Stanford School of Medicine
High-Throughput Bioscience Center (HTBC)–

High-Content Imaging

We have updated our filters to Semrock filters. See below for new filter configurations. The ImageXpress 5000A is located in CCSR 0116. Please contact us for key card access to this room.

For high-content automated cellular imaging and analysis we are utilizing the Molecular Devices ImageXpress 5000A with the live cell option.

This instrument contains a fully automated inverted epifluorescence microscope that is capable of scanning a 6-, 24-, 96- or 384-well microplate and recording multiple fluorescent images in a few minutes. The ImageXpress Micro is then able to quantify morphological properties through image analysis software. Our current ImageXpress configuration permits the visualization of both fixed cells and live cells, as the instrument has a temperature-, humidity-, and carbon dioxide-controlled chamber. We plan to add an external automated incubator to permit long-term or large-scale studies of cellular events, and a robotic plate stacker, thereby increasing the throughput of this workstation.

ImageXpress 5000A Brochure

ImageXpress 5000A Schedule. Analysis Computer1, Analysis Computer2

To use the ImageXpress 5000A, please contact David E. Solow-Cordero (x5-6002)

Technical Information
Objectives Excitation Filters Emission Filters Dichroics
40X 0.6 Plan Fluor ELWD 427_10nm 472_30nm Triband1
10X 0.3 Plan Fluor 504_12nm 542_27nm 444/521/608
20X 0.75 Plan Apo 589_15nm 632_22nm  
20X 0.45 Plan Fluor ELWD 387_11nm 440_40nm Quadband1
40X 0.9 S Fluor 485_20nm 525_30nm 410/504/582/669
4X 0.2 S Fluor 560_25nm 607_36nm  
  650_13nm 684_24nm 400DCLP
  D340_X D510-80nm Q455DCLP
  D360_10X ND0.5  
  D380_X ND2.0  

Filter Sets
Label Name Excitation Emission Dichroic
DAPI 387_11nm 440_40nm Quadband
Cyan GFP 427_10nm 472_30nm Triband
Yellow GFP 504_12nm 525_30nm Triband
Texas Red 560_25nm 632_22nm Quadband
optimized yfp 504_12nm 542_27nm Triband
Fura-2-340 D340_X D510-80M 400DCLP
Fura-2-360 D360_10X D510-80M 400DCLP
Fura-2-380 D380_X D510-80M 400DCLP
GFP 485_20nm 525_30nm Quadband
AlexaF430 427_10nm 542_27nm Triband
Alexa 594 589_15nm 632_22nm Triband
Alexa 594Quad 560_25nm 607_36nm Quadband
Alexa 647 650_13nm 684_24nm Quadband
Dronpa 387_11nm 525_30nm Q455DCLP
FRET 427_10nm 525_30nm Q455DCLP

 


 
 
CFP/YFP/HcRed-3X3M-A Triple-band "Sedat" Filter Set
 
 Optimized for these fluorophores
Cyan: CFP, AmCyan, SYTOX Blue, BOBO-1, BO-PRO-1
Yellow: YFP, Calcium Green-1, Fluo-3, Rhodamine 123
Red: HcRed, Cy3.5, Texas Red®, Alexa Fluor 594
Complete list of fluorophores for this filter set
 
Actual measured data from typical filters
     
 

Exciter 1

 Dichroic Emitter 1
   
Exciter 2   Emitter 2
     
Exciter 3   Emitter 3
     
 
 
 Set description
This 7-filter "Sedat" set is ideal for high-speed sequential imaging CFP, YFP, and HcRed with high brightness, extremely low crosstalk, and superb signal-to-noise ratio. The set is also ideal for samples triple-labeled with CFP, YFP, and an orange or red dye, like Texas Red. It is comprised of three single-band exciters and three single-band emitters, along with a triple-band beamsplitter, and thus works best when used with both exciter and emitter filter wheels.
 
Filter Average Transmission Center Wavelengths Bandwidths *
 Exciter 1 > 90% 427 nm 10 nm
 Exciter 2 > 90% 504 nm 12 nm
 Exciter 3 > 90% 589 nm 15 nm
 Emitter 1

> 90%

 472 nm 30 nm
 Emitter 2 > 90% 542 nm 27 nm
 Emitter 3 > 90% 632 nm 22 nm
  Average Reflection Reflection Bands Transmission Bands
 Dichroic > 95%
> 95%
> 95%		 420 - 430 nm
496 - 510 nm
579 - 596 nm		 451 - 480 nm
530 - 561 nm
618 - 664 nm

 

fluor
DA/FI/TR/Cy5-4X4M-B Quad-band "Sedat" Filter Set
 
 Optimized for these fluorophores
Blue: DAPI, Hoechst, AMCA, Alexa Fluor 350
Green: FITC, GFP, rsGFP, BODIPY, Alexa Fluor 488        
Orange: TRITC, Cy3™, Texas Red®, MitoTracker Red,
  Alexa Fluor 568 & 594
Red: Cy5™, APC, TOTO-3, TO-PRO-3, Alexa Fluor 647 & 660
Complete list of fluorophores for this filter set
 
Actual measured data from typical filters
     
graph
 
blue Exciter 1 g Dichroic r Emitter 1
     
b4 Exciter 2   r4 Emitter 2
     
b3 Exciter 3   r3 Emitter 3
     
b2 Exciter 4   r2 Emitter 4
     
 
 
 Set description
This 9-filter "Sedat" quad-band set is designed for high-speed, sequential imaging of DAPI, FITC, TRITC, and Cy5 (and other key fluorophores – see complete list above). The complete set is comprised of a quad-band beamsplitter with four single-band emitters and four “no burn-out” single-band exciters. The single-band filters are intended to be mounted in filter wheels.
 
Filter Average Transmission Center Wavelengths Bandwidths *
 Exciter 1 > 85% 387 nm 11 nm
 Exciter 2 > 90% 485 nm 20 nm
 Exciter 3 > 90% 560 nm 25 nm
 Exciter 4 > 90% 650 nm 13 nm
 Emitter 1 > 90% 440 nm 40 nm
 Emitter 2 > 90% 525 nm 30 nm
 Emitter 3 > 90% 607 nm 36 nm
 Emitter 4

> 90%

 684 nm 24 nm
  Average Reflection Reflection Bands Transmission Bands
 Dichroic > 95%
> 95%
> 95%
> 95%		 381 - 392 nm
475 - 495 nm
547 - 572 nm
643 - 656 nm
 420 - 460 nm
510 - 531 nm
589 - 623 nm
677 - 722 nm
 

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