Photometry and photometric systems attempt to quantify the results of biological experiments. Systems can be integrated with a wide range of common electrophysioloy / patch clamp packages such as Axon Instrument's P-Clamp and HEKA's Pulse package. Complete systems require a ±10 vdc signal to select the wavelength of the excitation source, and the detectors provide a 0 to 10 vdc output to quantify emission intensity. With only one D/A and one A/D channel needed, integrating the system with any existing system is quite easy.

These systems are usually used with a scanning monochromator such as a TILL Polychrome™ illumination system as the excitation source. However stand-alone packages are also available that contain a control unit, a ViewFinder assembly, and a detector. The detectors can include a photomultiplier tube or a solid state photodiode. ASI offers a photomultiplier, as well as our FluarQuant software package. A brief overview of the various system components and system packages is listed here.

Excitation Source

Monochromator

A monochromator is commonly comprised of a xenon light source, galvanometric scanner-mounted grating, and mirror optics. Monochromatic light is coupled to specially designed epifluorescent condensers via a solid quartz fiber. Specially designed condensers are available for most microscopes to provide optimal illumination.

Why deal with multiple sets of excitation filters, filter wheels, epi-fluorescence illuminators and shuttering devices? A scanning monochromator takes the place of all of these components, and provides nearly a 400 nm range with a resolution of about 1 nm, in the case of a TILL Polychrome™. Just imagine all of the standard filters that this would require with conventional methods. By selecting a wavelength not transmitted by the microscope's optics, artificial shuttering is achieved to prevent photo bleaching. The monochromatic light is coupled to the microscope via a quartz light guide and condenser assembly. These UV achromatic epi-fluorescence condensers are available for virtually all major microscopes. Monochromators are usually controlled by either a ±10 vdc analog voltage or standard RS232 commands. Filter wheels at best can only provide 40 ms switching times between filter slots -- some monochromators can reach any wavelength in 1 ms. This high speed makes them an ideal excitation source for calcium and other ratioing experiments. Mechanical filter wheels can create a substantial amount of vibration. This is not the case with a monochromator unit.

ViewFinders

Pre-select detection area for precise measurements

Low level light detection with optional sensitive B&W camera

The basic function of a Viewfinder is to allow one to select a predefined exposure area for the photomultiplier or photodiode assembly that is attached to it. The exposure area is selected via four knobs that allow you to place a square aperture of a selectable size anywhere within the field of view. The image of the slit/aperture can be viewed directly through the binoculars of the microscope or via a separate monitor. If you look through the binoculars/eyepieces of the microscope you will see a light square rectangle imaged onto the specimen. This image, both the specimen and the illuminated aperture, can also be viewed on a black and white monitor via the CCD camera that is attached to the viewfinder. The basic camera is only used for selecting the exposure area and is not designed to acquire a low light image. However, more sensitive cameras with greater resolution are also available.

There are typically two different types of detectors: a photomultiplier or a photodiode detector. Make your own decision and choose which detector will best fit your application from this comparison.

Basic PMT Specifications (Detailed data sheet is available.)

Quantum Efficiency versus Dark Noise: a consideration

The major advantage of a photodiode-based detector over a PMT tube, besides easy handling, is the extremely high quantum efficiency. For most applications you will gain signal-to-noise ratio, since you are able to collect 3-5 times more photons, your detected signal will increase while excitation intensity stays constant. The trade-off is the higher dark noise of the photodiode. When your signal is small (in the range of the dark noise of the photodiode), then the PMT might come up with a better signal-to-noise ratio, since the dark noise of the PMT is very low.

You can get a rough estimate of the signal to noise ratio using the following formula:

S/N = #signal photoelectrons / sqrt (#signal photoelectrons² + #dark noise electrons²)

Signal-to-Noise Ratio vs. Number of Fluorescence Photons

• PMT: (20% QE, 20 photons dark noise)
• Photodiode: (80% QE, 200 photons dark noise)
• Therefore, for fluorescence signals larger than 1000 photons, the photodiode detector would give a better signal-to-noise ratio.

Which Detector To Choose For Your Experiment

If you will be planning on using an uncaging flash with your system we strongly recommend that you chose a solid state photodiode as your detector, as it can not be damaged by over exposing it. Even though the PMTs that we use are protected by over exposure circuitry this circuitry has been designed as a safeguard, and is not guaranteed to protect the sensitive PMT against repeated over exposures.

PMT

• Low dye concentration or small volumes: (<2 x 10⁹ dye molecules, 2 µm diameter cell with 100 µm dye)



• Kinetic measurement (single wavelength) with response times < 1.5 ms. (Note that for most fast biological processes the time constant is limited by the kinetics of the dye and by diffusion.)

Photodiode

• Typical dye concentration (<2 x 10⁹ dye molecules)
• higher signal-to-noise ratio than PMT


• If the signal-to-noise ratio reached with a PMT is sufficient, then the photodiode detector gives room to optimize the experimental design. For example:
• lower the dye concentration (less Ca²⁺ buffering)
• lower excitation light intensity (less bleaching)


• Measurements in which the signal is independent of the dye concentration. For example:
• measurement of fluorescence decrements used in estimation of Ca²⁺ currents and Ca²⁺ buffer capacities

Uncaging Flash

An uncaging flash to release caged compounds can also be easily added to our system. The flash offers a wide range of features and is combined with the excitation light through a specially designed a dual port epi-fluorescence condenser. The condenser contains adjustable apertures that allow you to easily select both the uncaging and excitation areas.

If you will be planning on using an uncaging flash with your system, we strongly recommend that you chose a solid state photodiode as your detector, as it can not be damaged by over-exposing it. Even though the PMTs that we use are protected by over-exposure circuitry, this circuitry has been designed as a safeguard, and is not guaranteed to protect the sensitive PMT against repeated over-exposures.

Flash Unit

The flash unit is an integral unit that provides a bright flash of UV light for the photoinduced cleavage of caged compounds and offers several advantages over similar products including:

Bright Optimized Output - the unit utilizes proprietary optics to deliver optimal photon flux in the specimen plane.

Low Electrical Noise - due to the integral design the unit products considerably less RF noise than other units. The device even utilizes an optical fiber for triggering to help eliminate noise.

Long Lamp Life - the flash has a life expectancy of over 10,000 flashes compared to the several hundred flashes that other systems offer.

Strobe Mode for Easy Alignment - the unit offers a strobe mode which provides a fraction of a Joule per flash and provides an easy way of aligning and selecting the exposure area.

The UV Flash unit contains two functional components:

1. The control module (FPC) contains the power circuitry, the microprocessor based control circuitry and the operator control panel. To minimize electromagnetic interference the external trigger line is made from an optical fiber connectable to a standard TTL trigger source via a special BNC to Lightguide Adapter.



2. The flash light source module (FLS) contains the complete light source consisting of a pulsed Xe arc lamp. A concave mirror forms an image of the arc within the arc itself. This yields a remarkably increased brightness of the arc. Using a toroidal mirror, the light from the arc is focused onto a quartz fiber which couples more than 90% of it into the epifluorescence condenser of the microscope.

This configuration allows for easy servicing of the system, especially for simple lamp exchange done by the user.

The pulse width of the flash can be adjusted in a range from 0.5 to 5 ms.

External triggering of the UV Flash unit is possible up to 10 Hz repetition rate, depending on the pulse width. For repetition rates above about 0.2 Hz the number of single flashes per sequence is limited to 30 shots. After this number of flashes the unit needs a short thermal recovery period with an automatically set duration. For repetition rates below 0.2 Hz no limitations apply.

The UV Flash unit can be run in different modes:

1. Single flash:  delivers more than 10¹⁵ photons per shot in the microscope



2. Strobe Mode:  very low intensity mode for alignment and preparation

The discharged energy and the corresponding number of photons/m² are dependent on the selected mode:

A full shot in the single flash mode will yield the following spots and photons/m² in the specimen plane, depending on the used objective:

With a given fiber diameter of 1.25 mm and a numerical aperture of the fiber of 0.25, the given spots and number of photons/m² can be achieved. The light at the exit face plate of the fiber may be demagnified (concentrated) to a 240 µm spot with a NA = 1.3 objective (1.25 x 0.25/1.3), while with a NA = 0.90 objective a spot of 1.25 x 0.25/0.9 = 347 µm is the minimum. Any further demagnification would only over-illuminate the objective's pupil and will not increase the brightness.

A full shot will yield more than 10¹⁵ photons between 340 nm and 390 nm in the back focal plane of the microscope. Depending on the objective used, this corresponds to 2 x 10²³ photons/m² (Fluar 40x, 1.3 Oil) or 5 x 10²¹ photons m² (Achroplan W 63x, 0.9W) in the specimen plane.

Note: The discharge of 80J of energy under high voltage and rapid trigger conditions during the ignition of the Xe arc lamp is a source of Rf noise. Although TILL Photonics uses a double shielded casing for the UV Flash unit and a non-metallic trigger line (lightguide), sensitive experiments (e.g. patch clamp measurements) may be influenced by the discharge.

Dual Port Condenser

Installation of Epi-Fluorescence Attachment for Two-Port Olympus BX / UV Flash

FluarQuant Software

The FluarQuant package is a simple and easy to use photometry package built under National Instrument's LabView. FluarQuant allows the user to set up complex real-time experiments where fluorescently-labeled cells can be excited at any wavelength and the quantity/intensity of the emitted photons measured via a sensitive Photomultiplier Tube (PMT) or photodiode.

The FluarQuant package can also be integrated with one of ASI’s automated XY stages and µ-Inspector microscope to provide a fully automated, microwell screening system for micro arrays, as well as, 96, 312, or 1024 microwell plates.

The complete FluarQuant package is comprised of the following components:

• A high speed scanning monochromator for fluorescent excitation
• A sensitive photomultiplier tube (PMT) for fluorescence detection & quantification
• A Viewfinder device for precisely pre-selecting the exposure area for the PMT
• A high speed D/A & A/D board mounted in a Pentium Windows-based computer that controls the monochromator and accepts analog data input
• The easy-to-use FluarQuant software running under LabView

TILL Polychrome™ Monochromator Specifications

Photomultiplier Features

• High Sensitivity
• Manual or externally programmable PMT Gain
• Automatic overexposure shutdown
• Wide Dynamic Range
• C-mount fits most camera ports

Photomultiplier Specifications

ViewFinder Specifications

The Viewfinder assembly provides a convenient means for selecting, viewing, and adjusting a rectangular spot of exposure for the photomultiplier tube. The unit consists of four manual adjustment knobs for adjusting the spot size, and a video camera for viewing the exposed area on a video monitor. 50% of red light and 80% of infared light reaches the CCD, while 95% of the fluorescence light reaches the fluorescence detector.

FluarQuant Software Features / Specifications

• Acquire the analog data from the PMT at a rate of 10Khz (0.1 ms) – This is system dependent actual acquisition speeds will vary from system to system depending upon the speed and configuration of the computer being used
• Operates under any Windows software package NT, 98, or Windows 2000Ò
• Display raw data or ratios in real time
• Utilize the high speed monochromator to provide an excitation wavelength from 330 to 680 nm to work with any fluorescence probe
• Synchronize the data acquisition with electrophysiology packages or other devices via TTL pulses
• Transfer data to any software package via standard tab delimited, and Excel files
• Modules are available to control ASI’s line of precision automated stages and µ-Inspector video microscope to provide a complete system for rapidly screening micro arrays, micro wells, and any other area of fluorescence detection / quantification

Summary / Complete System Description

The complete photometric system consists of a ViewFinder for selecting the exposure area, a photomultiplier or photodiode as a detector, and an excitation source which is usually a scanning monochromator unit. A separate control unit is provided to house the control cards for the detector(s). The detector assembly provides an analog output 0-10 vdc to quantify the emission intensity, while a TILL Polychrome™ monochromator requires ±10 vdc to select the excitation wavelengths. These simple analog controls allow the system to be controlled by virtually any software package. This includes HEKA's Pulse program to provide simultaneous patch clamp recording and photon detection. An uncaging flash can also be easily added to the system as well as a number of other options such as programmable D/A-A/D devices which make the ASI and TILL Photonics photometric systems truly versatile tools for research.