Taking photographs using cartograph 0 and the z-stepper




Дата канвертавання26.04.2016
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TAKING PHOTOGRAPHS USING CARTOGRAPH 6.2.0 AND THE Z-STEPPER


  1. Turn on focusing column. You must do this prior to starting the software for correct communication between the computer and the focusing column.

  2. Turn on Archimed. When loaded, choose the Image Processing icon from the choices on the left.

  3. Turn on Cartograph. When loaded, choose the Video icon from the choices on the left.

  4. Place the specimen on the stage, either within the BUFO or the Dome. Adjust focus and desired size of the specimen within the field of view.

  5. One must next enter the lighting source and other parameters of image appearance. These can be changed either by: 1) clicking on the Display Camera Control Dialog icon in the tool bar or 2) going to the top tool bar and clicking on “Image’ and then ‘Adjust camera’. A dialog screen will appear with the following options:

Exposure: This is the shutter speed for the camera, and perhaps the most useful way to adjust light intensity. If this setting is used to adjust light intensity, it will not be necessary to alter the default settings for “Gamma” or “Gain.” Hitting the ‘control’ key and using the mouse to move the slider allows a continuous range of shutter speed adjustment. One is otherwise limited to preset shutter speeds.

Light source: either Halogen for the fiber optic and the BUFO, or daylight for the Dome.

White balance: Rebalancing WB at the beginning of each session is probably prudent and is mandated when switching light sources. Move the specimen from the field of view so that the field is completely gray (and the background need not be in focus). On the vertical shading oscilloscope, the bands should be tight and in the right quadrant when the background is empty and gray (between lines 2 and 3, counting from the right). Do not adjust vertical shading. Push the WB button once. Note the WB values on the right change.

With both the Dome and fiber optic light sources, changes in light intensity do not necessitate a reset of WB. CAUTION: The iris diaphragm (left knob) of the fiber optic light source changes light intensity. The right knob changes rheostat voltage AND WB – this should never be moved from the value of 80, the optimal setting for the system.



Gain: This is the amplification of signal from the camera. Raising the value results in a brighter image (with the introduction of some noise). Default is ‘0’

Master black: There is no fixed value, and the setting depends upon the taxon and specimen size. A value of or near -10 works well for many specimens that are not predominately black, whereas a value of or near 0 is good for many specimens which are predominately black.

Gamma: This affects midrange tonality, brightening or darkening the image. It can be an alternative to adjusting camera shutter speed. Default is 0.45; leave it there.

Sharpness: The default is ‘0’. Positive values sharpen, negative values soften. In general, do not go below ‘0’; fine setae and delicate surface sculpture can be enhanced by increasing the value. Most users find that values of 5 or higher produce digitization of the image. For clarity, this use of sharpness will be henceforth referred to as α-sharpness (as opposed to the sharpness value used in building extended focus images).

Noise reduction: Default is ‘0’; leave it there.

  1. After settling on image size and orientation from the live image, go to the toolbar, select ‘Measure’, ‘Calibration’, and then the appropriate calibration for the current magnification (objective and zoom position). Make sure that correct objective value (1.0x or 0.5x) is entered. [See section on focusing and magnification values in ‘General notes’, below]. IMPORTANT: The calibration must be adjusted every time you switch lenses or change the zoom position, or scale bar annotations will have incorrect measurements.

  2. Go to the ‘File’ menu, select ‘New document’, verify that ‘Stack’ is selected under Document and ‘Field Depth’ under Fields layout, then select ‘Ok”. THIS IS A CRITICAL STEP WHEN DOING MORE THAN ONE RUN!

  3. In some cases it may be desirable to change the number of images Cartograph will capture for a given range of field depth. This can be done under “Measure” and “document properties.” Under “Fields Layout” you can adjust the number of images Cartograph will capture for a given range of field depth by selecting “Z Step” and changing the value. By cutting the value given for Z Step in half, Cartograph will double the number of images captured for a stack. Experience has shown that cutting the value of the Z step from the default setting will result in superior images in some cases. If one obtains an image that is not in as sharp of focus as desired after using Cartograph, it may be possible to obtain a better image by reducing the Z step value, or by manually capturing more images with Archimed.

  4. Select the ‘New scan’ (scissors) icon – this deletes the previous top and bottom focus positions.

  5. Adjust the focus of the microscope so that the top of the specimen is in sharp focus, then select the Set first field of scan icon.

  6. Adjust the focus of the microscope so that the bottom of the specimen is in sharp focus, then select the Add field to scan icon.

  7. Select the Record all fields icon: the z-stack will now be captured automatically.

  8. If you wish to change the ‘Build Sharp Image’ parameters, go to the ‘Measure’ menu and select ‘Set sharp image building’; otherwise, proceed to step L. Drag the viewer window over ‘difficult’ areas, such as overlapping structures or severely rounded structures (e.g., dorsum of head in lateral view) and compare how the various settings are resolving these areas. Estimation area and sharpness can then be changed to overcome stacking errors such as the halo effect. [See section on “Sharp Image Building”, below]

  9. Select the Build sharp image icon – this will create the extended focus image. Note: occasionally Cartograph will fail and building process will not proceed – there is only a momentary flash of the menu. The only cure is to shut down the programs and reboot the computer.

  10. Go to the icon list on the left side of the screen and select ‘Document’. Then mouse click on the extended focus image, and then enter ‘Control-C’. Switch to Archimed, select the Image processing icon from the list on the left side of the screen, then paste in the extended focus image with ‘Control-V’. The image can then be further manipulated in Archimed.

  11. To continue making Z-stepper images, simply go back to Cartograph, go to the icon list on the list on the left side of the screen and select Video, then repeat the above steps. Note: bringing a new image into Archimed Image Processing will not clear out the previous image – it is still available by using the ‘previous’ or ‘next’ buttons to view them. ‘Control-w’ will close images.

  12. NOTE: Experience has shown that at times Cartograph will make an image that is only in sharp focus for a fraction of the field depth range selected. In these cases, the desired image can be obtained by manually capturing images for the stack in Archimed.


TAKING PHOTOGRAPHS MANUALLY WITH ARCHIMED 5.3.1


  1. Turn on focusing column. You must do this prior to starting the software for correct communication between the computer and the focusing column.

  2. Turn on Archimed. When loaded, choose the Real Time Video icon from the choices on the left.

  3. Place the specimen on the stage, either within the BUFO or the Dome. Adjust focus and desired size of the specimen within the field of view.

  4. One must next enter the lighting source and other parameters of image appearance. These can be changed either by: 1) clicking on the Display Image Acquisition dialog icon in the tool bar or 2) going to the top tool bar and clicking on ‘Video’ and then ‘Adjust camera’. A dialog screen will appear with the following options:

Exposure: This is the shutter speed for the camera, and perhaps the most useful way to adjust light intensity. If this setting is used to adjust light intensity, it will not be necessary to alter the default settings for “Gamma” or “Gain.” Hitting the ‘control’ key and using the mouse to move the slider allows a continuous range of shutter speed adjustment. One is otherwise limited to preset shutter speeds.

Light source: either Halogen for the fiber optic and the BUFO, or daylight for the Dome.

White balance: Rebalancing WB at the beginning of each session is probably prudent and is mandated when switching light sources. Move the specimen from the field of view so that the field is completely gray. On the vertical shading oscilloscope, the bands should be tight and in the right quadrant when the background is empty and gray (between lines 2 and 3, counting from the right). Do not adjust vertical shading. Push the WB button once. Note the WB values on the right change.

With both the Dome and fiber optic light sources, changes in light intensity do not necessitate a reset of WB. CAUTION: The iris diaphragm (left knob) of the fiber optic light source changes light intensity. The right knob changes rheostat voltage AND WB – this should never be moved from the value of 80, the optimal setting for the system.



Gain: This is the amplification of signal from the camera. Raising the value results in a brighter image (with the introduction of some noise). Default is ‘0’

Master black: There is no fixed value, and the setting depends upon the taxon and specimen size. A value of or near -10 works well for many specimens that are not predominately black, whereas a value of or near 0 is good for many specimens which are predominately black.

Gamma: This affects midrange tonality, brightening or darkening the image. It can be an alternative to adjusting camera shutter speed. Default is 0.45; leave it there.

Sharpness: The default is ‘0’. Positive values sharpen, negative values soften. In general, do not go below ‘0’; fine setae and delicate surface sculpture can be enhanced by increasing the value. Most users find that values of 5 or higher produce digitization of the image. For clarity, this use of sharpness will be henceforth referred to as α-sharpness (as opposed to the sharpness value used in building extended focus images).

Noise reduction: Default is ‘0’; leave it there.

  1. After settling on image size and orientation from the live image, go to the toolbar, select ‘Measure’, ‘Calibration’, and then the appropriate calibration for the current magnification (objective and zoom position). Make sure that correct objective value (1.0x or 0.5x) is entered for the session [See section on focusing and magnification values in ‘General notes’, below]. IMPORTANT: The calibration must be adjusted every time you switch lenses or change the zoom position, or scale bar annotations will have incorrect measurements.

  2. On the left side of the screen, click on the Extended Focus icon. When the menu comes up, click on ‘new’ in the tool bar. Otherwise, the new images will be added to the image stack from the previous session.

  3. Go back to Real Time Video. Use the coarse or fine focus to go above the specimen so that it is out of focus. Focus back down on the specimen; when the first desired structure is in focus, click on the image and drag it to the Extended Focus icon. A brief purple highlight indicates that the image was successfully captured. Go down through the focal plane until done.

  4. Click on the Extended Focus icon. You will see the focal plane captures. If you wish to change the ‘Build Sharp Image’ parameters, go to the ‘Focus’ option in the top toolbar and menu and select ‘Settings’; otherwise, proceed to step I. Drag the viewer window over ‘difficult’ areas, such as overlapping structures or severely rounded structures (e.g., dorsum of head in lateral view) and compare how the various settings are resolving these areas. Estimation area and sharpness can then be changed to overcome stacking errors such as the halo effect. [See section on “Sharp Image Building”, below]

  5. Then click on the Build a sharp image icon in the toolbar. A dialog box will appear, tracking the process of the building procedure. The extended focus image will appear beneath the captured images.

  6. Drag the extended focus image to the Image Processing icon on the left, then click on the icon.

  7. The scale bar and other annotations can be added at this point if desired. Save the image in the desired folder with the appropriate name by going to the top tool bar and clicking on ‘File’ and then ‘Save image as …”.

  8. To start over again, go to Extended Focus, click on ‘new’ in the tool bar to clear the previous stack, and then go back to Real Time Video.


SHARP IMAGE BUILDING
Settings:

Position compensation = box should be checked

Magnification compensation = box is checked or not depending on subject matter

Brightness compensation = box should be blank

Method = standard

Sharpness = no default value.

Estimation area = ‘Small’ is default
Enabling the magnification compensation option will often result in the ‘plasticization’ of surface detail: fine structures (granulation, small punctures) that are visible in individual focal planes are lost and the result is a featureless surface with setae). The user is strongly encouraged to turn off this feature when photographing fine surface detail! However, magnification compensation is almost always necessary in low magnification imaging, and also to correctly resolve some broad three-dimensional structures such as pectinate antennae.
Sharpness is the tightening of pixel boundaries along the edges of focal planes [note: Buffington says that this sharpness is not quite the same as that of Photoshop, which modifies the brightness of adjacent pixels along edges of contrast]. The sharpness used for sharp image building will be henceforth referred to as β-sharpness (as opposed to the α-sharpness value used in the ‘Camera Control’ dialog box).
The interaction of α- and β-sharpness is an important consideration. An α-sharpness of 4-5, combined with a β-sharpness of 10 or higher, will result in a ‘noisy’ digitized background.
Typically, one can increase the estimation area to overcome stacking errors; the cost of increasing the estimation areas is a decrease in overall resolution. This resolution can be salvaged somewhat by increasing the sharpness level.
Stacking is an algorithm whose job is to combine in-focus areas into one in-focus image. Changing the estimation area changes the tolerance of the algorithm for sharpness vs. non-sharpness of adjacent pixels. For a moth, there are lots of sharp areas and contrast – no challenge for the algorithm. Contrast this situation with that found in a small shining cynipoid.
The estimation area is the tolerance in the algorithm for neighboring areas of contrast. Changing it changes the tolerance for what the stacking algorithm considers to be in focus. Small has the lowest acceptance of what is in focus. If small produces a halo problem, changing to medium or large may reduce or eliminate the halo, but it makes the image less sharp. Increasing the sharpness value will help restore some loss of resolution.
Estimation area size: Take the situation of an ichneumonid at medium magnification (up to 3.2x on the zoom ring) on the 1x objective. There is a head and mesosoma in lateral view. Examine the dorsal margin of the head for a halo. The sharpness level = 2. Go to the estimation area and move it around to see how the halo disappears. It might go away if the estimation area is set to large and the sharpness increased (say, 15 or so). Go to the dorsal margin of the mesoscutum and look at the setae for a halo. Increasing sharpness might get rid of it but at the cost of getting the head halo back.
A large estimation area increases the tolerance for what the algorithm considers to be focus, so the image won’t be as sharp.
When checking for halo, look at the anterior and posterior ends (where magnification compensation often fails, particularly with the 0.5x objective and 0.57x setting of the zoom ring).
All this being said, there is no cut-and-dried approach to taking quality photos. Individual taste is a further complication. Below are our preferences for different taxa:
Cynipoidea (by Matt Buffington, April 2008)
α-sharpness = 2

master black = -3 to -7

estimation area = small

β-sharpness = 10


Most cynipoids are small, black and shiny. Resolving delicate surface sculpture is a major challenge, and having the master black too high will preclude the user from ever even seeing certain sculptural elements, much less photographing them. This section is applicable to Ceraphronoidea, Platygastroidea and Proctotrupoidea as well.
I prefer a very tight halo around the edges of specimen rather than adjust the estimation area to compensate for the halo. With a neutral gray background the specimen on a clear point, editing the halo out in Photoshop is an easy task. The real focus needs to be on resolving structures of interest, leaving the asthetics of the image to post-production in Photoshop.
The LED dome works fairly well for heavily sculptured black wasps (e.g. Scelionidae, some Megaspilidae) but for more shiny individuals (e.g. eucoiline Figitidae) the raking light one is capable of producing with the BUFO is needed. A slight amount of glare can be very informative and result in an image that is not only informative but also rather attractive. The BUFO requires more adjustment than the dome, and this takes time, but once a user has some experience, the process with be faster. Remember, the goal is to obtain excellent images, it is not a race.
Ichneumonidae (by David Wahl, April 2008).

For dark-colored ichneumonids with a ‘normal’ complement of setae and surface sculpture (granulation and/or punctures), I find that the following settings usually give the best results:

α-sharpness = 2

master black = -8 to -10

estimation area = small

β-sharpness = 10


Note the caveat about magnification compensation in the preceding section on ‘Sharp Image Building.’
With a small estimation area, I can’t find any difference between β-sharpness values of 10 and 20. There is a definite quality drop at 5; although a value of 2 will certainly reduce the halo, it is not worth the overall loss of detail. Given the ease of use of Photoshop tools, for critical applications it is better to remove the halo manually.
For light-colored ichneumonids, particularly those with little or no surface sculpture (such as Mesochorinae or Ophioninae), a medium estimation area and a β-sharpness of 20 will give better results.
One cannot reliably predict which specimens will present problems (such as a strong halo, anomalous areas of poor resolution, etc.). For these, it is best to try a second extended focus image from the stack, varying the estimation area and β-sharpness value.
While the GT Vision ‘Dome’ gives a good lighting solution in most situations, it can fail to adequately resolve low but prominent carinae (such as the epicnemial or postpectal carinae). While these structures are easily visible under a regular light microscope, they can be practically invisible under the Dome. The Buffington UFO (BUFO) is a lighting arrangement that usually solves this particular problem [see: Buffington, M.L., R.A. Burks, & L. McNeil. 2005. Advanced techniques for imaging parasitic Hymenoptera (Insecta). American Entomologist 51: 50-54.].
Lepidoptera Adults (by Hugo Kons Jr., Feb. 2008)

I find the following settings usually give the best results:

α-sharpness = 2

master black = -10 (except for predominately black specimens)

estimation area = small

β-sharpness = 20

Position compensation = ON

magnification compensation = ON

Light Source = GT Vision Dome

Background Color = Grey



Do NOT include labels in the photo with the specimen.
Many examples of adult Lepidoptera habitus images taken with these settings can be viewed at http://www268.pair.com/hktownes/Lepidoptera/FLLepGuide.htm under the sections for Nolinae, Hypenodinae, Rivulinae, and Hypeninae. These web pages may not contain exclusively GT Vision pictures, but the GT Vision pictures can always be recognized by the scale bar and/or text annotations in the photo. Note that to currently to view these images one must use Microsoft Internet Explorer with a PC.
The main adjustment to make is the “exposure” setting, which I change for almost every individual specimen. The appropriate setting is very specimen specific, and sometimes trade-offs must be made. Dark specimens generally need a higher exposure setting, whereas too high of an exposure setting can alter (wash out) the natural appearance for colors such as yellow/orange and green. For yellow/orange- or green-colored specimens, it is often useful to have a second reference specimen handy when adjusting the exposure setting, in order to get the color just right. Some of the most challenging specimens are those that are patterned with yellow/orange and black or green and black. To get the natural color one may need to settle for a darker than optimal exposure for dark head, body, and appendage features.
A word of caution: sometimes images which look excellent on the high-end monitor (Dell 24” flatscreen) that comes with the GT Vision system may look darker than ideal on a cheap monitor. For most specimens there is a range of exposure settings that look good on the Dell monitor, and selecting for the upper end of this range can be helpful if the image will later be viewed with a cheaper monitor or posted on the web.
For some predominately black specimens, it is often optimal to raise the master black setting from -10. This requires experimentation with individual specimens.
For specimens with broadly pectinate antennae, it is often important to make sure magnification compensation is enabled. While this setting may result in the loss of surface detail in Hymenoptera, it generally is not a problem in Lepidoptera specimens where the body is covered with scales and hairs.
The halo effect sometimes occurs around the antennae, or around other structures, particularly when a broad field depth range is used to create the image. This problem is merely aesthetic and does not detract from the scientific value of the image. The halo can sometimes be eliminated by changing the estimation area from small to medium, but this can detract from the overall sharpness of the image.
GENERAL NOTES


  1. The lower left corner of the screen gives the magnification values of the objective. Magnification calibration may be set either by: 1) clicking on the microscope icon in the top toolbar and entering the correct values for the objective (0.5x or 1.0x) and its zoom magnification value (found on the knurled ring near the bottom of the Leica optical body), so that you have something like “1.0 @ 0.57” etc., or 2) go to the toolbar, select ‘Measure’, ‘Calibration’, and then the appropriate calibration for the current magnification (objective and zoom position). Make sure that correct objective value (1.0x or 0.5x) is entered for the session.

  2. The magnification values on the objective must be aligned with the XXXX line, and not be between the values, for the scale bar to work properly.

  3. On the Z-stepper control, the red button toggles between coarse and fine adjustment. One turn of the coarse setting = six turns of the fine setting. Use the coarse focus to bring the specimen into view, fine focus for finding the proper focal planes for manual and automatic image capture.

  4. The Z-stepper has an independent power supply and motor. It should be shut off when not used for an extended period (say, several days).

  5. Stack capture is from top to bottom.

  6. The 1.0x objective gives a maximum field of view of 12 mm.

  7. The fiber optic unit uses an EKE bulb. The iris diaphragm (left knob) is used to adjust the light intensity – do not adjust the rheostat’s setting (right knob), which should be left on ‘80’.

  8. Saving the session images as TIFF files is recommended.

  9. Easy examination of Z-stack images in Cartograph is possible with the Viewer function. Once the stack is made and the combined image is built, select the Viewer icon from the list on the left side of the screen. The controls at the top of the window allow you to move amongst the images.

  10. When changing specimens under the focusing column, be sure to raise the column high enough so that that the objective lens is out of the way. The lens needs to stay absolutely clean, and you do NOT want to drop the BUFO or the Dome on to your specimen!


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