How to Optimize Imaging with Nikon Elements Software, Part 1

May 28, 2020

Optimizing your microscopy imaging with Nikon Elements software will streamline your workflow. In this webinar, the first part of a three-part series, Technical Sales Representative James Bristol presents an overview of this exciting software platform. Part 1, here, covers the basics.






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Transcript

Charles Zona (CZ): Good afternoon, and welcome to another McCrone Group webinar. My name is Charles Zona, and today, our presenter is Jim Bristol, of McCrone Microscopes & Accessories. Jim is going to talk to us today about How to Optimize Your Imaging with Nikon Elements Software, and this is a three-part tutorial. This is the first installment in the three-part series, with Part 2 and Part 3 to be presented in the coming weeks.

Before we get started, I would like to tell you a little bit about Jim’s background and experience. Jim is a technical sales representative with McCrone Microscopes & Accessories, with over 30 years experience in optical instrumentation and software sales. Jim has successfully sold and supported complex imaging systems, such as laser scanning and spinning disc confocal microscopes, integrated systems for cell sterology and neuron tracing, as well as scanning electron microscopes and thermal microscope systems.

Today’s webinar is a little bit different in that it is a recording, and is not taking place live. However, you can still ask questions by typing them into the questions field. We will answer all of your questions individually in the coming days. And this webinar is being recorded and will be available on The McCrone Group website under the Resources tab. And now, I will hand the program over to Jim.

Jim Bristol (JB): Thank you Chuck, and hello, everyone. Thank you for joining us today, as we present to you the Nikon Elements Imaging Software System.

NIS-Elements is an integrated software imaging platform which delivers comprehensive control of microscopes and peripheral devices, image capturing, documentation, image analysis and data management, thereby contributing to experimental efficiency with intuitive image analysis features, and database and archiving management.

Please note that Elements is not a software solution dedicated only to Nikon microscopes. Elements can be used on any microscope system that can accept a digital camera, and Elements has motorization drivers for many of the third-party peripherals on the market today, providing motorization to non-Nikon microscopes.

For today’s presentation, we will place our focus on Elements D, as many of the capabilities that we will discuss today are also part of Elements Ar and Br, which stand for Basic Research and Advanced Research.

It all begins with the camera.

Nikon currently offers three Digital Sight Series cameras to be used with Elements. We will discuss these three cameras in more detail with the next slide.

Elements is compatible with many third-party cameras. There are eight third-party digital camera manufacturers that Elements currently functions with. These cameras are mainly monochrome, highly sensitive systems for low light applications, such as single cell fluorescence. We have a complete list of these manufacturers later in the presentation.

The DS-Fi3 is a 5.9 megapixel CMOS color camera, with a C mount connection to the microscope. The C mount is the most common connection type in microscopy. The preferred C mount for the Fi3 has a 0.5X magnification factor. The Fi3 has a very high frame rate of 15 frames per second at full resolution, and 30 frames per second at half resolution, or 2 x 2 binning.

The DS-Ri2 is a 16.25 megapixel CMOS color camera, with an F mount connection to the microscope. The F mount is a less common connection type in microscopy. Due to it’s large format sensor, derived from the Nikon digital single lens reflex camera, the Ri2 F mount is recommended to be a 2.5X magnification factor. You’ll notice that at 16.25 megapixels the frame rate of the Ri2 at full resolution is a slow six frames per second, but the frame rate increases to 45 frames per second at one-third resolution, or 3 x 3 binning. When working with large, very large, megapixel cameras, one must be aware of file size and storage requirements.

The DS-Qi2 is also a 16.2 megapixel CMOS Camera, but the Qi2 provides only monochrome images. The CCD is thermoelectrically cooled to provide improved quantum efficiencies for higher sensitivity and low image noise. The Qi2’s main market is low light applications, such as fluorescence.

Each of these Nikon cameras has CMOS technology, which improves energy consumption, reduces the overall size of the camera and provides for greater frame rates than non-CMOS systems.

The graphical user interface, or GUI, is the face of the software application. To operate the software, you must navigate the GUI to select the features and functions, from the tabs and the windows, that you wish to utilize. Elements has two main GUIs that a user can work with.

The Routine GUI is a fixed layout. The tabs and windows cannot be rearranged on the screen. The Routine GUI is designed for the casual, infrequent user. The basic imaging processes are set up in an easy step-by-step process. The selections are contextually relevant to the user selection.

The Standard GUI is much more flexible. The tabs and windows can be arranged and are fully customizable. Multiple users can have individual GUI configurations. There are three main toolbars, allowing for efficient image documentation processing and analysis. Within the Standard GUI there are two main options: docked, where multiple function tabs and windows are laid out for operator preference, and full screen, which maximizes the image size for post image processing and easier collaboration with colleagues and clients.

The tabs and windows of a GUI allow the user to select the specific function or operation they require for their individual imaging application. At the very least, the most common function of any imaging software is to allow the user to annotate and measure the image’s characteristics.

Elements’ Annotations and Measurements Tab provides for these functions. The Annotations portion allows the user to place descriptions and highlight image characteristics with arrows, squares, etc. The Measurements portion allows the user, once his system’s objectives have been calibrated, to measure in lengths, areas, angles and radii, as well as count and classify the image characteristics manually. The text can be adjusted for size, style and color as any normal Windows application. All of this is data is exportable to Excel files or tables for computation. In addition, this data can be exported into reports, that along with the images, will provide a permanent record of the operation.

Presets, or Optical Configurations can be saved for each observation method, such as brightfield or polarized light, to include the objectives, contrast method, microscope components if motorized, and the camera settings. They can then be displayed in the toolbar of the software interface. These Optical Configurations make adjustments to the microscope or camera much more efficient and repeatable.

The image in this slide depicts the operations of a fully motorized Nikon microscope, including nosepiece, light path, Z drive, lamp, filter, and condenser. Once each Optical Configuration is created, it can be named, and a descriptive button placed onto the toolbar of the software application.

Elements provides automated control for the family of Nikon motorized microscopes as well as many third-party peripherals that provide motorization including, but not limited to X-Y stages, Z motors, filter wheels and shutters, nosepieces, etc.

Image Stitching ( or large image) generates a single, wide field of view image by stitching multiple adjacent frames together. This function can be performed automatically utilizing a motorized X-Y-Z stage and can also be easily performed manually without motorization. Generating a large image manually is quite easy with the adjustable offset feature that eases the alignment process from image to image.

The Extended Depth of Field or as some say Extended Depth of Focus module selects the in focus area from multiple Z-stack images and produces one all-in-focus image. The composite image can be viewed and rotated as a virtual 3-D image if it contains Z-axis information. EDF can be performed automatically with motorization or manually on either a compound or stereo microscope. EDF is an optional module in Elements.

HDR, or High Dynamic Range Image Acquisition, creates an image with appropriate brightness in both the dark and bright regions of the sample by combining multiple images acquired from different exposure settings. This function can be performed manually or via a one touch function of the software. HDR is an optional module in Elements.

AVI Acquisition automatically captures live data into an easily exportable and viewable AVI format. File type, location, image quality, image size, frame rate, speed, and duration are all selectable.

Z-Series: Through motorized focus control, Elements reconstructs and renders 3-D images from multiple Z-axis planes.

Time Lapse imaging is easily configured by setting the time interval and duration.

Shown here are Image Filtering and Color Adjustment examples of Image Processing in Elements that allows the user to make changes to the image for better viewing and processing.

Merge Channels allows the user to combine up to four image channels (red, green, blue and brightfield) into a single overlay of full depth and separately-scalable images.

Volume View: Z-series images (motorization is required for this function) can be displayed in various formats, rotated, and converted to AVI or MOV formats for display. Selectable views are Max/Min Projection, X-Z Axis & Y-Z Axis, Cross-Sectional Slice view and 3-D Volume views.

Database Management: Using the organizer function, captured images can be organized in thumbnails for easy retrieval of the desired image. Sorting and filtering of the database of images and datasets can be performed using acquisition details, such as objective settings, date and author.

Report Generator: Images captured in Elements can be exported to a Report Generator, which is customizable in form and with your company logo, if you desire. Captured images have acquisition details and analysis results. These images and the associated image header and data information can be exported into the report template and easily converted in a PDF document.

These are some of the additional optional functions and operations that are available in NIS Elements. If there is interest in any one or more of these, please do contact us here at McCrone Microscopes.

  • Filter Particle Analysis
  • Concrete Measurements
  • Weld Measurements
  • Multipoint Imaging
  • Metallurgical Analysis

As promised, this is the current list of approved manufacturers of third-party peripherals that communicate with NIS Elements. Not all of these manufacturer’s products are approved for Elements. Besides cameras, they include motorized stages, Z focus drives, high speed shutters, piezo drives, triggering devices, illuminators—both white light and wavelength changeable, as well as thermal stages, scanners and incubators.

CAMERAS:

  • Photometrics/Qimaging
  • Andor Technology
  • Hamamatsu
  • Imaging Source
  • pco
  • Princeton Instruments
  • Inspectis
  • PixelLINK

THIRD-PARTY DEVICES:

  • Prior
  • Scientifica
  • Sutter
  • FLI
  • Cairn
  • Marzhauser
  • Vincent Associates
  • Ludl
  • ASI
  • Luigs & Neumann
  • Linkam
  • LCI
  • CoolLED
  • Excelitas
  • Agilent
  • Lumencor
  • 89 North
  • Physik Instrumente
  • MCL

Again, NIS Elements is not an imaging solution just for Nikon microscope products, but with their approved third-party peripherals, for many makes and models of microscopes.

LIVE DEMO

We will now show you a short video clip of the application functions we have just presented to you. This video was captured with one of the many remote meeting applications available today. The audio/video quality is the result of the limitation of the application and not that of the microscope or camera.

This is the Routine layout. The tabs and windows are fixed in place on the left side. There are tabs for camera controls, and Capture and Store functions.

Across the top and down the right side, there are icons for tool bars, such as bringing up your lookup table, or placing a graticle onto the image.

And then on the right side, the far right side, you can see all of these basic functions. These are the contextually relevant selections we spoke of earlier in this presentation. You may not see all of them, depending on which software options you purchase.

We are now looking at the Standard layout. The tabs and windows are moveable and each user has his own custom layout, so you can move windows around, place them where you’d like them to be.

In the Standard layout, you have three main toolbars: on the left side, across the top, and also down the right side. Here is a comparison of a simple custom layout, where we made changes by eliminating the number of toolbars, or limiting those numbers of changes. And also, you have the full screen option, which you can work with, with the toolbars, to process a captured image, or use strictly for collaboration because it has a larger window than the Standard layout.

Each objective is calibrated with a stage micrometer, and their identifiers are place in this Manual Microscope Pad. As you change objectives, you must update the Pads once you are sure that you have correct calibration. If you are using an intelligent or a motorized nosepiece, this Pad will automatically change the calibrations for you.

Color settings for the camera, as you can see here in the Color DS-Fi3 Settings, we can adjust the resolution for our focusing and for our capture.

We have exposure settings that are manual, and auto-exposure, or Automatic Exposure (AE), and a Continuous AE, which allows the camera to automatically change based on the changes in the field of view. Analog Gain is here so that you can brighten or darken your entire image based upon what you’re trying to frame, and AE Compensation gives you slight adjustments up and down when you’re in Auto Exposure.

Auto White Balance is something that you should do each time before you begin an image capture session by placing a white object in the optical path, and imaging it. You can also select one of the probes, move it to a particular area to white balance, and click Auto-White—you can perform the function, also, that way.

Scene Mode gives you the ability to make slight adjustments to the camera’s algorithms based upon the type of specimen that you’re looking at, and whether it might be industrial or biological in nature, or just a straight linear. And under Commands, you have the ability not only to define, and save, and load regions of interest for processing in the future, but also an advanced set up where you can control camera exposure setting, Live Acceleration for live movements, and other image settings, including White Balance and Resolution Mode

Annotations and Measurements are just what they are: the ability to place descriptive information and measurements on your image. Annotations come in the form of text boxes or arrows or squares or circles. Your measurements come in the form of Length, or Area, or Angle, Radii, Axes, or Count & Taxonomy. The measurements are placed in the image and tabulated in the customized table at the bottom of the tab. The image and measurements can then be exported into a report that is also customizable, or exported to an Excel format.

We have done a pre-annotation here for you, and we’re going to place a micron bar on our image, and then we’re going to place a small image on it—just a cross, nothing fancy, just a line with a designator. So what we have here now are some annotations, we have a micron bar for reference, and we have an actual measurement. We can now take this information, and we can actually export it to a report. And here we’re just going to type in the type of material that it is; I’m working with a very simple quartz permamount, and we can move this over, and then we can make sure that we are exporting to a report. We can export our measurements to the report. And there is a very simple export to a very simple report that we’ve put together that shows our annotations, our measurements, and our reference bar. You can always save as a PDF or print, if you have interest in that. We’ll close this out and move on. We’ll remove our annotations, we’ll eliminate our measurements, and we’re back to our clean image.

We’ll now talk about Look Up Tables (LUTs). Look Up Table give you the ability to make adjustments to your images, and if we wanted to work with one individually, we just click the Shift button and we make the move there. If we do happen to make something and we don’t like what we see, we can just hit the Reset button. Optical configurations can be saved for each observation method, such as brightfield or polarized light, to include objectives, contrast method, the microscope components—if they are motorized, and the camera settings. These optical configurations can then be displayed in the top toolbar of the software interface. You see I have one here for brightfield, and if I go to polarized light—I make the adjustment to polarized light, we’re automatically in the right camera configuration. Now if we had microscope components or filters that were motorized, they could be included in this same process.

I’m going to move up not to a higher magnification, and I’m going to do an Automatic Exposure and to make sure that I’m in good shape, I’m going to stick with what I think is here, 4, and I’m going to go to my polarized light. So here we are, if you look and focus live, we can see that there are portions of this sample that are thicker than the depth of field for the objective. So we’re going to do something called an EDF, or Extended Depth of Field, and what that does is, it allows you to select an in-focus area from multiple z-stack images and produce a single, all-in-focus image.

I’m going to focus on this area here, which seems to be my highest. I’m going to go to Applications, I’m going to go to Real Time EDF Manually, and I’ll move this start button off of the image plane, and then I’m going to Start. And you will see that we will end up with two windows, and I’m going to, slowly now, focus up through that image—again, this is a manual microscope—this can be performed with a motorized Z, where you set the top and bottom as well, but as I go through and continue to focus on, down through, I then come to a certain position. I’m going to then finish that function, and the computer is going to process that image for you, and it’s going to display a single, all-in-focus image from top to bottom. And there is that image, now.

The next function we want to look at is High Dynamic Range (HDR), where we have bright and we have dull. So if I go here and I click about a 60 second exposure, or maybe even a little bit higher, and I begin to focus, on the right-hand segment of the image, you can see I’m getting some detail, but over here, it’s washed out. So what I’m going to do is, with these images, I’m going to again go to my Applications and I’m going to capture an HDR image. I can set a high exposure, and a low exposure. A high exposure allows me to get information in the dark areas, if I were to knock this down, and then also, if I go to the low areas, I’ve now picked up some of this information, here.

We’re going to go with five individual images, we’re going to click on HDR, and you now see the image before you balanced out. If I hit this one function, I now have a window that gives me what the original image looked like, and you can see how it was balanced out. We’ll go back to a live image, and as you can see, we’re back.

I’m going to slide back down to a different magnification here, which in this case is 10X. I’m going to do an Automatic Focus and make sure I’ve got an image.

And the last thing we’re going to do here is talk about a large image, or image stitching, which generates a single, wide field of view image by stitching multiple adjacent frames together. This function can be formed automatically using a motorized X-Y-Z stage, but can also be done very simply in manual mode.

So down here in Manual Large Image (Grabbing), we’re going to make an adjustment to show a side-by-side comparison. Here’s our live frame, and here is where we will generate our large image, or image stitch. We hit start, and we now move over to the right edge, and the software automatically picks up and takes the next image. We move over one more time, and it automatically takes the image. And if I say Finish, go ahead and say Finish, and there is our finished large image of a 1 x 3 taken now. Or, I simply could have dropped down, or gone backwards, or in a snake-type position. With a good X-Y stage you could do a 3 x 3 or 4 x 4, of even larger, in just a few minutes.

As we get ready to close today’s presentation we want to be sure to offer our thanks to Ryan McGaha. Ryan is our Nikon Metrology representative and provided valuable support in the development of this presentation.

Plan to join us for our next segment of this 3-part tutorial, focusing on more advanced imaging and metrology operations. This upcoming segment will highlight more EDF Imaging applications, 3-D Volume Measurement, Movie Maker, additional High Dynamic Range Imaging, Image Stitching, Live Compare, Auto Measure, Auto Thresholding, and Macro Commands.

We look forward to having you present for this next segment. We will announce the date via our website and emails to you very soon.

Please email any questions that you may have or requests for further information or clarification to me, at the above email. We will answer your questions promptly.

Thank you for joining us.

Charles Zona: Okay, thanks, Jim. I’d like to thank everyone again for tuning in today. We really appreciate your time. We’re going to leave the questions field open for a little bit longer, so again, if you have any questions, please go ahead and type them into the questions field, and we will get back to you with answers to your questions. And as I mentioned earlier, this is part one of a three-part series, so please check out our Webinars page for upcoming McCrone Group webinars information and for parts two and three of this imaging series. Thank you.

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