CQ1. Confocal quantitative image cytometer. Riccardo Pasculli Field application specialist GSM:

Transcription

1 CQ1 Confocal quantitative image cytometer Riccardo Pasculli Field application specialist GSM:

2 Agenda Introduction and key advantages Applications

3 Agenda Introduction and Key advantages Applications

4 Development of life science business 2010 Incubation Microscope Confocal Unit CV Basis Research Start CSU10 CSU21/22 Drug Discovery System CSU-X CV CV7000 CSU-W Image Cytometer CQ1

5 CQ1 Key features Cell clusters are directly measured by 3D imaging Gently 3D image acquisition Easy to use to measure the feature data of cells Bench-top and no need for darkroom

6 Cell clusters are directly measured by 3D imaging Gently 3D image acquisition Easy to use to measure the feature data of cells Bench-top and no need for darkroom

7 CQ1 confocal technology Microlens enhanced dual Nipkow disk scanning high-speed, low photo toxicity and low photo bleaching Microlens array disk Light source Conventional Single beam scanning Pinhole Pinhole array disk (Nipkow disk) Dichroic mirror Yokogawa confocal Objective lens Multi-beam scanning with 1,000 laser beams Sample

8 CQ1 confocal technology Microlens disc Light source Camera Microlens disc Pinhole disc (Nipkow disc) DM Objective lens Sample DM Pinhole disc Light efficiency is 70% improved!

9 Fluorescent intensity(%) Advantage of multipoint confocal scanning Example of Photo bleaching CSU *depend on sample and condition Time (minute) LSM

10 Wide field fluorescence vs. confocal Wide field fluorescence Image Confocal Image ポウディスク と Sample:Horsetail spore (about φ30um) Lens: 60x oil Pinhole: 50um Z: 1um step Total 100um

11 3D imaging of Embryonal Carcinoma cells Z slice images The nuclei of canceration stem cells dyed with Draq5 Confocal is able to go deep inside of spheroid. Information of not only entire colony but also individual cell of colony Analyzed nuclei in 3D

12 CQ1 confocal technology 1) Good quality images Accurate analysis 2) 3D imaging and analysis Available on thick sample (spheroid) 3) Low photo-bleaching and low photo-toxicity Good for Live cell imaging 4) High-throughput Happy for various experiments

13 Cell clusters are directly measured by 3D imaging Gently 3D image acquisition Easy to use to measure the feature data of cells Bench-top and no need for darkroom

14 Easy workflow CQ1 Main unit (benchtop size 600X400X298) 1 set sample Image data 2 Input the measurement /analysis parameters (protocol) Workstation Confocal images (2D 3D) 4 On the fly analysis 2.5 min in 96-well Numerical 数値データ数値データ data Area Volume Intensity 3 Confocal image capturing with the image cytometer 5 Save data

15 Compact and easy to set-up Main head 60cm x 40 cm x 30 cm Workstation Laser combiner

16 Multi-vessel compatibility Sample Holder 3-35mm Dish Set the sample Door opens Sample Holder 4 Slide Glass 60mm Dish Cover Glass Chamber

17 Measurement software Image Viewer Control panel: Set the conditions of measurement/analysis Wells table: results( number of cell) Chart Viewer: Scatter graph Histogram

18 Breakdown of output data Item Unit Dimension Area um 2 2D Volume um 3 3D Shape Intensity Circular - 2D Sphere - 3D Perimeter um 2D Surface um 2 3D Average - 2D 3D SD 2D 3D Sum - 2D 3D Max - 2D 3D Min - 2D 3D

19 Breakdown of output data 3D imaging Image analysis Draw charts Gate analysis CQ1 TIFF OME-TIFF ICE FCS CSV FCS Express 5 Image Cytometry FlowJo(no images) Excel, SpotFire etc.(no images) StrataQuest MetaMorph etc. For Slide Scanner For HCA

20 CQ1- key features Spinning disk confocal -low photobleaching scmos camera (2560x2160) Bright field, phase contrast 6 objectives 4x, 10x, 20x, 40x, 60x Laser lines, 405nm, 488nm, 561nm, 640nm Precise automated X-Y Stage, setting resolution ±0.1um

21 CQ1- key features Hardware and software autofocus Multi format sample holders On the fly analysis software Environmental control Temperature, CO2, humidity, Hypoxia

22 Agenda Introduction and Key advantages Applications

23 CQ1 applications Regenerative medicine 3D cell culture(spheroid /Colony) Cell sheet and Tissue Cancer research Cell cycle analysis Cell proliferation check CTC Screening : NFκB translocation, granule analysis Toxicity test

24 Trend of live cell imaging These days, ips/es cells have become popular and it has become necessary to measure 3D cultured cell such as spheroids. Not only flow cytometer but also conventional microscope can t measure the thick structure. Moreover, exhaustive experiments with a lot of parameter require high throughput instrument. ips cells (Kyoto University) Cell sheet (Oosaka University)

25 Cell proliferation The number of cells in each well High-speed Quantitative

26 Cell count Evaluation of cell differentiation Red; nucleus Green; ctnt (differentiation marker) Amount of the differentiation marker in individual cells Negative cells 分化マーカーが高発現の細胞 CQ1 ではこの部分の細胞のみを表示することも可能です Z-slice images 3D analysis Positive cells Fluorescence intensity Undifferentiated Partially differentiated Differentiated High-speed Quantitative Multicolor 3D Page.26

27 Sum vs MIP images of thick samples Sum (Summed image) MIP (Maximum Intensity Projection) Blurred image Clear image High quality High-sped image Multicolor 3D

28 Quality check of spheroid Lens: 2x 400 Spheroid High-sped Multicolor High-speed Quantitative Lens: 40x 4 spheroid 400 um Single spheroid 100um

29 Area Cell cycle analysis G1 phase Nucleus area vs. labeling intensity G2 phase S phase M phase M; Cell Division Sample; Hela cells Nuclei were labeled with Hoechst dye. Total intensity G2 S; DNA Synthesis G1 Quantitative High High-sped Multicolor 3D quality image

30 Number of cell Cell cycle analysis-3d Amount of DNA G1 G2/M 3D imaging of confluent A549 cell (Lung carcinoma) culture Total intensity of Draq7 (DNA) Quantitative High High-sped Multicolor 3D quality image 3D Multicolor Quantitative High quality image Image acquisition and analysis Page.30

31 Count Cell cycle analysis- FCS express sw 400 DataDirectoryFile.ice 300 G1 51.1% 200 S 26.8% G2 22.1% x x x x10 5 (Object1) TotalIntensity CH1 Cycle G1 Mean G1 CV %G1 G2 Mean G2 CV %G2 %S G2/G1 %Total B.A.D. Diploid Quantitative High High-sped Multicolor 3D quality image

32 Area of Detection Zone Migration assay 0uM Control 1uM Cytochalasin D (1 μm) 1 2 HeLa cells (Fucci, a cell cycle marker was introduced) were cultured in Oris TM Universal Cell Migration Assembly Kit (Platypus Technologies) Effect of Cytochalasin D Area of Detection Zone CytochalasinD 3 Time lapse imaging for 72 hrs uM 1uM Quantitative Time-lapse High High-sped Multicolor 3D quality Quantitative Multicolor image Time (hr)

33 Scratch wound assay MitomycinC + MitomycinC ー Both cells in S/G2/M and G1 phase increased in the control (MMC-). The scratch was filled with the migrated and the proliferated cells MMC+ MMC- MMC+ MMC- Cells in G1 phase decreased while those in S/G2/M phase increased in the MMC-treated culture. Number of cells in S/G2/M phase Number of cells in G1 phase The scratch was filled with the migrated cells. Quantitative Time-lapse High High-sped Multicolor 3D quality Quantitative Multicolor image

34 Number of nucleus Mean intensity Apoptosis- toxicity tests Apoptosis Control Apoptosis Control Area of nucleus Area of nucleus Six hours time-lapse movie of HeLa cell cultures. Staurosporine (0-10uM) was applied to induce apoptosis. Nuclei were stained with Hoechst Control cells survived for 41 hours. Staurosporine (apoptosis) Control Time-lapse Quantitative Multicolor

35 Intensity ratio Translocation of NF-κB TNFα 0ng/ml Nucleus NF-κB Marge & recognition Localization of NF-κB Ratio of nucleus to cytoplasm TNFα IL-1β TNFα 100ng/ml Concentration (ng/ml) HeLa cells, 96-well plate TNFα or IL-1β was applied for 20 min. Fixed sample, labeled with Hoechst and a specific antibody to NF-κB. 10x objective, 1 z-slice, 16 field Quantitative Quantitative High High-sped Multicolor 3D quality Multicolor image

36 Neurite outgrowth Sample PC12 cells, cultured overnight in collagen IV-coated well plate then for three days with/without nerve growth factor (NGF) Red line; neurite area/ cell (um 2 ) Blue line; branch number / cell Green bar; average neurite diameter (um) Dose response Response to NGF Staining Nuclei by Hoechst33342 β-tubulin by Alexa635 Image acquisition 40x dry lens, 25 z slices with 12 μm interval, mip analysis Quantitative High High quality Quantitative High-sped Multicolor 3D quality Multicolor image Quantitative image Multicolor Concentration of NGF Page.36

37 Angiogenesis Effect of suramin (an inhibitor of angiogenesis) 0 μm 30 μm 50 μm Total length Num. of branch Suramin (μm) Suramin (μm) Num. of junction point Total area Suramin (μm) Suramin (μm) Quantitative High High-sped Multicolor 3D quality image tiling High quality image HUVEC labeled with CellTracker Red 96 well plate (IWAKI:EZ-VIEW) with Matrigel coating. Suramin was applied after the tube formation was confirmed.

38 Calcium imaging-drug screening CQ1 supports high-speed time lapse image acquisition (maximum 20 frame per second) Sample: icell Cardiomyocytes 2, CMC (Cellular Dynamics International, Inc.) Quantitative High High-sped Live Quantitative Multicolor 3D quality image High quality image High-speed

39 Tissue section 3D reconstruction 3D analysis Mouse Kidney Slice Quantitative High High-sped Multicolor 3D quality image 3D tiling High quality image High-speed

40 Tissue section Bright field image by CQ1 Fluorescent image by CQ1 3D reconstruction from fluorescent z-slice images 40x lens, 1 field 31 z-slices with 1um step Bright field image taken in a conventional microscope 4x lens, tiled image of 4 5 fields, 1 z slice Quantitative High High-sped Multicolor 3D quality image 3D tiling High quality image High-speed

41 Key resources //accela.eu

42 THANK YOU FOR YOUR ATTENTION accela s.r.o. Služeb Prague 10 Czech Republic Tel.: Fax:

43 Nipkow disk The device is a mechanically spinning disk of any suitable material (metal, plastic, cardboard, etc.), with a series of equally distanced circular holes of equal diameter drilled in it. One of the advantages of using a Nipkow disk is that the image sensor can be as simple as a single photocell or photodiode, since at each instant only a very small area (a pixel) is visible through the disk (and viewport), and so decomposing an image into lines is done almost by itself.

44 % of maximum count Scale - making spheroids transparent Z-distribution of cells recognized by CQ1 (normalized) Scale Control 15 μm z-step, 10x lens Z position ( 15 μm) High-sped Multicolor 3D High-speed High quality Quantitative image Quantitative

45 Scale 0 μm 60μm Control 120 μm 180 μm 240 μm