Kodak Image Station 440CF SOP

 

This SOP is intended for 2 Kodak Image Station 440CF, serial number 907103 and 907105 located in Rm. 4C04 and 3B24, bldg. 49.  The document contains: A) 3 procedures for chemiluminescence, fluorescence, and transmission mode (regular light), respectively; B) a reference table on filter selection; C) a set of saving options; D) technical contact and training resource information

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A.                 Image Acquisition

 

Fluorescence Imaging

 

·      Set f-stop to 1.2 or 2.0 (for clearer image use 2.0)

     Filter position (see Table for filter guide or IS440CF

     Manual.)

 

    Use the back side of the compression pad (or no pad)

 

    Check “preview” mode

 

    Center sample and maximize zoom

     uncheck “preview” mode

     UV lamps are ON

 

    Close the lid

 

    Binning is generally OFF, unless very high sensitivity is required.

     For EtBr gels, capture time is generally 15 sec-2 mm.

 

    Set exposure time and take image

 

    Submit to analysis software by accurately entering

     information in the 1S440CF submit window.

 

Chemiluminescence lmaging

 

·        Set f-stop to 1.2 Filter in position “0’

      Black side of compression pad (or no pad)

·        Place sample-side down on platen

·        Check “preview” mode

·        Center sample and maximize zoom uncheck ‘preview” mode

     Close the lid

     Binning is ON (X & Y) UV lamps are OFF

Start with “pre-capture” of 15-30 sec Use image histogram (see protocol) to estimate exposure time required for imaging

     Set number of captures to 1 or 2

·        Set exposure time and take image

     Submit to analysis software by accurately entering information in the IS440CF submit window.

Transmission Mode Imaging

(For imaging of visible stain dyes and autoradiography films)

 

     Set f-stop to appropriate position depending on light in room (usually between 4.0 and 8.0)

 

     Check “preview” mode

 

      Center sample and maximize zoom, uncheck ‘preview’ mode

 

      Open lid

 

      Place diffuser plate over sample

For flat sample (e.g. X-ray film) - feet side up

For raised sample (e.g. Coomassie Brilliant BIueTM stained gel) - feet side down

 

      Set filter clockwise to position “0”; UV lamps are OFF; Binning is OFF

 

      Set exposure time and take image (usually less than 1 second in length)

 

      Submit to analysis software by accurately entering information in the 1S440CF submit window.

 

 

 

 

B.       Reference Table

                                

Position

Color/Wavelength

Application

Comments

0

 

Transmission mode, chemiluminescence

Densitometry, Coomassie-stained gels, all chemiluminescence substrates

1

Blue band-pass filter, 380~500 nm

Coumarin

 

2

Green band-pass filter, 480~600 nm

Fluorescein

 

3 

Yellow high-pass filter, > 520 nm

Fluorescein, AttophosTM, cyanine 3, SYBR Green and all fluors at higher 

emission wavelengths   

Excellent blue attenuation, good general high pass filter, high speed (short exposure times)

4

Red high-pass filter, > 580 nm

Ethidium bromide, Texas Red

Excellent blue and green attenuation

5

Dark red high-pass filter, > 620 nm

Cyanine 5

Excellent blue and green attenuation

 

                  

Note:   Wavelengths (nm) at which high-pass filter optical density is 3, 2 and 1 respectively, or low-pass filter optical density is 1, 2 or 3. Both apply to band-pass filter.

For fluorochromes of known emission maximum, the following guidelines may be of some benefit in optimizing signal over background fluorescence. Use a high-pass filter whose optical density is less than 1 OD at a lower wavelength than the fluorochrome emission maximum, but not too much lower (perhaps ~2O nm). Ideally, choose a band-pass finer whose mid-band matches the fluorochrome emission maximum. For example, ethidium (emission 595 nm) is compatible with position 4; fluorescein (emission 524 nm) is compatible with position 3, but expect better results with position 2 (mid-band is approximately 534 nm).

 

 

C.                 Saving Options

 

After acquiring an image and submitting it into Kodak 1D Image Analysis Software, save a copy of your image with the following saving options:

 

Save your image onto your desktop by opening the ’Shortcut to NHGRI’, click open your computer, enter password and drag the image to your desktop.  The .bip file format maintains the integrity of the image and preserves history and file information with the image. This file type should always be used. If you would like to view your image using another software program, you can save a 2nd copy as described below:

 

1.  To view the image using Microsoft Word or PowerPoint:

 

Save as .emf file. This will save both the original image as well as any annotations or lane and band information that has been added to the image.

 

2.    To view using Adobe PhotoShop or other drawing or analysis programs, there art two options 8 or 16 bit TIFF..

 

            For quantitative analysis, you want to choose the “scaled to image min/max” option.

 

        For qualitative analysis, you want to choose the “scaled to display min/max” option.

 

3.  You also have the option of saving images as .jpg or .bmp files for using with any type of imaging software

 

 

D.                 Technical Contact & Training Resource

 

Victor Cao,  Lab Manager, (571)276-3634 (cell), or vcao@smenih.com

 

XXXXXX, PerkinElmer Technical Support at xxx-xxx-xxxx