Biological Microscopes

• The camera LED shows no signal and there is no ventilator sound.

• The software camware is installed as described and the LED at the camera rear panel is lighting green. When starting the program the message appears ‘No pci card found or no camera detected! Starting without camera control functions!’

• The camera can not be focused to infinity.

• When I work with some programs under Windows9x/ME/2000/XP and then call sensicontrol/camware again, there is much less space for the image recorder available compared to calling sensicontrol/camware immediately after reboot. Why does the system provides less

• When starting the camware software, the program returns "No camera detected or no PCI card found"

• Does the selection of a smaller area (e.g. with binning, ROI) improve the read out time and the imaging frequency?

• The picture shows transmission and shift errors.

• Can a too high light intensity (e.g. direct irradiation of a light source, sunlight) damage the camera?

• How many images can be made in a sequence?

• What is a "correction collar" of high magnification objective lenses for?

Most of objective lenses for biomedical microscopes are designed for observation of samples with a cover glass on. Sometimes the sample is not properly focused due to uneven thickness of cover glasses used. To fully display the performance of the objective, the thickness between the cover glass surface to the specimen can be corrected at the objective side, by this correction collar.

• I want to make a DIC observation of cells cultivated in the petri dish but failed.

The DIC microscope uses polarized light. Thus when the material which has a big optical distortion such as plastic is used for observation, it directly affects the optical system, which results in the remarkably lower contrast in DIC. To avoid this, for DIC microscopy, it is recommended to use a glass bottom culture dish on the market (a plastic rid is to be removed in observation), or glass petri dish (no rid is desirable during an observation). Or to get the relevant relief contrast using a plastic petri dish, the Hoffman Modulation contrast equipment is suitable as it is free from optical distortion of dishes, and available from us.

• I cannot get proper Epi-Fluorescent images. Is there any solutions?

(1) In case of weak Epi-Fluorescence
‧Increase excitation light by removing ND filters (Note that fading is accelerated in this case)
‧If you use a mercury lamp for light source, reconfirm if the lamp is properly centered. It may be off from the best position due to deterioration of the lamp.
‧Check if the suitable excitation filter is chosen. There is another choice of filter which has wider transmission range. (ex: Change from EX450-490 to EX420-490)
‧Check the barrier filter (emission filter) If you use a band pass type filter, try a filter with wider transmission range and see the result. Or the long pass type may do.
‧Check what objective lenses are used. Choose an objective lens with a higher NA, the same magnification. Oil/Water immersion lenses are recommended for Epi-Fluorescent microscopy rather than dry objective lenses.
(2) In case of low contrast of Epi-Fluorescence.
‧Check the barrier filter (emission filter). If you use a longpass type filter, try a bandpass type.
‧An objective lens may be dirty. Also an image quality deteriorates markedly if some oil adheres to the tip of dry objective lens, or some water mix with the oil at the tip of oil-immersion objective.

• Do Not Unplug or plug in the camera cables while either the computer or camera power supply are on

Do Not Unplug or plug in the camera cables while either the computer or camera This can destroy the board. You may still hear the shutter click but when you take a picture, you will get gray or black on the monitor.

• My colleague has the same type of Spot camera that I do but her images look much better than mine. Why?

Same make/model cameras give different quality images.
We have observed a dramatic difference in image quality between images taken with Plan Achromat objectives and those taken with Plan Apochromat objectives. Images taken with Plan Apochromat objectives are much crisper and have better colors.

• Interaction between the 3-Shot Kala filter and the phase objective’s light pattern results in mottling in non-specimen areas.

Kala filter mottling with phase contrast
The light phase shifts that normally occur in creating a phase contrast image depend on the proper alignment of a phase objective with a phase condenser as well as using a green filter matched to the phase set. The green filter is used to provide the correct wavelength of light to satisfy all the design and physical criteria for phase optics. By its nature, phase contrast is a monochrome contrast effect to enhance unstained tissue and cells or other low contrast objects. Using phase contrast with stained tissue, cells or other color high contrast objects can produce unintended optical effects or aberrations.
These effects are not obvious when observing high-contrast specimen areas that fill the field of view. However, when large, non-specimen background areas are encountered (for example at tissue edges or when cells/crystals are spread over an open field of view) then uncorrected chromatic and non-chromatic aberrations at the edges of these objects become visible. These aberrations are associated with uncorrected, phase shifted, 3-color (non-monochromatic) image capture and possibly with normal filter structure (adhesive between filters). Such background aberrations are normally not seen when using non-phase color capture or phase monochrome image capture. If you are using Phase Contrast and a 3-Shot camera here are ways to reduce the mottling. You may substitute a mosaic color camera but the resolution of the captured images may not be appropriate for your samples.
A flat field correction found in the Spot software can also be used to help remove the background problems. Please be aware that any corrections may not entirely remove mottling or improve any color variances. It is suggested that non-phase optics only be used to capture high-contrast color images.

• Which coupler should I use with Insight 4/12 MegaSample Cameras?

Which coupler should I use with Insight 4/12 MegaSample Cameras?
The Insight 4 &12 MegaSample Cameras use the Kodak KAI 4020 CCD. This CCD has a diagonal of 21.4 mm and it requires a coupler which can support this large field of view in a 1.0x magnification. The Diagnostic Instruments no lens C-mount and F-mount Direct Image Projection series of couplers support this requirement. Further, the C-mount versions were modified over 2 years ago in anticipation of the release of cameras with this CCD. This new version C-mount can be identified by measuring the inside diameter of the through-hole at the C-mount thread. It should measure 0.850” (21.6 mm), compared to the previous version’s 0.750” (19.0 mm). Please note that other manufacturers’ optical couplers with the correct 1.0x magnification may not support the larger 21.4 mm diagonal CCD format.

• I have noticed that the lower the power of the microscope objective, the greater the difference in resolution between the camera image and the image seen through the eyepieces.

Difference in resolution between eyepiece and camera
This effect is also well documented. The sharpness of an image formed by an optical system is proportional to the NA of the objective divided by the overall magnification. With high magnification objectives, this ratio is low and the image is not so sharp. In this case, the microscope optics, not the camera, become the resolution bottleneck. You perceive that the camera image is as sharp as the image seen through the eyepieces. At lower magnifications, the NA to magnification ratio is high and the image is very sharp. In this case, the camera resolution becomes the bottleneck and the camera image will not be as sharp as the image seen through the microscope eyepieces. The transition occurs around the 20x objective, depending on the quality of the objective.
You can improve matters by turning on the Image Doubling feature in the Setup. Image Doubling uses a sophisticated bicubic interpolation algorithm to create an image with twice as many rows and columns as the CCD chip. It will quadruple your file sizes but will increase the perceived resolution of images taken with lower magnification objectives.

• The camera LED shows no signal and there is no ventilator sound.

Check the main of your 12V power supply.

• The software camware is installed as described and the LED at the camera rear panel is lighting green. When starting the program the message appears ‘No pci card found or no camera detected! Starting without camera control functions!’

Be sure that all installed devices in the computer are working without conflict. Please check all interrupt settings in the system BIOS and in the Windows diagnostic menu. You are not allowed to use the interrupt of the pci slot where the PCI-interface-board is placed, for other devices.

• The camera can not be focused to infinity.

Adjust the back focal length as described in the manual.

• When I work with some programs under Windows9x/ME/2000/XP and then call sensicontrol/camware again, there is much less space for the image recorder available compared to calling sensicontrol/camware immediately after reboot. Why does the system provides less

When you close a Windows application, the used memory is released by Windows, but under certain circumstances this memory will no longer be available as continuous free memory - a crucial precondition to allocate the memory space for the recorder. All you can do about this is to restart Windows.

• When starting the camware software, the program returns "No camera detected or no PCI card found"

This message might have a number of reasons:
‧ The camera is not switched on.
‧ The link between PCI-interface-board and the camera is defective. The camera LED shows a permanent red signal, either since the wire is not plugged in, since the coaxial connections RX and TX have been interchanged or since, in the fibre optics version, the plugs are dirty. When properly linked, the camera LED should show either a green signal (ready) or flash red / green (ready, but operation temperature has not been reached yet). The PCI-Interface-Board is not properly placed onto the PCI slot.
‧ Wrong PCI/PnP definitions in the BIOS of your computer.
‧ The driver does not get a non-shared interrupt.

• Does the selection of a smaller area (e.g. with binning, ROI) improve the read out time and the imaging frequency?

Vertical reduction of the exposed area (e.g. by binning) reduces CCD read out time. Setting an ROI (region of interest) has the same effect. The read out time for the non-exposed lines (skipped lines) is scaled down by a factor of 4. Reading out a VGA line takes 64 μs, where reading out a SuperVGA line takes 117 μs. Horizontal binning or horizontal ROI does not affect the read out time.
Example: For a VGA 640x480 sensor, a ROI of 400(H)x200(V) is selected. At full resolution, the read out time would be 32 ms. The ROI reduces this time. The resulting actual read out time is displayed in the info window.
Calculation:
200 lines are read out with 64 μs per line. This gives 12.8 ms. The remaining 480 - 200 = 280 lines which have not been exposed (skipped) are read out at quadruple speed, thus 280 : 4 = 70, 70 x 64 microseconds = 4.48 ms. Summing up we obtain a read out time of 12.82 ms + 4.48 ms = 17.3 ms. The imaging frequency is therefore 57.8 Hz.

• The picture shows transmission and shift errors.

A defective or too long coaxial cable is used.

• Can a too high light intensity (e.g. direct irradiation of a light source, sunlight) damage the camera?

No! The CCD sensor can not be destroyed in this way, unless, of course, thermal destruction...
Caution: This does not apply for image intensifier cameras!

• How many images can be made in a sequence?

The number of images depend on the size of the RAM. When starting the program camware the available memory will be allocated. The image of a VGA camera requires about 0.6MB memory at full resolution, a SuperVGA camera requires about 2.6MB. With binning or ROI a data reduction can be achieved. In the status line of the main window (bottom) you can always see the actual available memory, the actual image size and the number of images which can be written to the allocated memory.