin Cell Biol 20: 609-6150
Glycosan Biosystems & SigmaAldrich
AFM in Cell Mechanics 生物機械特性
• 細胞は‘力’を感知し、反応する
• それぞれの組織でさまざまな弾性を示す
• 病気は細胞の機械特性に影響を及ぼす
• AFM フォースカーブが測定できるのは: • Elasticity/Modulus
• Adhesion
• Molecular (un)folding
• Binding interactions
• Response to mechanical stimulus
Bruker Nano Surfaces Division 9/7/2015 13
z Δz
F= -k(Δz)
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Bruker Nano Surfaces Division 9/7/2015 15
Exposure Time
• 選択したスキャン範囲上で、2次元のアレイ状の各位置でフォースカーブを取得
イメージングスピードと分解能はトレードオフの関係をもつ
• 繊維芽細胞にサイトカラシンBを投与した後に
アクチンフィラメントがほどけていく様子
Radmacher et al. (2000) Biophys. J., vol 78: 520-35.
Force Volume Imaging 細胞の弾性マッピング
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New
Increased flexibility & productivity for force mapping
9/3/2015 16
• 高速, 定量的フォースマッピングが <1Hz から最大 300 Hz のランプレートで測定可能
• データ取得が改良
• ランプ中のZ-sensorデータを保存 よってクローズドループはもはや必要なし
• 時間の無駄なく、コの字型の両方向のスキャンも選択可能
• 軟らかいサンプルのための低荷重のトリガー
• 新 FASTForce Volume ウィンドウ
• 複数のランプデータチャンネル
• 複数の特性マッピング: force modulus, stiffness, adhesion, force slice (and height)
• 高分解能測定
• (256x256)x2048 or max pixels (956x956)x256
• 解析の改良
• “Real Height” データ
• 密度プロット, 輪郭プロット 等
Bruker Nano Surfaces Division
Movie 1: Traditional Force Volume image acquired at 1Hz (16x16 pixels)
Movie 2: New FASTForce Volume image acquired at 156Hz (128x128 pixels)
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PeakForce Tapping Technology Controls and measures force as feedback
Z motion
Deflection
Z position
Probe motion resembles a typical
force curve…
PeakForce Tapping モード:
• プローブが低い周波数、小さい振幅で振動する (125-2kHz).
• フィードバック信号にはチップとサンプル間の最大フォースを使う
• イメージングとーすを直接制御し、弱い荷重で測定が可能 (<100pN).
• 高分解能の計測が一般的なスキャン速度で取得可能 (1000’s force curves/sec).
Bruker Nano Surfaces Division 9/7/2015 17
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PeakForce Tapping Imaging Accurately Measuring the Diameter of DNA
9/3/2015 18
イメージングフォースを低くし、かつ精密に制御することが可能なPeakForce Tapping技術を使えば、B-DNAの約2nmの直径を正確に計測することができる
50nm
Bruker Nano Surfaces Division
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Quantitative Nanomechanical Property Mapping
Fo
rce
Separation
Sneddon Model
DMT Model
Adhesion Imaging
Dissipation
Modulus
Deformation
• 形状測定と同時に、定量性のある二次元のナノ機械特性データが取得できる
• 広範囲での材料物性が計測可能
(kPa-GPa).
• DMT と Sneddon 弾性モデルに対応
• PeakForce キャプチャー機能で全フォースカーブを保存し、計測後に解析することが可能
Modified from Cox and Erler. Dis. Model. Mech. (2011) 4:165-178.
Bruker Nano Surfaces Division 9/7/2015 19
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of Live Cells
再現性の良いデータ、高分解能の細胞機械特性
9/3/2015 20
• PeakForce QNM マッピングで生細胞観察に供給するのは:
• 高分解能機械特性マッピングの高速データ取得(最速2kHzまで)
• 定量的な、再現性の高い測定
• Achieved Through:
• サンプルを非常に安定に固定できる機構を有する
• ブルカーの PeakForce QNM-Live Cell プローブ (k~0.08N/m, 17µm tip, ~65nm 先端曲率固定)
• 新 PeakForce Tapping live cell バックグランド除去 アルゴリズム
PeakForce QNM topography image (top) and modulus image (bottom) of living MDCK cells.
5µm
5µm
Bruker Nano Surfaces Division
~130nm
PeakForce QNM-Live Cell Probe
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9/7/2015 22 BRUKER CONFIDENTIAL
Glioblastoma (isogenic cell line) Low Res (but faster) imaging with PFQNM
Topography (z: 0-250 pN) Elasticity (z: 0-1.2 MPa) Adhesion (z: 0-800 pN)
Deformation (z: 0-250 nm)
• 128x128 images (5 min per image): averaging on a high number of images
• Highly quantitative
• No damage of the sample
75x75 µm
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9/7/2015 23 BRUKER CONFIDENTIAL
Glioblastoma (isogenic cell line) Results
Elasticity (kPa)
0
20
40
60
80
100
120
140
Ctrl IND Ctrl non-
IND
tp53 non-
IND
tp53 IND pTEN non-
IND
pTEN IND
Deformation (nm)
0
50
100
150
200
250
Ctrl IND Ctrl non-IND tp53 non-
IND
tp53 IND pTEN non-
IND
pTEN IND
Young’s modulus (kPa)
Deformation (nm)
TP53 and PTEN induced are
significantly stiffer and less
deformable than the other
cell types
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定量性のある、再現性の高いデータ
9/3/2015 26
PFT -1kHz (kPa) PFT -500Hz
(kPa)
PFT - 250Hz
(kPa)
Sample: 1.25% Agarose Gel
Average* 24.2 13.1 15.2
STD 3.1 3.5 1.8
2.5% Agarose Gel
Average 283 180 120
STD 51 27 24
5% Agarose Gel
Average 485 258 213
STD 75 26 12
• 複数のユーザーが複数のプローブで複数のサンプルを測定
• 広い周波数範囲で、 PeakForce QNM が非常に安定したデータを示した
* Average represents 2 users, alternating with 3 probes and 3 samples on many sites to perform measurements
Bruker Nano Surfaces Division
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• 周波数範囲を拡張し、フォースボリュームとPeakForce QNMの計測領域がオーバーラップ
PeakForce QNMの最低周波数を125Hzまで下げ、かつ、FVの速度を300Hzまで増加
• 生産性を向上させ、高分解能FVマッピング実験が可能
• 物性マッピングの時間依存性を調べることができる
Most Comprehensive Package for Biomechanics 物性の時間依存性の研究に貢献
9/3/2015 27 Bruker Nano Surfaces Division
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倒立型顕微鏡上に組み合わせたAFMでは最高の分解能
9/3/2015 28
10nm
DNA二重らせん構造の主溝、副溝を分離するサブ分子分解能.
画像は倒立顕微鏡の上で撮影
10nm
バクテリオロドプシン(bR)の格子構造を倒立顕微鏡の上で撮影。挿入図はbR三量体の1分子の平均画像を示している。緑の丸は1格子欠陥を示し、青の丸は1格子構造を示す。Z scale 0.6nm
20 nm
Bruker Nano Surfaces Division
• ScanAsyst Fluid-HiRes プローブ (k ~0.05N/m or 0.4N/m, <2nm 先端曲率)
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PeakForce Tapping イメージング 生きている細胞
9/3/2015 29
XY= 95µm
Z = 4.5µm
XY = 5µm
Z = 0.7µm
• アーティファクトなしに細胞の全体の構造を計測できる
• XY-Scan 範囲 ≥100µm; Z-Scan 範囲 ≥15µm
• Bruker PeakForce QNM-Live Cell プローブ (k~0.08N/m, 17µm tip高さ)
• 新 PeakForce Tapping 生細胞バックグランド除去アルゴリズム
• 細胞イメージングのための新 ScanAsyst モード
Bruker Nano Surfaces Division
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Bioscope Resolve with PeakForce Tapping 生細胞上の微絨毛の高分解能イメージング
9/3/2015 30
5µm
生きているMDCK細胞の1kHz PeakForce Tappingイメージ, Dr. Hermann Schillers, University of Münster. (Inset: SEM image of MDBK cells at
2400x mag.)
Bruker Nano Surfaces Division
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PeakForce Tapping of Cell Structures 細胞の上の微絨毛の‘そのままの’構造を、低荷重で分離
PeakForce Tapping of Living MDCK Cells. BioScope Resolve using PFQNM-LC probes (0.1N/m)
170pN
0.3Hz
170pN
0.3Hz
28pN
0.2Hz
28pN
0.45Hz
左: 微絨毛の電子顕微鏡イメージ 右: 固定したMDCK細胞のPeakForce Tapping イメージ
• 上皮生細胞に微絨毛は存在し、複数の過程に関与する
• PeakForce Tappingは初めて生きている細胞の上で微絨毛の観察を可能にした.
• <30pNの低荷重イメージングにより、微絨毛が立ち上がった円柱状構造を持っていることがイメージングできる structures.
Schillers et al. (Submitted to J. Mol. Rec.)
Bruker Nano Surfaces Division 9/7/2015 31
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Bruker BioScope Resolve BioAFM Unrivalled BioAFM Imaging Resolution and Biomechanics Enabled by PeakForce Tapping
9/3/2015 32
• 細胞と分子機械特性測定のための 最も包括的なシステム
• BioAFM史上、最高イメージン分解能に到達
• AFMと光学顕微鏡を完全に融合させ、高度に相関が必要な研究に用いることが可能
Bruker Nano Surfaces Division
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