Here are few basic concepts one should know before starting your acquisition:
The objective lens is one of the most important piece of equipment of your microscope, it will determine the size of your field of view, your resolution, your sampling and depending of the quality of your objective, it will help with some optical correction.
Key parameters that you can see on the markings on the barrel of an objective lens:
It is the angular aperture of objective NA given as:
Where is the half angle of the cone of specimen light accepted by the objective lens and n is the refractive index of the medium between the lens and the specimen.
The higher the NA the more diffracted rays are collected from the specimen and then the higher the spatial resolution you could expect.
Because sinθ can never exceed 1, the NA can never exceed n, which itself has fixed values (e.g. around 1.0 for air, 1.34 for water, or 1.5 for oil).
For an interactive tutorial on the effect of using different NAs, see http://www.microscopyu.com/tutorials/java/imageformation/airyna/index.html
A higher magnification will not give you a higher resolution, however, the magnification is critical for sampling (see bellow) while working with a camera. Moreover, the ratio of numerical aperture to magnification determines the light-gathering power of a lens and hence the image brightness B:
Where M is the magnification and NA the numerical aperture.
Finally you should keep in mind that a higher magnification will yield smaller field of view.
As we have seen before, in the equation the NA is directly proportional to n then, the higher the refractive index of the immersion medium, the higher the resolution you could have. However, if the immersion refractive index of the objective does not match the refractive index of the medium surrounding the sample we get spherical aberration. This is a phenomenon whereby the PSF becomes asymmetrical at increasing depth and the blur becomes worst. Therefore, matching the refractive indices of the immersion and embedding media is often strongly preferable to using the highest NA objective available.
For example, for fixed cells, if you use ProLong® Gold Antifade Mountant which as a refractive index of 1.47, an oil () objective will be great or, if you have the option, a glycerol () objective would be perfect. For live cells, you will probably be working with a live cell imaging solution (see here for more info on how to prepare your sample) with a reflective index and therefore should work with a water objective.
This is more or less an indication of the quality (and the price) of your objective. It is indicative of the amount of colors that are corrected for spherical and chromatic aberration as well as if the objective provide flat-field correction, here is table that summarize the different optical correction available:
|Objective Type||Spherical Aberration||Chromatic Aberration||Field Curvature|
|Achromat||1 Color||2 Colors||No|
|Plan Achromat||1 Color||2 Colors||Yes|
|Fluorite||2-3 Colors||2-3 Colors||No|
|Plan Fluorite||3-4 Colors||2-4 Colors||Yes|
|Plan Apochromat||3-4 Colors||4-5 Colors||Yes|
Even the best objective will not make good image if it is dirty, ONE SHOULD ALWAYS START IMAGING BY CLEANING THE OBJECTIVE as well as the holder of your specimen.
Spatial resolution and sampling
The spatial resolution of the microscope is defined by the smallest resolvable distance between two points in an image.
Which define the Rayleigh criterion where d is the minimum resolved distance (in other words, your XY resolution), and NA is the numerical aperture of the objective lens. It's also the radius of the central diffraction disk also known at the Airy disk (see figure bellow).
On a confocal microscope it's possible to increase the axial and lateral resolution of a microscope: , However, this is possible by closing the pinhole bellow the size of the Airy unit inducing a important lost in emitted light.
Where n is refractive index of the immersion medium.
Sampling is the process of converting the continuous analog signals out of the objective into a numeric digital sequence. According to the Nyquist sampling theorem, in order to preserve the spatial resolution, the radius of the Airy disk () should be covered by a minimum of 2 adjacent pixels.
Concretely, what does that mean? Let's assume that you are working with an objective with , and with a the resolvable power of you objective is:
If you are working with a sCMOS camera, which typically have a physical pixel size, after magnification, your pixel size is:
, which will then give you a good sampling as illustrated is figure bellow:
The number of resolvable steps of light intensity, gray-level steps ranging from black to white, is called the dynamic range.
Ideally, especially if you plan in doing any image quantitation/measurements, the larger the number of gray levels the more accurate your measurement would be and also the easier the extraction of information will be.
Most of the computer monitors (and your eyes) have only 8bit gradient, so you won't be able to see the difference between an 8 bit or a 16 bit image on your screen.
After digitization (by an analogue to digital converter(ADC)) in the computer, the photon signal is displayed as shades of gray ranging from black (no signal) to white (saturating signal) on the monitor. The ADC are described in terms of their bit depth. Since a computer bit can only be in one of two possible stated (0 or 1), the bit depth is described as number of steps. Therefore, an 8 bit processor can encode steps or 256 gray levels and a 16 bit processor can encode steps or 65536 gray levels.
The higher the bit depth, the larger your image would be, an "8 bit pixel", will take 1 byte (1 byte is 8 binary digits) on the disk while a "16 bit pixel" will take 2 bytes.
Here the advice is relatively simple: "Use the lowest laser power to give an acceptable image". This is true especially working with live cells, too much laser power will be phototoxic to cells and will alter their behavior. Moreover, using low laser power will prevent photobleaching of your fluorophores.
What is an acceptable image? It's an image you acquire for quantification and chevk for signal-to-noise level.
Gain is a relative measure of the amplification one applies to the photon detection system. On a traditional PMT the signal voltage is amplified by multiplication by a factor, while on a camera, increasing the electronic gain reduces the number of photoelectrons that are assigned per gray level. In either case, increasing gain will result in brighter images. Particularly, gain does not make camera more sensitive. It boosts the noise as well as the signal and at some point as gain is increased (and the overall image gets brighter), the signal/noise ratio (S/N) starts to decrease. The overall aim is to get the highest S/N, NOT the brightest image.
Offset is an electron adjustment that shift the signal positively or negatively. In cameras, offset has to be adjusted to prevent the ADC to hit 0.
Caution: by decreasing the offset too far, very faint data will actually be lost - it is critical that this is not allowed to happen. New users are often tempted to decrease the offset value to try to eliminate sample's background or rather, non-specific signal. That should NEVER be done for the purpose of preserving data integrity.