C & L Instruments Multiwavelength Fluorescence Photometry Systems 

Multiwavelength Fluorescence Photometry Systems

Overview

We off several types of fluorescence photometry systems for inverted microscopes.  If you have an standard upright microscope, several of these systems may also be used.  

The components that make up a complete system can be configured in a manner to match your requirements and your budget.  A simple and inexpensive system consists of a controller (PC-DAQ) and the D48 Detector (System 1).  Using your existing microscope illuminator, this allows to quantitate fluorescence intensity using up to eight emission wavelengths.  The addition of a filter changer (Model MFW-4) allows you to automate the selection of the excitation wavelength without moving the microscope dichroic cube (System 2 and 3).   Finally, addition of our S48D Illumination Source provides a Xenon arc source for fully automated multiwavelength excitation (System 4). 

These components can also be used to measure fluorescence in cuvettes and with the addition of our cuvette accessory (Model CV1).  The addition of a camera allows multiwavelength and ratio imaging.

C&L's design allows for:

• High speed and multiwavelength capability.  The Dye Fluorometer can measure eight separate measurements at eight wavelengths in eight milliseconds and display the data in real time.
• Optical purity in the selection of excitation and emission wavelengths, resulting in very low background signals.
• The ability to use combinations of dual wavelength excitation and dual wavelength emission dyes. Two or more dual wavelength dyes can be monitored in the same experiment.
• Software control of illumination intensity.  Illumination intensity can be precisely varied over a 50-fold range for any wavelength pair.
• Simultaneous acquisition of 8 channels of analog data to collect signals from other instruments. This eliminates the need for collecting other data with secondary recording devices and the difficulty in equating secondary events to changes in fluorescence.
• Low cost of ownership.  Get into multiwavelength, high speed fluorometry for much less than you would pay for less versatile instruments from other manufacturers.

·         New Mini Series Perfusion chambers are available.   The Mini Series Thin Window chamber forms a 100 micron thick chamber with one body part and two coverslips!

·         NEW 3D Interactive Model!

·         New C-Mount photomultiplier detectors, Models DPC-BA and DPC-MA provide simple and cost effective photometry.  See them here.

·         Incubate coverslips in standard fluorescence cuvettes using our CCH-1 and CCH-2 inserts.  Very nice.

·         New FluorMeasure^TM Software allows for:

o    external data acquisition triggering.

o    data synchronization with electrophysiology systems.

o    time-lapse fluorescence measurements.  See details!

·         Measure O₂ consumption simultaneously with fluorescence.   Details here.

 Applications

The fluorescence systems offered by C&L Instruments support a wide range of applications. Our fluorometer systems connect to fluorescence microscopes, a cuvette assembly and a bifurcated optical probe (for surface fluorescence measurements). The modular construction of our instruments, together with the ability to measure fluorescence from up to eight excitation-emission wavelength pairs, makes this a remarkably versatile system. The following data illustrate this versatility, as well as the sensitivity of our instruments. Click on the images for larger versions.

Fluorescence Imaging
Our fluorometer systems can now be used for fluorescence imaging and ratio fluorescence imaging! We support a variety of analog and digital cameras. You can use our Model S48D Illumination Source together with a camera and our FluorImage software to image the fluorescence emission from single cells. You can also use your existing microscope epi-illumination light source for imaging.

Ion Analysis (Ca²⁺, Mg²⁺, Na⁺, pH, DY, etc.)
There are numerous applications in which fluorescence can be used to measure ion concentrations or other biological parameters, such as pH and membrane potential (DY), using fluorescence indicators. The fluorescence from these type of probes can be monitored singly or, in some cases, together in dual-probe combinations in single cells and in solution. The following examples illustrate how different configurations of the Dye Fluorometer have been used in the research laboratory.

Calcium transients in isolated myocytes

In these experiments, the Dye Fluorometer was used to measure calcium transients in cardiac myocytes isolated from the ferret.  The effect of electrical depolarization and caffeine are shown in this figures.  Data were collected at the rate of 100 samples per second.  Myocytes were loaded with the calcium indicator fluo-3. 

Tissue Surface Fluorescence
The C&L Dye Fluorometer can connect to a bifurcated silica fiber optic probe for sensitive fluorescence measurements using the technique know as surface fluorescence. 

Calcium in the isolated perfused heart.  In this application, alternating 340 and 380 nm light from the Model S48D Illumination Source is used to illuminate the specimen using one "leg" of a bifurcated fiber optic probe.  The common end of the probe serves to illuminate the specimen (the left ventricle of the heart in this case) and collect the fluorescence emission from the heart.  Emission light is collected and travels to the Model D48 Detector using the other leg of the bifurcated probe.  The absence of any dichroic mirrors in this arrangement allows the experimenter to use any wavelengths for excitation and emission.

Estimation of cardiac oxygen tension by surface fluorescence using the inner filter effect.

In this application, hearts were loaded with two fluorescent dyes that either absorb (coumarin carboxylic acid) or emit (rhodamine) light at the same wavelengths as endogenous oxygen-binding proteins in heart.  Myoglobin absorbs light at the excitation wavelength of coumarin carboxylic acid, while cytochrome aa₃ absorbs light at the emission wavelength of rhodamine.  Changes in the oxygenation state of these proteins cause their wavelengths of maximum absorbance to change, which can be observed as a change in the fluorescence properties of these probes due to the inner filter effect.