BackgroundWith the increasing number of LED excitation light sources for fluorescence applications now being offered, the User needs a method of assessing their relative performance. The information below attempts to explain some of the issues and provides performance information on the CoolLED pE-series of products using the pE-100 unit as an example. Measurement is addressed in two ways:
a) Numerical data based on optical measurements
b) Subjective performance based on feedback from Users
This should help the User to decide which light source to select and when they can start to take advantage of the many operating and performance advantages that LEDs provide.
Method for Numerical ComparisonCarrying out comparisons of quoted light power figures for different light sources can be confusing and misleading. If Users want to perform numerical comparisons, then it is important that they understand how the numbers are generated.
Unlike most other LED excitation sources, CoolLED builds its pE products from bare semiconductor die to create arrays of LEDs which are actively-cooled to maintain wavelength and intensity stability whilst driving the LEDs for maximum intensity.
Manufacturers will often quote the light power being generated at the source. This can be irrelevant as only a very small proportion of the photons being generated at the source pass through the light source optics and the overall microscope light path to the sample. This is due to Lambertian dispersion from the LED surface and the limitations of optics.
Optical powers are often quoted in two ways: Total Power measured in Watts (W) and Intensity in W/cm2. For example, the intensity at the 470nm LED array source within the pE100 is 94W/cm2 with the total light power output being generated being 3.8W.
The optics within the light source, are designed to generate a collimated beam to couple with the internal optics of the microscope. Some manufacturers will quote total power output from the light source but the usefulness of this power is determined by the level of the collimation.
Ideally the most important intensity values for selecting a light source are those which are a measurement at the sample.
However the intensity at the sample is directly related to the quality and type of objectives and filters within the microscope. This makes it impractical to compare data light source data measured on different microscope systems.
In order to assist the User, CoolLED has chosen to quote intensity and power values based on measurement of the collimated beam at a distance of 200mm from the source so that non-collimated light is not included in the measurement.
Measurement Results for pE-100*
* Total power at the sample plane for the pE-1 & pE-2 will be approximatley 10-15% greater
based on a dedicated single LED wavelength module (LAM)
Method for Subjective ComparisonWhile comparing power values between different light sources might be helpful, the User is more interested in how a particular light source compares with a source they are familiar with. Unlike the broad spectrum of peaks from mercury and metal halide ‘white light sources’, LEDs produce single peaks of energy around a specific wavelength. The intensity of these LEDs varies with wavelength which is governed by the semiconductor manufacturing processes. Fluorophores also vary in their brightness and so it is difficult to provide a clear subjective comparison.
Independent tests have shown that the CoolLED 470nm LEDs through a standard GFP filter cube give 20% higher intensity at the sample compared with a 100W Hg bulb. We can state with confidence that this arrangement will give good results in the majority of applications. LEDs in the green and yellow regions are less intense and so for weak fluorophores their use may not be appropriate. However many fluorescence microscopy systems use cameras for imaging and this lower intensity can be balanced by increasing the integration times.
LED Suitability GuidesCoolLED has many hundreds of customers using their light sources in a wide variety of applications worldwide. From feedback from these customers we are able to judge the suitability of the technology in different applications.
Specific wavelengths are graded for use for applications using either a camera imaging system or visual screening. For guidance on a specific product, click on pE-100, pE-1 or pE-2.
NOTE: CoolLED operates a customer-satisfaction refund policy . If you find that the excitation is insufficient for your application, based on selection from the LED Suitability Guides for pE-100 and pE-1 & pE-2, CoolLED will provide a full refund for the purchase price of the product (see quotation for details).