Microscopy always brings a challenge for imaging truly 3D subjects; since the depth of focus of most optical elements is small. Thus one choice you have as a microscopist is to squeeze something really flat in a slide – but this option will most likely kill and distort the sample you are trying to image. Another trick; which I describe briefly here – is focus stacking. This is a very common technique used in macro photography (you can read more about it at many places 0 including here: http://www.cambridgeincolour.com/tutorials/focus-stacking.htm or https://iso.500px.com/tutorial-focus-stacking-for-landscape-photography/).

To achieve a high depth of focus image using Foldscope; firstly you need to take images at different focus with a foldscope and use simple tools to “blend” all the images with focused regions to form a single image which is in focus everywhere.

A) Mount your sample in usual ways. Choose a sample that has some three dimensionality to it – for example an insect. I choose an unidentified insect that was walking in the root system of a patch of grass growing in my balcony.

B) Keep the foldscope and your cellphone to record the images from on the table (I had described a simple way using A4 sheets of paper before – see an old post: http://microcosmos.foldscope.com/2015/01/20/simple-way-to-keep-fixed-focus-on-foldscope-use-paper-stack/)

C) The goal is to take a few images at different focus; and we will merge/blend them together to form a single image. I am using an iPhone6 right now; and in camera->photos; when I select a timer – the phone naturally collects 10 or 11 images in a quick succession. So all I need to do is change focus while these images are taken. I simply achieve this by putting a small weight on the phone; and quickly but gently removing the weight while the pictures are being taken. This allows me to translate the focus very gently but precisely in a quick succession. You can also do this using the standard imaging by putting the phone on the edge of the table.

Here is an example of two of the total 10 images I captured. The different focal planes is clearly visible.

D) I also tried an automated way to generate a focus stack. Since an autofocus feature exists in most common phones now a days – I used a a feature to move the focus in an automated manner. This is exposed in many apps – one that I tested and got to work well is “Stay Focused”. I am still not happy with the results for the same (since it collects the images but does not save them). Once I have the exact processed worked out – I will post it again here.

http://petapixel.com/2015/01/21/stay-focused-app-brings-focus-stacking-iphone-camera/

E) Now once you have your stack of images – you can use several simple tricks to make a focus stack.

1) Use Photoshop to just automatically blend the images. See video tutorial here:

https://www.youtube.com/watch?v=gIh4v4RPQ70

Here is my result from the same.



2) Since not all of us have access to Photoshop – you can also use free tools; such as gimp or imageJ to blend images to make a focus stack. See video tutorial for the same here:

https://www.youtube.com/watch?v=wnPRom4xtvA

Here is my result for the SUM and AVG techniques described above. I am yet to try GIMP.

3) For some really good results – several professional (but expensive) tools exist – one of them is helicon focus. People can comment how much they like and the quality of results obtained. Link here: http://www.heliconsoft.com/heliconsoft-products/helicon-focus/

cheers

manu

ps: Now, can anyone identify this special bug. It’s very small (roughly 2mm in length); and has a very peculiar pattern on the head and on legs (stripped red legs). I found this inside the mud in roots of grass. I will post videos of trachea and pumps in the live organism in my next post.