Fluorescence is used as a major tool in biological research to provide contrast formation in imaging applications. Quantification of fluorescent signals has become an important tool to analyze cellular structure and function. One reason for the success of fluorescent imaging in cell biology, besides the excellent availability of labels including fluorescent proteins, is the cost-efficient use of equipment. A standard fluorescent microscope is equipped with a number of filter sets, consisting of exciter, emitter and dichroic, and a lamp for excitation. However, the power output of light sources changes over time and the total energy delivered to the specimen will depend on the optical path of the microscope and the filter sets and objectives used, as well as on their alignment. Changes in the environment, photo damage to the filter sets and run time of the light source provide effects that will lead to a deterioration of the transmission characteristics of the microscope, adding to the biological variability of the results and making comparison of experiments difficult. Intensity standards have been used for calibration and quantification1–4
and light-emitting diodes (LEDs) have been used to generate an adjustable signal. Imaging of the LED output through the microscope provides a calibration method for the optical system5,6
. Microscopes used for single-molecule detection are often equipped with laser lines for illumination. This allows calibrating the excitation intensity to compare results based on this parameter using calibrated photodiodes7,8
Currently, most labs use beads and dyes as the standard way to calibrate a fluorescence microscope because they are easy to use and are widely available.1,2
The only limitation is that the actual amount of light used to excite the sample is still unknown.1,2
The amount of light can be translated into a power density that allows a direct comparison of experiments, independent of the equipment, for example, objective lenses or filter sets. In the case of photobleaching or photoactivation experiments, a defined amount of applied power is crucial for repeatability of the experiment9
. For imaging, knowledge of this parameter can be used to define the detection threshold and is necessary for quantitative analysis of the image brightness10
In summary, we describe a rapid way to calibrate the amount of light/heat delivered to the specimen for any configuration of a standard research grade fluorescent microscope which we have used in our studies on single-molecule mobility in living cells7,8
. This method offers a tool to directly compare results of experiments performed using different optical equipment and microscopes. The major limit to the precision of this method is the variation between the transmission of the objective as provided by the producer and the real transmission. The simplicity and ease of the measurement make this calibration feasible for labs that do not have an extensive background in physics.
This section contains background information on the optics and concepts related to this protocol. gives a schematic of the light path and where to perform the measurement described in this protocol. shows a photograph of the components used for this protocol. Because laser light is monochromatic, its intensity can be measured using calibrated photodiodes. However, to measure the intensity of light that covers a certain bandwidth, for example, 40 nm passing through an excitation filter, a thermo-coupled detector is needed. Thermal detectors measure the temperature increase that results when the detector surface absorbs light energy, which provides a wavelength-independent measure of the power of light. The goal is to obtain a measurement of the intensity used to excite fluorescence in a given experiment.
Figure 1 Principle setup and components. (a) Beam path of an inverted fluorescence microscope. Fluorescent light is provided by a lamp (LH) and delivered to the objective by the tube lens (TL). The spectral region of interest is defined by a band pass filter (F), (more ...)
Each objective has a back opening of a defined diameter, acting as an aperture to limit the beam diameter that is allowed to enter the objective. Light delivered to the objective is focused into the back focal plane of the objective by the tube lens in the microscope stand (see ). The power measurement is best done above the objective turret without an objective in place. One reason for this is that if a high numerical aperture (NA) immersion objective is used, immersion media would be needed between the objective and the power detector. In addition, many objectives will not fit into the detector head. Removing the objective, hence, helps to avoid damage to the objective’s lens. Placing an adjustable iris centered on the turret opening allows adjustment of the beam diameter to the same size effectively seen by the objective (). Chopping the beam diameter eliminates the need to measure the actual power profile of the beam (see ). A lens with a short focal length is used to focus the light from the objective turret onto the power detector that can be mounted on the microscope stage ( and ). The focused spot should have approximately the size of the active detector area to provide accurate power measurements. Using the transmission curve and field of view of the objective, the intensity measured at the turret can be translated into a power density at the sample. presents data taken on an Olympus IX-81 stand for different standard filter sets.
Figure 2 Beam profile along the optical axis of the microscope. (a) Photograph of an IX71 microscope stand for orientation. Instead of an objective, a screen is installed to visualize the beam profile. A scale (metal ruler) is installed on top of the objective (more ...)
Figure 3 Accounting for differences in the objective back aperture. (a) Calibrated iris with 5-mm opening. (b,c) Photograph of two different high numerical aperture objectives. The open apertures of the objectives are largely different. (d) Calibrated iris with (more ...)
Figure 4 Lateral intensity profile of the excitation light. (a) Photograph of the beam profile on a screen 12 cm above the objective turret. The position is arbitrarily chosen to optimally present the intensity distribution within the beam. (b) Line profile plot (more ...)
Figure 5 Assembling of the refocusing unit. The assembly of the iris and lens is shown. (a) Thread adapter (here RMS to SM1), (b) calibrated iris that is mounted to the thread adapter, (c) lens tube that holds the lens centered on the iris, (d) short focal length (more ...)
Figure 6 Installation of refocusing unit and power meter on the microscope. Overview of the installation on the microscope. (a) Total view of an IX71 stand. (b) The iris/lens assembly is installed in an open position of the objective turret. (c) The detector head (more ...)
The power density describes how much light is passing through a defined area and might be pictured as the ‘flow’ or light flux. It directly describes the effective amount of light applied to the sample and is commonly used in laser-based applications. The unit of the parameter is kW cm−2
. The illuminated area on a fluorescent microscope is in the range of hundreds of square micrometers and less, depending on the field of view (FoV) of the objective lens, which is expressed in the field number (FN). The field of view is the field number divided by the magnification of the objective; for example, an objective with a field number of 26.5 and a magnification of ×60 will illuminate an area in the object plane of 442 µm in diameter according to equation (1)
Using the radius of the FoV it is straightforward to translate the power measured into a power density as done for using equation (2)
For instance, a 60× objective with a field number of 26.5 mm, as used in , will provide a field of view of 441.7 µm (FoV = 26.5 mm/60 = 0.44167 mm). Accordingly the illuminated area is ~3.14 × (220.85 µm)2 = 0.0015 cm2 (when scaling from µm2 to cm2 keep in mind the square so the total is 10−8). In an intensity of 0.0011 W (or 1.1 mW) is reported, resulting in an power density of 0.0011 W/0.0015 cm2 = 0.73 W cm−2, which multiplied by the transmission of 80%(as provided by the producer of the objective) leads to the power density of 0.6 W cm−2 as given in .
The intensity reading at the turret also provides a convenient way to monitor the transmission and alignment performance of all the components in the excitation path such as the lamp, light guide, lenses, filters and mirrors.