Cairn Research Limited

Imaging

When configuring any fluorescence imaging system it is useful to identify the critical elements and consider how these can be combined effectively to solve specific experimental needs. At Cairn we understand that the choice of components depends not only on scientific and budgetary requirements, but also on other issues such as compatibility with existing equipment, departmental expertise, and the flexibility to combine imaging with other experimental techniques. As designers, manufacturers, resellers and users of a wide range of fluorescence imaging equipment we are in a strong position to integrate complete systems. If your experimental requirements cannot be fully met using readily available hardware and software then we can design or source the missing elements. Imaging systems are usually based around an epi-fluorescence microscope, but can also be configured with macro lenses or using an objective housed in a dedicated chamber. The following discussion assumes the former, but please contact us with Cairn if you have any more specialist requirements.
In most cases a fluorescence imaging system comprises of the following.

Illumination source

The energy to excite fluorophores is typically provided by a mercury or xenon arc light source. The major considerations when selecting a source are intensity, stability, uniformity of field, and the wavelengths of interest. As an alternative to arc lamps it is sometimes possible to use conventional halogen lamps, lasers, or light emitting diodes (LEDs) to provide the excitation energy. We have many years experience in the design of various light sources and are always happy to advise.

Excitation wavelength selector

In the simplest time-lapse applications, where a single non-ratiometric indicator is used, the excitation wavelength can be selected using a fixed bandpass filter in conjunction with an electronic or manual shutter. More commonly the researcher might need two or more filters mounted in a filter wheel or other optical switcher. If a greater flexibility of wavelength selection, or very high switching speeds, are required then a monochromator is often a more appropriate choice. A monochromator also allows spectral scanning of samples to assist with the optimisation of signal / noise and dynamic range.

Coupling optics

The excitation light can be introduced to the microscope either with a direct optical coupling or via a fibre-optic. A direct coupling will usually result in higher intensity, but a fibre couping isolates the microscope from thermal, electrical and mechanical interference from the light source and wavelength selector.

Microscope

In most instances fluorescence imaging systems are centred around a classical (upright) or inverted epifluorescence microscope. These are broadly categorised into laboratory and research grades with the larger and more versatile research grade instruments being the usual choice for fluorescence imaging. The objective lens is the most important element of any microscope and because it is used for both excitation and emission its Numerical Aperture (NA) is even more critical for epifluorescence applications. In addition to a high NA objective it is important that the microscope is equipped with an epi and brightfield condenser, a stable stage for holding the sample, and appropriate c mount (or similar) output port(s) to couple the detector(s) of choice.

Filter cube

Excitation light is usually diverted into the objective lens using a dichroic mirror at 45 degrees immediately prior to the objective. The dichroic mirror reflects the shorter wavelength light towards the sample and passes the longer wavelength fluorescence to the detector. Selectivity of the excitation and emission wavelengths is usually enhanced by using optical filters which can be positioned in the cube if no other wavelength selectors are fitted.

Emission wavelength selector

In the simplest case, for single wavelength studies, this will be a longpass or bandpass filter fitted into the microscope filter cube. However if multiple probes, Fluorescence Resonance Energy Transfer (FRET), or ratiometric emission are required then it is necessary to monitor different emission wavelengths during a single experiment. This can be achieved either by using an electronic filter changer, or an image splitter to divert the different wavelengths onto different regions of a camera chip (or even different cameras). A filter changer is more versatile as it can accommodate more filters, but an image splitter has the benefit of being truly simultaneous and vibration free.

Camera

In most fluorescence imaging applications the detector comprises of a digital CCD camera. There are a wide range of cameras available with varying speed, sensitivity, dynamic range, pixel size, resolution etc. Typically a useful camera will have 12-bits of dynamic range, flexible binning and region of interest control, and some degree of cooling to reduce dark noise. If the application requires extremely low light levels or high speed then an image intensified or electron multiplied camera is usually used. In simpler applications it is possible to use frame-rate CCD cameras which lack the dynamic range and flexibility of fully digital cameras, but are usually inexpensive. In some specialist applications photodiode, or photomultiplier arrays are used instead of CCD detectors.

Workstation

Data acquisition and analysis is usually performed by a dedicated PC or Macintosh computer. Real-time imaging can be memory intensive so the computer should be equipped with plenty of RAM and hard disk memory. A large monitor and CD or DVD writer are also useful, but the processor speed and graphics card of any modern computer should be more than adequate. Depending on the number of peripherals to be controlled by the computer it might be necessary to have a large case to accommodate multiple control boards.

Software

The acquisition and analysis of fluorescence data is usually handled by dedicated real-time imaging software. There are a wide range of commercial packages available which will manage the control of all aspects of the experiment and present and analyse data in a user-friendly way. There are systems to suit a range of budgets and most adopt a modular approach so that the end-user can purchase only those elements that he or she needs.
Off-line analysis of fluorescence data can also be performed by inexpensive (or free) general purpose imaging applications, but this will require a degree of expertise within the laboratory, and possibly some basic programming skills. Rudimentary timelapse acquisition software is also supplied at little or no cost with most digital cameras, but this is only suitable for the simplest of fluorescence applications.