54Summer 2011Digital InSight #5

DigitalInSightTHE MAGAZINE OF THE DIGITAL IMAGING INSTITUTE

5

Lifescience digital imaging

Lifescience digital imaging

Eric Wexler

Introduction to the use of a flatbed scanner and Photoshop in biomedical research

Flatbed scanners are still an important research tool, especially when combined with scanner soft-ware used with the correct set-tings, image processing software to prepare the images, and image analysis software to collect data.

Over two decades the develop-ment of other imaging technologies (like slide scanning systems and phosphor plate readers), has pro-vided researchers with tools that are focused on specific uses, albeit at a much higher price.

The availability of relatively in-expensive flatbed scanners, in the early 90s, for consum-

ers to scan photographs opened the door for biomedical research-ers of all levels to digitize research samples. At first, the obvious materials to scan were photomi-crographs, blots, autoradiography films and other thin flat materials. As with many tools, additional uses became evident and the scanner took on broader utility.

In my own lab, wet samples, like brain sections and soft agar colony multiwell plates, have been imaged for data analysis. Scanners also increase productivity by allowing multiple microscope slides to be scanned simultaneously, when the resolution of the scanner is appro-priate for the samples and regions of interest. Figure 1. Artixscan M1

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Benefits of using a flatbed scanner: X A flatbed scanner provides a large imaging area with limited distortion. X Many scanners can be calibrated using a standard Kodak Q-60 target allowing repro-ducibility over time using different scanners. X Flatbeds have a large footprint that gives stability and are less likely to walk away, as digital cameras unfortunately do. X Scanners save images as TIFF images, which are preferred for scientific analysis. X Scanners are not updated as quickly as digi-tal cameras. As such, a scanner does not go out of date quickly and can be used for many years. One big downside for flatbeds can be that

the manufacturer sometimes abandons software update support and the original software will no longer work with newer operating systems. The original controller software may become unus-able. If this happens, a program called Vuescan (found at www.hamrick.com) might be able to provide help, as it supports hundreds of scan-ners that work on the PC and MAC platforms. Vuescan can be used in a demo mode to see if it will work with your particular configuration. It is updated frequently, and I have used it for relatively old scanners. Vuescan offers a single application, which can be learned and relied upon for the majority of scanners in use.

General use settingsFor research tissue samples and microscope

slides it is important to capture as much raw color data from the scanned samples as pos-sible. Built into the scanner software is quite a bit of automatic processing—turn it off. The control interface provided is basic and needs to be switched to the expert or advanced mode. Turn off the automatic adjustment based on a white and black point that the software detects. This will permit scanning using the full dynamic range. Set the gamma at 1.0 so the scan in-tensities are linear and therefore suitable for analysis.

My setupIn my labs I use Microtek Scanners because

they have two separate scanning methods. I am currently using the Artixscan M1, introduced in 2007, that has an upper glass flatbed with a large 8.5 x 14 inch area and a direct glassless slide scan area of 8 x 10. Its resolution is 4800 x 9600 pixels, which equates to a 34 megapixel DSLR image sensor. The optical density rating is 4.4 DMAX, giving a large dynamic range.

I can use one of three scanner applications—Scanwizard (Microtek), Vuescan (Hamrick), or Silverfast (Lasersoft Imaging). I use Scanwizard most of the time. If higher end features like HDR

Figure 2. Scanwizard screen shot Figure 3. VueScan screen shot

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tures can be viewed. By using an adjustment layer, any modifications are displayed but do not permanently change the underlying image. This makes the core of the fetus much easier to observe.

scans are needed, I use Silverfast. If I need multiple automated scans and I simultaneously save multiple formats, Vuescan is best.

Scan of a clarified mouse for tetralogy examination

One of my lab’s first scans of a three-dimen-sional object was a fetal mouse stained with Alcian blue and Alizarin red to identify malforma-tions of the skeletal structure. At that time, digi-tal cameras had limited resolution. The sample was floating in PBS solution in a petri dish. Covering the sample with thick white paper for a backdrop, the scan produced a very nice image.

that the color is the default by pressing the D shortcut, then paint on the area where the detail needs improvement.

This technique makes the core details much more voidable, i.e., capable of being nullified, so if I want the details look more natural I can modify the mask adjustment blend better by de-creasing the opacity of the Adjustment Layer. To do so, in the layers panel change the oPaCiTy by moving the opacity slider to make the transition less obvious. The Opacity effect can be seen easily as you adjust the slider.

To permanently commit to the changes you make you must flaTTen layers and then save in an appropriate file format. I always duplicate the layered file first and make a copy, I then Flatten the copy and use it for output to slide or print.

Figure 4. Original scan: fetal mouse for tetralogy examination

Figure 6. Fetal mouse: finished image

Figure 5. Fetal mouse: levels adjusted

In the original scan the thicker part of the body seems to lack detail. By using Photo-shop’s Contrast Enhancement additional struc-

To apply the Levels change only to the core and leave the rest of the scan displaying the original, use the Layer Mask that is provided with the Adjustment Layer. First select the aD-jusTmenT layer. Then by using shifT-baCksPaCe the fill Dialogue box comes up. In this case se-lect Black for the contents. The levels mask is now black instead of white. The black obscures the adjustment and white would allow the modi-fication to come through completely. Now for the portion of the scan that we want to see the level adjust on we will paint white on the mask overlaying the area. Grab the paintbrush tool and use a brush with a good size, approximately one fifth of the entire image area to be modified. Set the brush to have soft edges. Make sure

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Myocardial infarct in cardiac myocardial sections

A scanner can be used to image tissue sec-tions on microscope slides. In this case the measurement of the infarct area of the rabbit's left ventricle could be done using the resolution of the scanner since we are looking at a larger multicellular region. The benefit was the ability to quickly image an entire set of 12 slides.

stained with Hematoxylin & Eosin, Trichrome, and Picro Siris red. Only minimal image adjust-ment was required. A horizontal image flip and 180-degree rotation arrange the image correctly. To see the slide labels more clearly two rectangu-lar selections around the labels were created and the levels were adjusted to see the writing more clearly. Using the Levels control, the white point was set on the level and the black point was set on the writing.

Analysis consisted of measuring the length of the infarct (red portion) of the Picro Sirius red and the perimeter of the left ventricle. Surprisingly even with CS5 Extended that particular mea-surement cannot be easily done. Any dedicated image analysis program can measure the two lengths. In this case Image Pro Plus was used.

Perfusion and infarct in rabbit heartSamples can be placed directly on the glass

scan bed to image sections of a rabbit heart that has a blue dye to identify a perfused area and a red dye to demonstrate viability. The white area is the infarct area that is not perfused. The sectioned face lies flat, giving a nice representa-tion of the tissue and staining pattern. Around the edge the tissue depth is more evident and would not be used in any analysis.

Figure 7. Original rabbit slides scan

Figure 8. Adjusted rabbit slides scan

Figure 9. Original three-color rabbit heart

Figure 10. Auto-improved three-color rabbit heart

The heart was collected at diastole using an injection of potassium chloride. The ventricle was filled with an embedding media and frozen. Then the heart was sectioned grossly into multiple cross sections and placed into tissue cassettes for cryosectioning.

For histological evaluation, sections were

Imaging permeability of blood brain barrier

This next example involved the scanning of 2mm thin sections of a rat brain and arranging the tissue sections to compare treated and un-treated areas in modeling a compound penetrat-ing the blood brain barrier. A Curves aDjusTmenT was created and applied to the scans of the two conditions. Then, using the magiC WanD, the tissue was selected and layer-by-layer the composite image was created. Text was added

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along with a crop of the scale bar to create the finished picture. Later the sections were ho-mogenized. Finally, the amount of Trypan blue was measured using a spectrophotometer. With Photoshop CS3 and the introduction of the QuiCk seleCTion Tool and refine eDge it became even easier to select the tissue areas and recre-ate this image.

Measurements of TTC-stained rodent brain sections

In developing a neuroprotective agent the medial cerebral artery occlusion model was commonly used. An artery in the rodent brain would be blocked mimicking what would hap-pen in a stroke—depriving a portion of the brain its blood supply. This would kill the brain cells in particular regions. Then, sections of the brain would be stained with a compound, TTC , that was only taken up by the brain cells that were still viable. The area of the infarct would remain unstained.

The brain sections would be placed in a 12-well tissue culture plate. To conduct the analy-

sis the sections needed to be imaged. A flat bed scanner would quickly scan the sections simultaneously at a very usable resolution. By using the scanner the imaging was consistent and repeatable. The analysis work was done in Photoshop Version 6, prior to Camera Raw or built in Analysis capabilities. Then we used Media Cybernetics Image Pro Plus Version 4.0 to measure the samples. With Photoshop Extended the workflow can be replicated and the analysis can be done entirely within the one program. The TIFF Image from the scanner can be optimized in Adobe Camera Raw. These op-timization settings can be applied to every other sample scan.

Figure 11. BBB brain section scan

Figure 12. BBB brain section layout

Figure 13. Original infarct scan

Figure 15. Red: entire tissue area Figure 16. Green: infarct area

Figure 14. Adjusted infarct scan

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Analysis of soft agar colony modelThe scanner can also be used to image the

colonies in tissue culture plates. Here a six-well plate is scanned. The image is processed in Photoshop so the colonies can be analyzed in Image Pro Plus. To increase productivity, an aC-Tion was developed to isolate the center 50% of

each well. Then those regions were moved into a tighter layout. The colony images were invert-ed and a threshold was applied. The final image was used for analysis of the number and size of colonies. This analysis was able to determine the effectiveness of a potential chemotherapeu-tic compound on different types of cancer.

I hope these examples demonstrate the ben-efit of incorporating a flatbed scanner into your research. By using another imaging tool addi-tional value and increased productivity will help you meet your goals better. It is possible that in a future article I can elaborate on the work-flows and cover their validation. Also I plan on

Figure 17. Colonies in six-well plate original scan Figure 18. Colonies image

processed for analysis

expanding the coverage of the various applica-tions used in this research, including the scan-ner software, Photoshop analysis tools, and additional image analysis programs. So please feel free to reach out to me and share what you may want to learn.