The Importance of Power Meters in Quantitative Fluorescence Microscopy

By AZoNano Staff Writers

Table of Content

Introduction
Spinning-disk Confocal Microscope
Laser-Scanning Confocal Microscope
Conclusion
About Lumen Dynamics Group Inc

Introduction

In fluorescence microscopy, the significance of the fluorescence intensity in the images obtained is limited unless they are compared with control specimens.

For this reason, majority of users prepare a number of slides simultaneously to reduce changes in the labelling, and then proceed to obtain images from several fields-of-view on each slide.

The same microscope is used week after week, without any additional control samples. They then proceed to choose the same imaging parameters and microscope components as they used in their last imaging session.

This article highlights the common problems associated with illumination in a multi-user microscopy facility and shows how the measurement of light power at the sample plane delivers critical information on the performance of microscopy systems used in creating critical data for research publications.

Spinning-disk Confocal Microscope

For the study, an old spinning-disk confocal microscope was used; the system had four lasers integrated into an optical fiber for delivery into the microscope. The microscope earlier had problems with the laser powers reducing practically to zero.

The lasers were later re-aligned into the fiber and experiment was carried out. With the help of an X-Cite® Power Meter, laser power that was reaching the sample was calculated over a period of weeks.

First, both microscope and lasers were started up and left undisturbed for a minimum of one hour to warm up. Then, while the lasers were warming up, the microscope computer was installed with the X-Cite® power meter software.

Following this, the X-Cite® XR2100 power meter was coupled to the USB port of the computer, and the four laser wavelengths were entered as the “favourite wavelengths” in the software to facilitate selection.

A low-power objective (10x) was selected to make sure that most of the light emerging from the objective reaches the sensor. Since an inverted microscope was used for this experiment, the X-Cite® XP750 Power Meter sensor was placed face-down on the specimen stage (Figure 1).

Then, using the transmitted light illumination, the sensor was laterally placed until it was centered on the cross-hairs which are opposite the window that gathers light. One at a time, the laser shutters were opened and power was noted down at the respective wavelengths. Finally, the four logged power readings were obtained and copied to a spreadsheet.

Figure 1. X-Cite® XP750 in use on an inverted microscope

The results illustrated in Table 1, shows that the laser power at the sample reduced instantly following re-alignment. After a period of six weeks, the power was so small that fluorescence excitation was not possible in most samples. It was also found that mounts utilized in the system’s optics are extremely sensitive to touch and cause shifts in alignment. Later, the X-Cite® power meter was used to re-align the lasers and improve signal.

Table 1. Laser power measurements (µW) on an older spinning-disk confocal microscope.

Date 561nm 488nm 405nm 636nm Notes
May 25 12.1 11.4 31.0 18.0 Freshly aligned
May 27 12.3 11.2 29.1 17.7 No problems yet
June 9 8.0 5.0 25.4 14.8 Starting to weaken
July 13 5.8 3.3 0.5 14.5 405 is nearly gone!
July 14 12.1 12.3 30.1 15.3 Re-aligned

From the above experiment, it is evident that the quality of laser systems was inconsistent. In multi- user facilities, it is a well-known fact that researchers depend on these systems for their data, and poor performance of systems will lead to wasted samples and loss of time. To this end, users should monitor the laser power and adjust the alignment on a regular basis as needed.

Laser-Scanning Confocal Microscope

One of the users of the new laser- scanning confocal microscope informed that DAPI fluorescence looked good at the eyepiece, but the 405nm laser had to be cranked up higher than usual to achieve a suitable confocal image of the DAPI-stained nuclei. It is a usual practice where users blame the facility equipment, while a facility manager blames the sample or slide preparation.

The laser-scanning confocal microscope was expected to be more stable than the old spinning disk confocal microscope. The same protocol described above was repeated on the laser-scanning confocal using the X-Cite® XR2100 Power Meter with XP750 sensor.

This data was compiled with the data obtained from when it was initially procured and installed. The results of these measurements are summed up in table 2. In this case, the user was correct; the 405nm laser had actually dropped to one-third of its earlier measured intensity. In fact, all the laser powers had rapidly dropped to the same degree.

Table 2. Laser power measurement (µW) on the new laser-scanning confocal microscope.

Date 405nm 473nm 559nm 635nm Notes
July 14 70.1 240 254 153  
July 26 66.5 277 194 193  
Aug 23 12.1 92.6 109 74.9 Problem!
Aug 30 91.1 266 271 192 Re-aligned
Nov 1 90.7 280 250 166  

It is assumed that an accident relating to the fiber had knocked out all the alignments of the laser beam concurrently. With power measurements, the problem was diagnosed and the alignment was fixed, and the system was restored to its functional operation.

Conclusion

While most of the components on a contemporary fluorescence microscope are relatively stable over time, users cannot depend on the illumination to be completely constant from week to week. In order to prevent negative results, fresh control slides must always be prepared at each imaging session if possible, and users must ask the facility staff when the system power was last measured.

From the above experiments, it is clear that the X-Cite® XR2100 power meter with XP750 objective plane power sensor proved to be a suitable tool for monitoring the intensity of the illumination at the sample stage for the timely detection and correction of light-source alignment problems.

About Lumen Dynamics Group Inc

Lumen Dynamics, an Excelitas Technologies® Company is a global leader in the design and creation of innovative light delivery solutions inspired by close to 30 years of light expertise in Manufacturing and Life Science applications. We are a technology company propelled forward by our commitment to providing customer solutions through the innovative application of light.

Precision bonding and UV curing is at the heart of the company’s manufacturing solutions for electronics/optoelectronics and medical device manufacturing processes, in addition to digital printing; while Life Sciences focuses on illumination and measurement technologies for bioscience research, microscopy and instrumentation. The comprehensive family of Lumen Dynamics’ products includes its renowned brands: OmniCure® and X-Cite®.

This information has been sourced, reviewed and adapted from materials provided by Lumen Dynamics Group Inc.

For more information on this source, please visit Lumen Dynamics Group Inc.

Date Added: Dec 2, 2013 | Updated: Jan 10, 2014
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