Image Fluorescence With A Camera

This is still a draft

• Choosing a lens. In most cases if you’re doing fluorescence imaging, you want to collect as much of the light the atoms emit as possible. There are two factors that contribute to the amount of light the camera collects: the efficiency of the lens and how far away the camera is from the light source.
  • Lens efficiency. The light efficiency of many lenses varies widely. Most photographer’s lenses are designed to be used in situations with lots of light and tend to be optimized for other features such as a larger depth of field. This makes them less ideal for imaging atoms and molecules in dim settings. The main measure of how efficiently a lens collects light is given by the camera’s f/stop number.
    • F/stop number. Most compound lenses have irises inside them. The f/stop is the ratio of the diameter of the iris to the focal length of the lens. Small f/stop numbers (ie f/1.4) have a greater light efficiency than higher f/stop (f/22) since the relative aperture size is much larger. For most lenses, shifting the stop on a lens by one corresponds to reducing the light efficiency by half. Ideally one uses a relative low f/stop lens (ie f/2.0 or f/2.8 as f/1.4 are extremely expensive and can be harder to focus) and makes sure that the lens is in the lowest f/stop setting. The only other thing to consider when selecting an f/stop for a lens is how large a depth of field is required (see ii for explanation). Using a lens with a low f/stop will result in images with a relatively narrow depth of field. For most applications, one does not need to worry about this, but there may be cases in which one would need to trade light collection efficiency for a deeper depth of field.
    • Depth of field. The depth of field describes the range of distances that are in focus when the lens is in a fixed position. Lower f/stop numbers have narrower depths of field while large f/stops have wider depths of field. Anything in an image that is outside of the depth of field will appear out of focus in the image.
  • Minimum focus of lens. Due to geometrical constraints, the closer that one can image the atoms, the more of the emitted light one will be able to collect. The minimum distance at which a lens may focus will determine how closely one may place the camera to the light source. In the lab, we’ll often also have constraints of needing to place the camera outside of the apparatus. A good general rule of thumb is to use camera lenses for which the minimum focal distance is shorter than the distance from the atoms to the walls of the apparatus.
• Getting better images. Even if you see the atoms and molecules just fine in your image, optimizing your image leads to better data.
  • Blocking light leaks. When using the MaxIm DL 5 package, tracking down light leaks is a little more difficult because the software renormalizes the contrast of each displayed image. The most efficient way to track down light leaks is to block the imaging laser and attach light blocks to the windows of the dewar. Apply tape to the edges of the windows to reduce light leaking through the sides (black photographers tape works great - Thorlabs sells it). When most of the light leaks are blocked the images displayed by the software will be gray.
    • If you are looking for a wavelength that is output in some significance by room lights, black photographers tape will not work. You will have to use thicker blackout paper or cardboard; the Thorlabs foamcore board TB4 is the best - just score it with a razorblade and crack it to easily form shapes.
  • Illuminating atoms or molecules.
    • One thing to keep in mind is that you don’t want your laser to be broader than depth of field of your lens. This leads to some of the fluorescent light being out of focus and blurring of the image. It can be difficult to see that this is limiting your ability to focus on the atoms or molecules so it’s good to make sure that this will be the case when setting up the camera.
    • Reducing scatter of laser light into camera. While some scattered light from the laser is unavoidable, there are a few techniques to reduce scatter light from saturating the camera and/or overwhelming your signal.
      • If at all possible, try to send the light out through windows in the dewar. 
      • If the laser must shine on a surface, try to make it optically black and place the camera such that the scatter point is out of the field of view of the camera.
      • Place a black background behind the atoms or molecules that you want to image. We’ve used charcoal plates that have worked pretty well.
      • Never try to iris the light near the lens. This has the effect of raising the effective fstop of the lens and reducing its light collection efficiency.
  • Focusing the camera.  
    • Make an educated guess as to where to set the focus to look for the signal. If there is a screw or landmark near where you expect to see signal, focus on it to start searching for atoms or molecules.
    • After seeing some signal, take a few minutes to refocus the camera to make the image of the atoms as sharp as possible. This is easy to do in the continuous mode with long exposure times.