1. Microscope Calibration
Tools for evaluating the performance and setup
of fluorescent microscopes and digital imaging systems
Tools for evaluating the performance and setup of fluorescent microscopes and digital imaging systems.

2. Imaging Standards
Currently there are few standards routinely used in fluorescence imaging.
Fluorescence microspheres are generally good candidates as they are uniformly labeled and durable.
MPI offers a variety of microsphere products specifically targeted at imaging applications.

3. InSpeck™ beads are single color, relative intensity standards.
Imaging Standards
FocalCheck™ beads can be used for assessing registration, alignment, and distance calibration issues.
TetraSpeck™ beads are useful for registration and point spread function measurements.
PS-Speck™ beads are single color, >200 nm beads specifically for point spread function analysis.
InSpeck™ beads are single color, relative intensity standards.
FocalCheck™ Fluorescent Microscope Test Slides
Products offered by Molecular Probes/Invitrogen specifically for evaluating microscopes and imaging systems.

4. For general alignment and calibration
FocalCheck™ Beads
15 µm 3 ring
FocalCheck™ Beads
Green (ex 488)
Orange (ex 543)
Far Red (ex 633)
15 µm Blue-Green FocalCheck™ Beads Green (ex 488) Blue (ex 365)
Examples of two of the FocalCheck™ beads we offer.
For general alignment and calibration

5. Optical Alignment Through FocalCheck™ Beads
Diagrammatic illustration of the utility of the FocalCheck™ beads. FocalCheck™ beads with their unique surface ring labeling patterns provide ideal targets for assessing a variety of possible problems in fluorescence imaging. The ring pattern is clearly visible in confocal microscopy allowing easy alignment of color channels. The large diameter of the bead also has utility for confirming size calibration and z-axis slicing.

6. Four colors per bead TetraSpeck™ Beads
4 µm TetraSpeck™ Beads Blue (ex 365)
Green (ex 488)
Red (ex 543)
Far Red (ex 633)
1 µm TetraSpeck™ Beads
Blue (ex 365)
Green (ex 488)
Red (ex 543)
Far Red (ex 633)
0.5 µm TetraSpeck™ Beads
Blue (ex 365)
Green (ex 488)
Red (ex 543)
Far Red (ex 633)
Illustrations of 3 types of TetraSpeck™ beads.
Four colors per bead

7. FocalCheck™ Double-stained Beads
Green Beads
Orange Beads
Red Beads
Far Red Beads
Example of the spectral unmixing of the Double Green FocalCheck™ bead.
Bead 1 503/511
Bead 2 511/524
Bead 1 541/555
Bead 2 545/565
Bead 1 578/605
Bead 2 589/613
Bead 1 657/676
Bead 2 671/692
Ring 503/511
Core 511/524
Ring 541/555
Core 545/565
Ring 589/613
Core 578/605
Ring 671/692
Core 657/676

8. FocalCheck™ Fluorescence Microscope Slides
FocalCheck™ Fluorescent Microscope Test Slides combine multiple imaging bead standards into a signal, robust format. This gives every researcher a stable ready to use sample that can be used for spot checking and evaluating the performance of fluorescence microscopes and imaging systems. Makes a great training tool.

9. FocalCheck™ Fluorescence Microscope Calibration Slide #1
15 µm 3 ring
FocalCheck™ Beads
Green (ex 488)
Orange (ex 543)
Far Red (ex 633)
4 µm TetraSpeck™
Beads Blue (ex 365)
Green (ex 488)
Red (ex 543)
Far Red (ex 633)
1 µm TetraSpeck™ Beads
Blue (ex 365)
Green (ex 488)
Red (ex 543)
Far Red (ex 633)
0.5 µm TetraSpeck™ Beads
Blue (ex 365)
Green (ex 488) Red (ex 543)
Far Red (ex 633)
6 µm Blue-Green FocalCheck™Beads Green (ex 488) Blue (ex 365)
TetraSpeck™ for widefield (and confocal) for misalignment filters (and chromatic aberration in optics); FocalCheck™ beads for confocal (equator = 15µM diameter); size and z-axis calibration
For general alignment and calibration

10. FocalCheck™ Fluorescence Microscope Calibration Slide #1
6 um Green Fluorescent (ex 488) Intensity Bead Series
Current offering: InSpeck™ Fluorescence Intensity Calibration Kits in blue (350/440), green (505/515), orange (540/560), red (580/605) and deep red (633/660). Beads are 2.5 or 6 micron in diameter.
The InSpeck™ Fluorescence Intensity bead series can be used to generate a series of well-defined fluorescence intensity levels for constructing calibration curves and evaluating sample brightness.
100%
Relative
Intensity
33%
Relative
Intensity
10%
Relative
Intensity 3%
Relative
Intensity
0.667%
Relative
Intensity
For general alignment and calibration

11. FocalCheck™ Fluorescence Microscope Calibration Slide #21 2 3 4 5 A
Mixture of all 8 Reference Beads B
Mixture of all 4 Double-Stained Beads
6 µm
Green Beads
Orange Beads
Red Beads
Far Red Beads
Ring 503/511
Core 511/524
Ring 541/555
Core 545/565
Ring 589/613
Core 578/605
Ring 671/692
Core 657/676
Bead 1 503/511
Bead 2 511/524
Bead 1 541/555
Bead 2 545/565
Bead 1 578/605
Bead 2 589/613
Bead 1 657/676
Bead 2 671/692
Designed specifically for systems with spectral imaging capabilities. Each bead set has closely overlapping spectral properties that allow researchers to evaluate the performance of both spectral imaging hardware and software.
Spectral Unmixing Standards

12. FocalCheck™ Fluorescence Microscope Calibration Slide #3
Sample Position 1 2 3 4 5
Wavelengths
350/440
505/515
540/560
580/605
633/660 A
2.5 µm Blue
Bright
100% intensity
2.5 µm Green
2.5 µm Orange
2.5 µm Red
2.5 µm Deep Red B
Dim
1% intensity
2.5 µm Deep Red
General color slide offers two intensities of beads across a range of commonly used spectral bandwidths. FocalCheck™ slide #2 can be used for evaluation of bleed through of optical filters and as a general target for system intensity evaluation.
General Colors, Two Intensities

13. Issues Encountered in Imaging
Chromatic Aberration
Color Misalignment
Spherical Aberration
Z-axis Errors
Field Illumination
Intensity Calibration
Dimensional Calibration

14. Chromatic Aberration
Chromatic aberration occurs due to light of different wavelengths refracting differently through the optical path of the microscope.
Even expensive objective lenses may not be fully corrected when working across wide spectral ranges.
Chromatic aberration is most commonly found when trying to image across broad spectral ranges. Most achromatic lens are corrected for two or three wavelengths in the visible range but may not be corrected in the >650 nm range. Apochromatic lens are much more expensive but most current lenses of this type have broader correction curves. It should be noted however that different manufacturers do the correction in different ways. Some have all the correction in the objective while others use a “tube” lens within the microscope to do the chromatic correction.
Chromatic aberration can arise from any optical element in the light path and camera coupling mounts are often overlooked culprits.
25x 0.8 NA oil immersed objective (multi-immersion lens)

15. Color Registration
While chromatic aberration can be a serious issue, color registration is a more common problem in fluorescence imaging.
Misregistration is caused most particularly by alignment differences between the optical filters.
Any of the multicolor beads we sell can be used to diagnose and correct this problem.
Misregistration shown using TetraSpeck™ beads 1 µm
Color registration mismatch in widefield fluorescence microscope system caused by switching between different filter sets. This kind of misalignment is usually corrected using the imaging software as it can be difficult to align the filters physically.

16. Color Misalignment
Misalignment in a confocal system caused by using different light path configurations between scans
The problem in this case was tracked down to the primary dichroic mirrors used for the scans.
15 µm FocalCheck™ beads
Color registration mismatch in a confocal imaging system as shown using 15 micron 3 ring FocalCheck™ beads.

17. Spherical Aberration
Spherical aberration is the distortion in an image that occurs due to differences between the refractive indices of materials in the optical path. Because the refractive index of the specimen is rarely known, this can be very difficult to correct.
The most common manifestations are:
The fluorescent intensity degrades as you image deeper into the sample and…
Objects become “stretched” along the Z axis.
The greater the refractive index mismatch, the worse the problem becomes.
The extent of the problem can be assessed by mixing beads in your mounting media, imaging a volume, and examining the effects on the bead signal.
Spherical aberration. Illustrated on following slide.

18. Spherical Aberration Using 200 nm Beads
RI 1.5 mounting media
RI 1.38 mounting media
Images show XZ projections from a 25 micron deep stack of 200 nm microspheres distributed in either PVA mountant (RI~1.5) or Low Melting Point Agar (RI ~1.38).
Note that near the coverslip surface the intensity of the beads is very similar in each image. However as one moves away from the coverslip the beads in the low refractive index material start to get dimmer and look stretched in the Z-axis. This effect is not apparent in the RI 1.5 mounting media.
Coverslip Position
Axial stretching and signal degradation due to spherical aberration
100x oil immersion objective , 1.4 NA
25 micron depth, imaged at 0.25 micron steps, Zeiss 510 META

19. Focus Motor Calibration
Slippage of the Z-axis (focus) motor can lead to some very puzzling problems in confocal systems.
Z-axis calibration using FocalCheck™ Beads can be performed with a single XZ line scan through the bead, then measuring for the correct diameter.
These beads can also be used to establish the correct XYZ ratio for the image display.
A Z-stack of 15 micron focalcheck. The image set was rotated slightly on side without rendering so that the individual slices can be seen.

20. Pinhole Misalignment
Incorrect alignment of the pinholes in a confocal system not only degrades the signal it can also lead to some strange image artifacts.
Pinhole alignment can be performed using a small target object such as our TetraSpeck™ beads or PS-Speck™ beads.
This problem was first noticed by a researcher imaging tissue sections then confirmed using FocalCheck™ beads. The problem was due to a large X-axis misalignment of the confocal pinhole.

21. Field Illumination
Uneven field illumination is a serious matter particularly for quantitative imaging applications.
If the images are not corrected for unevenness in illumination, then segmenting the image may be very challenging.
Without field correction, the distribution of intensities of similar objects in the image will be much greater than expected, making statistical analysis difficult.
The best method for performing this correction is the use of a uniform fluorescent sample or thin layer of fluorescent material.
Slide describes the importance of correcting images for uneven illumination. Not so critical for morphometric measurements but even then can make segmenting (identifying) objects of interest very difficult.

22. Field Illumination 10x objective 100x objective Same scope, same lamp
Illustrates illumination pattern using two different objectives on the same microscope. Only difference is that the lens in the lamp was not adjusted when taking these images. Contour plots generated in MatLab.
Same scope, same lamp

23. Correcting Images Shading image, pseudocolored to show intensity range
Pseudocolored images of original, shading image and corrected image.

24. Correcting Images Uncorrected image of microspheres
Shading image, pseudocolored to show intensity range.
Pseudocolored images of original, shading image and corrected image.

25. Correcting Images Image after applying shading correction
Pseudocolored images of original, shading image and corrected image.

26. Correcting Images Before correction After correction
Intensity projection of field of beads before and after shading correction.

27. Shading Correction Considerations
Random noise in the shading image can result in large distortions in the values of certain pixels in the corrected image.
This effect can be reduced by using a correction image created by averaging multiple “shading” images.
If this is not possible, a median or other smoothing filter can be used to eliminate unusually high or low pixel values.

28. Intensity Variations
The recommended working life for a mercury arc lamp is about 200 hrs. Beyond this the performance of the lamp can degrade both in terms of output and stability.
Using any of our standard beads as a target one can follow the performance of the light source over time to help spot problems.
Similarly, variations in intensity due to an unstable arc can be detected by creating a time-lapse image series of a field of beads and plotting the intensities over time.

29. InSpeck™ Beads for Intensity Calibration
Calibrating intensities between microscopes is difficult due to variation between the system components.
We offer a series of intensity beads that provide a relative scale that can be established for each instrument.
Sample intensities can then be compared against this scale independently of the actual instrument intensity values.
Plots of representative data on following slide.

30. Intensity Bead Comparison Between Systems
Two plots showing the different slopes you obtain from intensity beads on 3 different scopes and imaging systems. When the raw intensity values are normalized and replotted the 3 systems have near identical curves. You can use this as a basis for comparing sample fluorescence independently of the imaging system.

31. Dimensional Calibration
An essential part of any microscopist’s tool kit should be a good stage micrometer. These are available from numerous sources.
Ideally all optical configurations should be calibrated (Objectives x Optivars x Camera mounts) to ensure that necessary measurements are available during imaging.
More sophisticated resolution targets are also available and can be a valuable aid to troubleshooting a misbehaving system.
Spatial or Dimensional calibration.

32. Dimensional Calibration
Stage Micrometer
The example shown here is 1 mm total length with 10 micron divisions.
Note. Shown for illustration purposes only. Not available from Invitrogen Corp or Molecular Probes.
All imaging systems should be calibrated for dimensional measurements using a stage micrometer. A stage micrometer is simply a printed ruler of known dimensions that is placed on the microscope stage and imaged typically with transmitted light. Stage micrometers come in a variety of types, sizes, and configurations. On confocal systems this is usually performed at the factory but most imaging software provide this capability.
Each objective lens needs to be calibrated along with any optivars or camera mounts as all affect the size of the image that is projected. If different camera systems are used, these also need to be calibrated separately as the pixel size and chip size are likely different. Once the calibration files are generated then all subsequent images can be calibrated so dimensional measurements will be give in known units rather than numbers of pixels.