Does Size Matter, Or Is Quality More Important?

Whenever we talk about imaging a sample with a customer, all too often it’s all about size.

Let me give you an example. Imagine customer A wants to buy a hotstage and he wants to make a movie of his birefringent sample morphology changing with temperature.

I think, hmm ok, so he’ll want fast frame rates so as not to miss the sample changing. He may need a large field of view to see more of his sample and polarized light so he can see the structural changes.

But he tells me,

‘I need to use 100X objective lens with a 10megapixel camera’.

He’s from the ‘more is more’ school of thought. ‘I need the sample as big as possible and I want as many pixels as possible’.

Here are the problems with this way of thinking. Using the highest magnification lens will not always give you the information you are looking for. A lower magnification lens with a higher NA and a flatter field with better colour correction may enable you to see far more detail and likely a significantly larger field of view.

What are you trying to observe with your optical set up? Are you looking for particles around 1 to 5um in size? If it’s the general change in morphology and birefringence of the sample, you may be better off with a lens with lower magnification that gives better colour correction and lets you see more sample area and depth of field. By only seeing a tiny part of your sample you may be missing the big picture.

What about imaging technique? Without phase contrast, many biological samples are nearly too transparent to see at all. Understanding when to use, phase, DIC and Hoffman modulation are critical to getting the right set of lenses for your sample.

So what about all those millions of available pixels? There are three key things to consider

1.Are you getting more detail/information by capturing more pixels, or are you just making an image big enough to cover the side of your house?

2. How well do the pixels represent your image? Is the senor you’re using to capture them, any good?

3. Are the amount of pixels more important than the frame rate of your live image?

You can’t turn a low resolution image into a high resolution image by capturing more pixels. All you are doing is capturing more of the same lousy pixels. You don’t get more information.

If you have a well resolved high resolution image then capturing as much detail as possible so that you can zoom in and perform image analysis can be a worthwhile investment.

However, if you are performing a dynamic experiment that involves your sample changing rapidly, your compromised frame rate may mean you miss capturing the crucial image. Not only that, but if your PC is also capturing other data such as temperature and sensor information, the significant strain on processing power may cause the system to fall over.

In our experience around 1.3megapixels is easily enough for any hotstage work and will give fast enough frame rates to observe dynamic sample phase changes. Make sure that those 1.3megapixels are the highest quality you can get. This means using a camera with a CCD sensor and not CMOS. If possible it should use multiple CCDs for red, green and blue and be peltier cooled to remove any noise from the image.

If you want to read some more and play with an interesting interactive tutorial that lets you try different lens magnification, NA and C-mount lens, take a look at this one on the Nikon MicroscopyU site. There's a lot tech stuff, but the tutorial is pretty simple.

Of course, there is no point in going for a high spec CCD digital camera if you have not first set up the microscope correctly. The greatest investment you can make is to learn the fundamentals of microscopy by taking a class at a recognized institution. McCrone College of Microscopy is arguably one of the best facilities in the world to learn all kinds of imaging and spectroscopy techniques. You can start by learning lots more about the basics at the excellent Molecular Expressions website which has many interactive online tutorials. I hear the McCrone College of Microscopy is also introducing a distance learning module that I’m sure will be excellent.

To take a look at which lenses we recommend for use with our hotstages, please take a look at our Optical Accessories page.