The device allows one to take multiple images of the slide under high magnification, which can later be stitched together on a local or remote computer, paving the road for more accurate image analysis.
Step 1: What You Will Need
• Lens assembly for the microscope – This can be newly purchased or can be salvaged from an old student microscope. We decided to buy a low cost student microscope ($50) and use the lens assembly for our project.
A 15X eyepiece and 45X objective lens were used for our project.
You will also need a 3.5 cm x 3.5 cm one way mirror to feed inside the periscope (as per 3D printed periscope design).
• Miniature stepper motors (2 nos.) from old CD-ROM drives – These can be easily sourced from second hand stores for less than $10.
One could also buy these along with a stage and movable carriage - http://www.aliexpress.com/item/New4-5V-2-phase-4-wire-stepper-motor-with-screw-drive-slider-slider-for-DIY-miniature/1139615226.html
The stepper motors are used to move the slide in a grid-wise manner.
• Microscope body – There are multiple options available for this
o Retain the microscope stand from the student microscope and use it to house the slide movement mechanism
o A good 3D model can also be found at http://www.thingiverse.com/thing:92355
o We created our own design to best suit our needs. The modified design eliminates the additional space under the slide base, making the design more compact and ergonomic. The design also incorporates a mounting arrangement for the motor assembly and allows for unobstructed movement of the slide holder.
• Webcam to capture individual slide images.
• Stitching of the captured images is carried out using Fiji, an open-source image stitching tool - http://fiji.sc/Image_Stitching
• 0.5W LED – This is placed under the slide and is used for illuminating the image under the microscope
Step 2: Hardware Setup
The hardware setup is fairly straightforward. As can be seen in the pictures, we utilized a motor assembly along with a movable carriage that slides along the length of the axis.
Stack the 2 miniature stepper motors (along with the stage) on top of each other so that the bottom motor moves the motor on top along its shaft axis. The axes of the 2 motors are at right angles to each other, which allows us to generate the X-Y movement of the slide.
We need a holder to mount the slide, which can be easily 3D printed (a very simple design is attached). The slide holder is attached to the carriage of the top motor, facilitating movement of the slide along the second axis.
Instead of using a linear lens assembly, as is found in all commercial microscopes, we decided to redesign the assembly and came up with a more compact setup. The new design attempts to have the same optical distance but manages to do in a smaller volume by utilizing a mirror that bends light at 90 degrees.
While setting up this assembly you will need to cut a piece of mirror into a 3.5 cm x 3.5 cm square. Please be careful while doing this (goggles, gloves and proper glass cutting tools recommended)
Step 3: Interfacing the Stepper Motors
Gridwise movement of the slide is achieved by using one motor at a time, as shown in the figure.
The arduino is also connected to the 0.5 W LED which acts as the light source for the microscope.
Be sure to use a multimeter to check all connections.
For your safety we recommend all wires are enclosed or wrapped in heat shrink tubing. It will be safer and also give the device a cleaner look.
The arduino will be connected through USB to a computer. Its voltage will be a constant 5V.
The stepper motor drivers need to be connected to an external power supply with a voltage range of 3-6V depending on the rating of the stepper motor used. For our purpose we found the best voltage to be 5.5V.
Be careful when trying different voltages as the stepper motor drivers are very easy to burn out.
Step 4: Image Capture
Note: The image might have to be cropped to remove any dark spaces around the region of interest. Also, ensure that there is significant overlap between successive images. This is done in order to ensure accurate stitching of the images in the next step.
The code for image acquisition has been developed in Microsoft .net 4.0. Microsoft Visual Studio 2010 and Microsoft Expression Encoder were used for developing the code.
Step 5: Image Stitching
We used Fiji's image stitching package (http://fiji.sc/Image_Stitching) to put together the different slide images. The software package offers different stitching plugins (pairwise, grid/collection stitching etc.) that can be readily incorporated into your code.
A lot of testing was carried out with the pairwise stitching plugin and we decided to use the same in our software. The code stitches two images at a time, creating a temporary image that is then stitched with the subsequent image. Although slower than grid stitching, this approach seemed to offer better results with the sample images.
The attached code can be used for stitching a set of linear images (as against grid), but can be easily modified to accommodate images arranged in a grid.