Project Scope Shootout

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Project Scope Shootout Fred Martin BIOSC. 3 Dr. Giorgi 5/10/2010 Specimen used: C. Elegans stained with DAPI, Alexa 488, Alexa 568 loaner slide Diana Chu lab(SFSU) Imaging systems used: Olympus CKX-41 Zeiss Axioimager Nikon 80i Leica AF6000 SD Leica SP5 Zeiss 710 Objectives used: Olympus Fl 20x/0.45 (using Phase contrast) Zeiss 20x Nikon 80i Plan APO 20x/ DIC Leica AF6000 SD Plan APO 20x/ Dry Leica SP5 HCX PL APO CS 20.0x/0.70 DRY UV Zeiss 710 Plan-Apochromat 63x/1.40 Oil DIC M27

description

Comparison of same FOV across different microscope systems

Transcript of Project Scope Shootout

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Project Scope Shootout

Fred MartinBIOSC. 3 Dr. Giorgi 5/10/2010

Specimen used: C. Elegans stained with DAPI, Alexa 488, Alexa 568 loaner slide Diana Chu lab(SFSU)

Imaging systems used: Olympus CKX-41Zeiss AxioimagerNikon 80iLeica AF6000 SDLeica SP5Zeiss 710

Objectives used:

Olympus Fl 20x/0.45 (using Phase contrast)Zeiss 20xNikon 80i Plan APO 20x/ DICLeica AF6000 SD Plan APO 20x/ DryLeica SP5 HCX PL APO CS 20.0x/0.70 DRY UV Zeiss 710 Plan-Apochromat 63x/1.40 Oil DIC M27

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Project SS: C. elegans on Olympus FX70 (room lights on made DIC-like look)

20x phase, Bin 1x1, 12 bit, Exp = 64.3ms, Gain 1.1 Offset 35

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Project SS: C. elegans on Olympus FX70

20x phase, Bin 1x1, 12 bit, Exp = 396.8ms, Gain 1.2 Offset -823

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Project SS: C. elegans on Zeiss AxioImager A1, DIC

20x DIC, Bin 1x1, 8 bit, Exp = 411.0ms, Bright -0.2 Contrast 3.31 gamma 1.0

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Project SS: C. elegans on Nikon 80i, DIC

20x DIC, Bin 1x1, 8 bit, Exp = 10.0ms, Black 495 White 4095 gamma 1.0

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Project SS: C. elegans on Nikon 80i, 3 channel RL + DIC

20x DIC, Bin 1x1, 8 bit, Exp = 10.0ms, Black 495 White 4095 gamma 1.0DAPI 2.59s, FITC 9.76s, TRITC 4.9s

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Project SS: C. elegans on Leica AFX6000 SD (MetaData at end of presentation)

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Project SS: C. elegans on Leica SP5Slice 37 of 87 from z stack

(MetaData at end of presentation)

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• Olympus: stage controls are very jittery / imprecise vs other scopes• Olympus: most affected by room lights; smaller FOV• Olympus: takes pretty great images for the ‘low scope on the totem pole’• Zeiss: Arc lamp was not to be used, so could not get RL images• Zeiss: fantastic resolution of details, but overall disappointing DIC images:

shear shadow was overwhelming, could not fix• Nikon: Great DIC and RL images: first set of good DIC images I got off the

Nikons

COMMENTS:

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Project SS: C. elegans on Zeiss 710 (63x oil, Max projection – 32 sliceArea showing meitotic divisions in sperm formation: capsules around sperm were the structure of interest in the study)

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Project SS: C. elegans on Zeiss 710 (63x oil, max projection 17 slice)Area near tail of male c. elegans showing spicuola

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Project SS: C. elegans on Zeiss 710 (63x oil)

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SP5 MetaData

• Pinhole [m]60.6 µm Pinhole [airy]1.00 • Size-Width238.5 µm Size-Height238.5 µm Size-Depth33.0 µm• StepSize0.38 µm Voxel-Width466.7 nm Voxel-Height466.7 nm • Zoom3.2 Scan-Direction1 SequentialMode0Frame-Accumulation1• Frame-Average1Line-Average1• Resolution8 bits Channels 2• Format-Width512 pixels Format-Height512 pixels• Line-Accumulation1• Sections = 87• PMT 1ActivePMT 1 (Offs.)-0.4 % PMT 1 (HV)747.7 PMT 1 (HV_Unit)VPMT 1 (Preamp)Direct• PMT 2ActivePMT 2 (Offs.)-2.4 % PMT 2 (HV)505.5 PMT 2 (HV_Unit)VPMT 2

(Preamp)DirectPMT • 3Inactive PMT Trans Inactive• Excitation Beam Splitter FWDD 488/543 • Laser (Argon, visible)On Laser (Argon, visible) (Power) 10 % Laser (HeNe 543, visible) On • Laser (HeNe 633, visible) On Scan • Field Rotation 0 degrees • Scan Speed400 Hz • ObjectiveHCX PL APO CS 20.0x0.70 DRY UV• Numerical aperture (Obj.)0.70• Refraction index1.00

CONFOCAL METADATA

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LEICA SD MetaData

• Camera Settings• System NumberAF 6000 LXCamera: DigitalCameraDFC Gamma1 • Binning1 x 1Resolution 12 bits• Black-Value 0 White-Value 4095• Microscope Settings• ObjectiveHCX PL APO CS 20.0x0.70 IMM UV • Numerical aperture (Obj.)0.70• Refraction index1.52• Dimension Logical Size Physical Length Physical Origin Voxel Size

X1392 pixels443.95 µm0.00 µm0.319 µmY1040 pixels331.60 µm0.00 µm0.319 µmChannels used : 1Name Cube Contrast Method Intensity Peak Emission Peak Excitation Channel 0 GFP WidefieldSD 8 400 nm400 nmChannel Name LUT Name Exposure Time Gain

• Channel 0 Green 8 ms 3.0

CONFOCAL METADATA

ZEISS METADATA:

to be retrieved from SFSU