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Evaluating Confocal Microscopy System Performance

  • Robert M. Zucker
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1075)

Abstract

A confocal microscope was evaluated with a series of tests that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, scanning stability, PMT quality, overall machine stability, and system noise. These tests will help investigators measure various parameters on their confocal microscopes to insure that they are working correctly with the necessary resolution, sensitivity, and precision. Utilization of this proposed testing approach will help eliminate some of the subjectivity currently employed in assessing the CLSM performance.

Key words

Confocal microscope Lasers Coefficient of variation Photo multiplier tubes Field illumination Axial resolution Spectral registration Laser stability Beads Microscope lenses Quality assurance Quantification Spectroscopy 

Notes

Acknowledgments

We wish to thank Jeff Wang of Spherotech for providing us a 10-μm bead that was uniform in size and fluorescence intensity and did not readily bleach with repeated samplings. Thanks to Gary Klinefelter for providing the sperm sample for comparisons of confocal with Normarski optics. We wish to specially thank Jeremy vLerner for providing us the light source to properly evaluate spectra in a confocal microscope. Figures 1, 4, 5, 7,and 11 and Tables 1,
Table 2

Comparison of relative dichroic reflectance

Wavelength

Dichroic

10× (0.3 NA)

63× (1.2 NA)

488

SD

0.95

0.97

DD

1.00

1.00

TD

0.84

0.80

568

SD

0.05

0.06

DD

0.62

0.72

TD

1.00

1.00

The relative laser power was measured with the 488 and 568 wavelengths using six different magnification objectives and three dichroics. The 10× and 63× data is shown for clarity. The table demonstrates the relative reflectivity of the dichroics in the system. The test was accomplished by measuring the intensity in a ROI of an image using either 488 or 568 excitation light, a fluorescent plastic slide, one of three specific dichroic and maintaining the PMT at a constant voltage. A ROI of the image yielded the mean value for each acquisition condition (wavelength, objective, and dichroic). The GSV of the highest value image is diveded into the other images to yield a ratio that is expressed in the table as a fraction. The value of 1.00 is the maximum reflection. The dichroic with the maximum reflection should be used when only one fluorochrome is required. Unexpectedly, the double dichroic (DD) yielded the best reflectivity with 488 nm wavelength light with most lenses and the triple dichroic (TD) yielded the best reflectively with the 568 wavelength light (30 % more light reflected than the DD) with most objectives. This table can be used to choose the dichroic that should be used with each excitation wavelength for optimized reflection

2,
Table 3

PMT comparison and noise

PMT comparison

Excitation

Emission

PMT #

PMT voltage

CV (%)

Relative CV

488 nm

505–555 nm

1

474

6.06

100

2

428

6.58

108.65

3

425

6.23

102.86

555–600 nm

1

471

6.02

100

2

432

7.00

116.25

3

421

6.46

107.17

568 nm

580–630 nm

1

439

4.00

100

2

411

4.88

122

3

393

4.49

112.11

647 nm

665–765 nm

1

802

20.30

100

2

732

22.70

111.68

3

675

20.30

100.12

The noise of the system was evaluated using a 10 μm bead (Spherotech) and a 100× Plan Apo (1.4 NA) objective. The intensity of a 10 μm bead was determined at a constant laser power, a zoom of 4 and no averaging using various PMT settings. The emitted light was measured in each of three PMTs. The pixels in each ROI were set to a mean of approximately 150 and the SD of pixel distribution was measured to determine the CV. The CV of the pixel intensity within the bead was measured at each PMT setting. PMT 1 is low noise blue sensitive while PMT 2,3 are far-red sensitive. The quality and the performance of each PMT can be measured with this test

3, and
Table 4

CLSM sensitivity

Relationship between laser power and CV

Laser type

Wavelength (nm)

Power (mw)

CV–Bead % (SD/mean)

(A, fixed power comparison)

Argon Krypton (75 mW, Leica)

488

1

4

568

0.2

4.6

Argon 25 mW (Zeiss)

488

1

1.3

HeNe 1 mW (Zeiss)

543

0.2

1.9

(B, maximum power comparison)

Argon Krypton (75 mW, Leica)

488

1.1

3.8

568

1.45

2.6

Argon 25 mW

488

3.2

1

HeNe 1 mW

543

0.23

1.9

The sensitivity from a Leica TCS-SP1 containing one noisy argon–krypton laser emitting three laser lines and a Zeiss 510 containing three individual quite lasers and a merge module are represented. The CVs were obtained from a 10-μm bead using a 100× Plan Apo objective (1.4 NA). The laser power was derived by using a 10× (0.3 NA) objective and a power meter situated on the stage. By setting the power to a fixed value of 1 mW of 488 nm laser light or 0.2 mW laser light on the stage, the sensitivity of two machines was measured (A, fixed power comparison). The CV of the bead was to be almost three times lower with the 488 nm and 568 nm laser lines using the Zeiss 510 system compared to the Leica TCS-SP1 system. By increasing the lasers to their maximum power the CV values were decreased (B, maximum power comparison). This test illustrates that different systems having different laser configurations can yield different sensitivity values using this test

4 have been previously published in Cytometry and the journal has allowed these figures to be reproduced in this book chapter.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Robert M. Zucker
    • 1
  1. 1.Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and DevelopmentU.S. Environmental Protection AgencyResearch Triangle ParkUSA

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