Scanning the fields of a microscope slide

Q: Do you have a preferred method/pattern for ‘scanning’ the fields of a microscope slide?  I have seen numerous patterns listed, but (like a telescope I imagine) moving the stage is counter-intuitive to what my brain wants to do when I move the image while viewing it through the
objective.  Any tips?

Lily flower TS photographed in tiles 7 X 8
starting top left to right, row by row

A: I'm assuming you have a mechanical stage.

• Start at a low power objective (10x)
• Find a random subject to focus on somewhere on the slide to be scanned
• Get your lighting set up correctly
• Then go to the top left corner of the coverslip or area to be scanned (as seen through the microscope). If your microscope gives you an inverted image, it will initially feel strange and counterintuitive (ie. you move the slide to the left, but the image appears to move to the right). However, if you concentrate on what the image does, you soon adapt.

If just scanning by eye:

• Scan towards the right across a predetermined width (e.g. coverslip width)
• At the end of your first scan, go down about three quarters of a field of view
• Scan back to the left across the slide till you reach the coverslip boundary
• Go down about three quarters of a field of view and scan to the right again.

I tend to scan at lower powers, then when I find an object of interest, I centre on it and zoom in with the higher objectives.

Lily flower TS photo panorama created using
Panorama Maker 8 using tiles as above

If you want to create a photo panorama you need to:

• Do one row scan from left to right, taking pictures every three quarters of the field of view
• Once you reach the end of a scanned 'lane', go all the way back to the beginning on the left 
• Go down three quarters of the field of view
• Scan the next row from left to right, taking pictures every three quarters of the field of view.
• Etc.

This ensures that your images are tiled in matching rows and columns. This makes for easier processing by a 2D panorama stitching program.

If you have a microscopy question, contact Chris at Chris@miltoncontact.com or go to www.usingthemicroscope.com

Finding low-contrast microbiological specimens under the microscope

My microscopy book, Understanding and Using the Light Microscope, has prompted some interesting questions, which we hope to address in later parts of the series. In the meantime, I thought I would share them on my blog.

Q: Do you have a preferred method on focusing on finding the focal plane of micro-biologic specimens when you can’t ‘see’ the specimen with the naked eye?  Do you prefer to begin by focusing on the upper surface of the microscope slide or lettering for instance?

Phase A contrast (a type of Phase contrast)
makes it easier to see very faint, transparent subjects

A: Well, there are several ways I might tackle this. In the absence of knowing what you are looking at, too many answers. But here are some to try:

Practice with a cheek scrape - you will have faint nucleated cheek cells, some of which will have bacterial colonies on them. Use minimal material and a coverslip, so sample mounted very thin in water (well, saliva actually).

1. Either
a) Focus on a known boundary (e.g. coverslip edge, dust on slide, scratch or ink on slide) and use the fine focus to search above or below that point to find your subject. OR

b) With a dried smear. Hold slide up so that you see light reflected on the surface with the sample - see if there is an area which looks less reflective due to more sample having dried there. Start trying to find something to focus on there. Then migrate away to a part of the slide where your sample is more thinly distributed.

2.  Use a method that increases the sample contrast against the background.

a) OBLIQUE ILLUMINATION: Start with a low power objective. Create oblique illumination by cutting off light entering one half of the condenser (a crude but effective way is to use a finger between the lamp and the condenser). Low contrast subjects are thrown in relief and stand out more. Once you find and focus on the subject, zoom in by going through to your higher objectives, again using oblique illumination. When at the highest magnification and focused on the sample, revert to standard optimum lighting.

b) DARKFIELD: Use of a central stop under the condenser creates a black background. Light hitting the samples from the ring around the central stop makes samples glow against the black background. You can create your own stops with ink or card discs on centre of a transparent filter. Many condensers have a filter holder. NOTE that the condenser iris diaphragm has to be opened wider to allow the ring of light outside the field of view to illuminate the sample.

c) If you have it, use PHASE CONTRAST at the higher power. Note that the phase contrast ring in the phase condenser for higher power will act as a darkfield illuminator with your lower power objectives.

d) Consider STAINING. Dilute fountain pen ink (a dye) stains protein (and fingers). Play. Samples stained against background.

e) Consider CONTRAST STAINING with drawing pen or other particulate ink. Ink particles are excluded by subjects, leaving them light against a dark background.

3. If your samples are too sparse and difficult to see - consider creating a new slide with more sample on it.

DO NOT SIMPLY CLOSE THE CONDENSER IRIS DIAPHRAGM TO INCREASE CONTRAST - YOU MAY APPEAR TO GAIN CONTRAST BUT YOU WILL HAVE LOUSY RESOLUTION!

If you need to go above a 40x objective to use an oil immersion one, life is a bit more problematic.

• Find a visible sample using one of the above methods (oblique, dark field or phase contrast) until you get to the 40x objective (bacteria are visible as tiny spots or rods).
• Centre on a good contrast object.
• Move objective away from slide
• Add small drop of immersion oil over sample
• Swing in oil immersion objective (Make sure it is a sprung lens so you do not damage slide, sample or lens if you accidently go too close to sample and touch the slide).
• Lower OI objective to touch oil drop on slide
• Get objective as close as possible to slide/sample by looking from side and lowering carefully with coarse focus.
• Look through eye piece and use fine focus to move objective away from slide.

Note that you should also use an oil immersion bridge between the condenser (if labelled with an NA greater than 1) and underside of slide for optimum resolution.

Do you have a microscopy question? Contact Chris at Chris@miltoncontact.com or visit www.usingthemicroscope.com

My brillig adventures in English

Guest Blog by Jane Thomas, Director, Milton Contact Ltd

Holding a brand new book in your hand is like walking in newly fallen snow or perhaps like discovering buried treasure. You will be the first to open it up and discover the delights inside. Imagine how this must feel if you wrote the words printed on those crisp white pages.

It is said there is a story in all of us; that we are all able to tell the tale of our lives, or that of others, or use our creativity in ways only limited by our imaginations.

This is my story.  As every other little girl, I had thoughts of what I wanted to be when I grew up. Definitely not a ballerina (that was cissy stuff) and definitely not a fairy princess! At age 6, I wanted to be a poet.  Where this urge came from I do not know, but that was what I aspired to.

At primary, I remember having to write stories and do projects. I recall eagerly recounting the Christmas story over and over and writing vast amounts about foreign lands, their flags, main exports and landscapes. The urge to study geography was strong even then!

Any spare moment was spent outside, building bonfires, planting seeds or even with my head under the bonnet of my father’s car as he tinkered with this and that. Reading was for the acquisition of information; I’d spend hours ‘reading’ maps and atlases or scouring encyclopaedias just for the fun of it. I could probably count on one hand the number of novels I had read from beginning to end.

By secondary school I remember spending many an English lesson watching the sport outside the classroom. I could speak English perfectly well. Why would I need to learn it?!  Two lessons stick in my memory, however. One was learning Carroll’s ‘Jabberwocky’, with its amazing jumble of rhyme and nonsense and the other was reciting ‘The Tiger’ by William Blake – the first verse of which is still with me.

Thankfully, I passed my ‘O’ Level English language, despite an oral exam that asked three of us to discuss marriage in Victorian times. That I can remember the topic means it must have been traumatic. I was fifteen and knew nothing about marriage and even less about the Victorians (I gave up history at 13). Thanks in part to reading a library book the day before the essay paper and regurgitating the content as best as I was able, I added English to my list of exam successes, which later allowed me to pursue my real interest in Geography.

However, just like many an avid reader and book worm, I always had an insatiable love of books. As a child, my own collection of ‘Puffins’ lined the shelves in my room and each was issued with a carefully written library ticket; even if it was never read, it was treasured. Libraries and bookshops are still like heaven to me: Rows of colourful tomes just waiting to be picked up; waiting to take the reader on an adventure.

This love took me to a variety of jobs involving the printed word, from cataloging old regional newspapers in a Welsh library, to running the book department in a well-known stationers’ chain, to checking manuscripts and proofs for a learned scientific society.

I now find myself as a co-director for a small publisher and love helping others realise their dream to publish their own words; to create their own adventure. Whether fiction or non-fiction, the shared journey from initial draft, through editing, design and formatting to the final printed copy is exciting for author and publisher alike. And to see the face of the author when they first hold that brand new book with pride is always a moment to treasure.

Will I ever write my own? Will it be poems as dreamt of by that little girl? Who knows? At least it will be a journey full of discovery and adventure from start to finish.

Milton Contact Ltd has been helping people self-publish since 2006. Chris and Jane offer a wealth of knowledge from a wide variety of disciplines and pride themselves on their friendly and sympathetic approach to every new project.  Jane has been a co-Director at Milton Contact Ltd since 2013. 
www.miltoncontact.co.uk