4 No Cost Ways To Improve Your Microscopy Image Quality
Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.
Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the formula R = λ / 2NA. Remember, this is theoretical because we don’t image our sample in a vacuum with perfectly aligned optics and materials with perfect uniformity. The iScope calculator is a fun way to try out different objects in a widefield microscope.
Often, we can’t just buy a new objective to solve our image quality problems. Therefore we need to explore other cost friendly alternatives to augment image quality. Here is a list of things that you can change to improve your image quality with little to no cost.
A bad sample can never be fixed by a good microscope.
Whenever you see an outstanding image in a publication, remember that it all started with a meticulous sample preparation technique. Aim to have a low background on the sample so the signal can be easily differentiated.
There are several causes of high background due to sample preparation with the two most common ones being: insufficient blocking or washing. Catch these issues by running proper controls, such as a sample with secondary antibodies alone. Check out a previous blog on controls for more details.
Figure 1. Comparison between contrast and resolution for image quality. High resolution without also obtaining good contrast dismisses the quality of the image. Image from Photography Life.
Choosing The Best Objective
All objectives of the same magnification are not created equally. You may not even need the highest magnification possible. Why?
There are many types of lenses in an objective. Based on the type and quality, they range from $100s to $10,000s. I will post a whole article on all the different features of an objective at a later time.
For our purposes, we will focus on the combination of numerical aperture and magnification on fluorescent images. Oftentimes you have a limited photon budget (amount of photons you can produce before bleaching), so you need to be as efficient at collecting the light as possible.
The image brightness is Image Brightness (Fluorescence) = NA4/M2.
This practically means that you want the highest numerical aperture with the lowest magnification that you can obtain. On our system, we have a 40x/0.95(NA) dry objective, a 40x/1.1 water objective, a 63x/1.2 water objective, and a 63x/1.4 oil objective.
Why do we have these different objectives?
40x/0.95 air is necessary for multi position time-lapse because water evaporates. The 40x/1.1 water is the most expensive as it can collect the most light (brightness) of all the objectives. Ideal for any dim samples. Our 63x/1.2 water is ideal for short-term live-cell imaging experiments. Use 63x/1.4 oil objective when you need the best resolution, but sample brightness is not an issue. If you are lucky enough to have a core facility you can visit, ask them what objective they might suggest for an experiment. Their answers might surprise you.
Figure 2. Comparison of light-gathering power (brightness) of different objectives. Two good examples that demonstrate how magnification and numerical aperture affect brightness is to compare the 20x/0.75 to the 40x/0.75 or the 40x/1.3 to the 100x/1.4. The 20x/0.75 objective will produce images that are 4-times brighter than the comparable 40x, but have the same resolving ability. Similarly, the 40x/1.3 oil objective is much brighter than the 100x/1.4 oil with only a minimal drop in the theoretical resolution. Table from Molecular Expressions.
Why does all this matter? If you have a dim sample and are not using the objective that collects the most light, you may have a high theoretical resolution, but low contrast (See Figure 1).
Noise from your detector hampers the ability to differentiate signal-to-noise in dim samples. Sources include read noise, dark current noise, photon shot noise, and clock-induced charge. Noise causes the camera or photomultiplier tube (PMT) to register a photon that was not actually there. The good thing about noise is that it is random, so unless there is a problem with your detector, two otherwise identical images would have a different noise pattern in the background. Averaging takes advantage of the randomness of noise. If you take two (or more) images in quick succession and mathematically average the image, where there is a true signal the large number will remain while noise will be halved. If speed (temporal resolution) isn’t important for your experiment, then averaging may be a way to improve your image quality. Molecular Expressions has a handy interactive tutorial for the effect of averaging on images with different noise levels.
Theoretical resolution assumes that all the light paths are correctly aligned and there is no dust, debris, or dirty objectives interfering with the light paths. The easiest step to augment image quality is by setting up a cleaning protocol for the microscope and keeping a dust cover over the microscope when it is not in use. A clean and well-maintained microscope will provide significantly better image quality (see Figure 3).
Many manufacturers are happy to provide detailed instructions on how to clean their instruments. Zeiss has an article on microscope care online that is very useful and comprehensive.
Widefield microscopes too require regular alignment of the light paths. Specific places to check are the condenser (Kohler illumination), lamp filament alignment, and the phase ring. Unless you are a very experienced user; leave the alignment of lasers to the service engineers . For this reason, it is critical to have a yearly preventative maintenance visit for any laser-based systems.
Figure 3. Comparison between a clean objective (a) and a dirty objective (b) when imaging the same sample slide. It is difficult to resolve the cells with a dirty objective, despite the theoretical resolution being identical. Image from Zeiss Campus.
Theoretical resolution is based on a perfect system in a vacuum, but as biologists, we know that this is not the reality. To obtain the best resolution and quality of images it is important to care for your microscope, as well as critically think about acquisition parameters before starting your experiments. You might not have the cutting-edge and most expensive systems at your disposal, but with good sample preparation and careful imaging, you can produce images that are on par with anyone.
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