Cairn Research Limited

Photometry

In many instances live-cell researchers are interested in monitoring fluorescence changes over time, but without needing the detailed spatial information offered by imaging solutions. By using single point detectors and an appropriate aperture, to frame a Region Of Interest (ROI), it is convenient and relatively inexpensive to record dynamic events with high time resolution and excellent signal-to-noise. Additional advantages of photometry are that it is very straightforward to combine with electrophysiological recordings and that it generates compact data files which are easy to store and process. Photometry systems are most 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.
The key elements of a photometry system are described below:

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, 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 photometric applications where a single non-ratiometric (or ratiometric emission) indicator is used, the excitation wavelength can be selected using a fixed bandpass filter in conjunction with a shutter. For ratiometric excitation probes or where multiple probes are used it is necessary to have a very fast method of switching excitation wavelengths. Stepping filter changers are typically not fast enough, so it is best to use a monochromator, continuous spinning wheel, or other custom device.

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. This is often important for photometric recordings which are typically combined with electrophysiology.

Microscope

In most instances fluorescence photometry 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 more advanced applications. 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 a c mount output port to couple the detector(s).

Filter cube

The 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.

Framing aperture

To make useful microphotometric recordings it is important to define the Region Of Interest (ROI) that the photodetector is measuring. This is usually achieved by fitting a rectangular diaphragm onto the primary image plane of the microscope and relaying this to the photodetector(s). An iris aperture can be used instead but this is clearly more restricted in terms of the shape of the ROI.

Photodetector connector box

In order to define and monitor the ROI for photometric measurements a basic CCD camera can be used in conjuction with the photomultiplier(s) or other photodetector(s). In a typical configuration the ROI will be projected onto one or two detectors positioned at 90 degrees to the image plane with a CCD camera in the normal position. The ROI can be monitored in realtime using far or infra red brightfield light without influencing the sensitive fluoresecence measurements. The wavelengths of interest are diverted to the appropriate camera or photomultiplier using dichroic beamsplitters. The relaying optics and beamsplitters are housed in a light-tight photomultiplier connector box.

Photodetector

For single-point photometric measurements a photomultiplier is the most common choice of detector. These are relatively inexpensive and have the high gain, and dynamic range useful for fast fluorescence recordings. It is important that a tube is selected with low dark noise and high sensitivity at the required emission wavelengths. Photodiodes are a viable alternative in some applications, but their enhanced Quantum Efficiency (QE) rarely compensates for the lack of inherent gain.

Viewing camera

This can be an inexpensive monochrome frame-rate (CCIR or RS170) CCD camera connected to an analogue monitor or basic frame grabber. Alternatively a firewire or other digital camera can be used and especially if combining photometry with fluorescence imaging. The most important consideration is to ensure that the camera is not fitted with an integral infrared blocking filter, which will be counter productive.

Photometry control rack

Because photometry is typically used to track fast dynamic responses, it is best to control experiments with hardware precision rather than relying on the vagaries of Windows software. This is typically achieved using a dedicated electronics rack to power and control the illumination and detection components.

Photomultiplier power supply

Photomultipliers require a stable high-voltage (-700 to -1200V) power supply. One or more power supply modules would usually be housed within a control rack.

Photomultiplier amplifier

The raw output of a photomultiplier is a small negative current. To be usefully processed by an acquisition system this signal will require inversion, amplification, and either integration (over a specific time interval) or current-to-voltage conversion with appropriate low-pass filtering. A flexible photomultiplier amplifier will provide for all of the above and should also provide an offset facility to compensate for background fluorescence prior to digitisation.

Demultiplexer

If a single detector is used to sequentially sample fluorescence at different excitation and / or emission wavelengths then it is important that the signal is sampled appropriately to give meaningful results. This can be achieved by synchronising the Analogue-to-Digital (A/D) conversion to the wavelength switching, or more flexibly by using sample-and-hold amplifiers to store the appropriate data for asynchronous digitisation.

Signal processing modules

Raw photometric data will often require simple processing in order to produce useful results. This would typically take the form of background subtraction and ratioing or averaging of individual channels. Dedicated photometry software will usually handle these operations, but it is sometimes preferable to carry them out prior to digitisation, in which case analogue processing modules can be used.

Workstation

Data acquisition and analysis is usually performed by a dedicated PC or Macintosh computer. Photometric acquisition is not demanding on computer resources so any modern PC will be fine, although a large monitor and CD or DVD writer are recommended.

Software

The acquisition and analysis of photometric data is usually handled by dedicated real-time fluorescence or electrophysiological acquisition software. There are a wide range of commercial (and public domain) packages available which will manage the control of all aspects of the experiment and present and analyse data in a user-friendly way.