When a camera or a device isn’t working there can be any number of things wrong. How do we narrow the potential list down? I’ve taken a page out of my Toyota service manual and made a basic diagnostic flow chart which one can follow. This chart will basically work with any device and any software. Just remember the “normal” arrangement of what it takes for software to run a device:
Control software that the user clicks to control device
Drivers that the software uses to talk to each device’s driver (or serial / parallel ports)
Windows Drivers (if used with device)
Hardware used to talk to device (serial, usb, parallel or PCI Card)
Device Brains (firmware)
Device itself
If any one of the links in this chain is broken or missing the whole arrangement won’t work. You can find the flowcharts which help identify the problem here.
Yes this is a bit late. Things are busy these days!
Elements B 548 can be found here if you need to download it. As usual back up all of your optical configurations and take screen shots of any filter configurations (i.e. if you are using sutter wheels) just to be safe.
So far this build has been stable. Only one or two bugs discovered. The major addition I see is we can now run High Dynamic Range processing from within elements. I can see why this would be a major advantage for stereo systems, as well as fluorescent images where the signal differences can be large. Haven’t tried it yet but it looks great.
Up for review is a new Infinity 3-1 camera from Luminera. The camera is a new venture for Luminera as it uses a high grade monochrome Sony IXC-285 CCD. This is the same sensor used in many of the Photometrics CoolSNAP cameras, Hamamatsu ORCA cameras and many others. This is my first review, so comments on the formatting are encouraged!
What Comes with the Camera:
Luminera cameras are all shipped in the same packaging. A cardboard box with foam inserts. Included in the box are:
-Camera itself
-USB cable to connect the camera
-Software on CD
-Documentation
Installation and Setup
Installation of the software was a snap. After installing the CD and following the on screen prompts the CD installed the software for operation and the camera drivers. Once that was complete I plugged the camera in and it identified in windows correctly. The usual WHQL driver warning popped up.
Image Quality
The system uses an “analog” type gain slider, so no true ADU analysis was possible. The following measurements were performed with the Gain set to 1.02:
-Read Noise = 49 Counts
-Dark Current @ 4.5 Sec = 261 Counts or 58 counts per second
-Frame Rate = ~15fps (unable to measure sequence timestamp in software)
-The camera read out “0” count values regardless of setting the gain to maximum and exposing for 4.5 seconds. (Bias offset incorrect)
-Gain control appeared non-linear. Images were acquired with no light to the camera, with gain changes from ~1 to ~11. Results are graphed below.
Conclusions
This is a great low cost solution for less than bright fluorescence when used for non-quantitative imaging. In order for the system to be a truly quantitative unit the bias must produce no “0” count values for each gain state used. Ideally some data would be provided with the camera showing the Electron to ADU conversion factor for each gain state used. These negatives aren’t really bad per se’. Considering the market space Luminera targets this camera should fit the needs for higher sensitivity, good performance across the normal fluorescence wavelengths, small size and simple operation. On top of all that this is the
best-priced camera that uses the venerable Sony ICX-285 CCD.
I’ve been asked by a few people what is the “Right Way” to export ND2 files to AVI. A lot of problems arise when the file is moved from the computer where the file is saved, to a different computer for playback (think your laptop for lab meeting or better yet your mac)
Here’s the scoop:
AVI as a format is simply a format with specified data dimensions intended to hold color video data (640×480 or 320/240 resolution , 30fps, 24bit). This data can be saved (and tagged with the *.AVI extension) as long as you save the file using the correct descriptions and dimensions as specified in the format of the file. The problem here is the inherent size of the file. Without any compression, the file size for a 100 frame video will be something like 240mb. The file gets to be too big for practical use once over 1gb, so what do we do?
This is where the choice for CODECs comes into the mix. A Codec is a “Compressor/Decompressor” layer that sits between the normal AVI playing programs like windows media player and the file. When the file is saved it gets processed through the compressor side of this layer and you end up with a smaller file. When the file is played the decompressor side of the layer is activated, the compressed data is stretched back out and displayed on screen.
There are a lot of options for the codec you use. Making this more complicated is the fact that you need the codec installed on BOTH the saving computer and the playing computer. So using this knowledge here’s the common scenario:
1. Lab member uses elements to save ND-2 data as AVI, and uses the defaultcodec. (Intel Indio Video 4.5 in Elements). The file saves and a check of the file on the computer shows the file plays back.
2. the lab member copies the file to his Mac and attempts to play the file. Quicktime doesn’t support Indeo Video, so the file doesn’t play.
In a nutshell the codec isn’t installed on the playback system. So what can we do?
For PC to PC playback the simple option is to download Windows Media Player 9 or 10 using Windows Update. Microsoft’s tech support site lists Indeo 4.5 as a supported format for WMP 9+.
For PC to Mac playback (or to Ubuntu playback) the simplest solution is to download the VLC player on the mac. VLC (or VideoLan) is a multi OS media player tool that will usually play any media file when everything else won’t. You will need to save the files as the previous generation Indeo (version 3.2) in order to play it in VLC on the Mac. Check out VLC’smedia compatibility page for more information.
Just keep in mind when saving to AVI that a LOT of information is lost using these formats. These are for DISPLAY ONLY and not for data analysis!!!
When acquiring images through a stereo microsocpe one of the most common problems to overcome is inconsistant lighting. Whether looking at a printed circuit or looking at a natural specimen such as a fly most of these samples have the potential to reflect light. Add to this the inconsistancy of reflection (think the fly eye vs. body, or the chip coating vs. connecting pins) and what we get is a dynamic range problem.
One way to overcome this is by using a cheap and simple copy of a softbox. Normal softboxes for photography are commonly used for macro shoots. We have two problems with a softbox for microsocpy: First, the bottom and top of our softbox must be removed, as we need to place the sample down, then place the softbox on top, then acquire images looking from above. Second, we need a softbox that can dynamically change it’s size depending on our magnification, working distance and specimen size.
Both of these problems can be overcome using either a piece of standard printing paper, or for better results a sheet of diffusing paper such as the type used for overhead projectors. In either case simply cut the paper to size, and form a cone with an open top. Tape the cone together and place it over the specimen. Assuming you are using bifurcated light pipes simply position the lights on either side of the cone and far enough back for even light distribution. What you’ll get is a uniform, diffuse light that drops glare and reduces the dynamic range!
Here’s an example image using a softbox from a sheet of printer paper. Enjoy!
A few weeks ago Photometrics released a new EM-CCD camera tagged the Evolve. After attending a webinar presentation on this camera I wanted to point out the major technological benefiets of this system. Time will tell how these additions impact the overall performance of the system, of course I’ll be posting my impressions here as soon as I have an opportunity to work with it.
Quant-View: This feature finally removes the need for users to back-calculate ADU’s to electrons. Normally the values seen on your screen in software programs are numbers generated in the conversion of millivolt values on the sensor into numeric values, with some conversion factor applied (for instance sensor / 4 = Value to image). This is normally controlled with Gain in the camera settings. What makes this a pain is when you want to figure out either how gain 1 compares to gain 3, or when you want to figure out how many electrons were actually collected. Using electrons collected you can determine how many photons were collected by the snesor. With this new feature from PM (Quant-View) you no longer have to make this conversion. The values present in the image equal the electrons measured in the ccd.
Rapid-Cal: This feature is used to quickly recalibrate the EM Register on the camera. The primary advantages to this system are the faster calibration (which will make it more likely for people to calibrate more frequently for additional accuracy in the gain register) and the addition of a physical shutter inside of the camera body. This shutter ensures all light to the camera is blocked during the calibration process. Standard microscope bodies do not allow for this, so this is a nice feature to have for the calibration process.
Background Event Reduction Technology: This feature basically runs a median filter on any pixels that show a single-pixel spurious charge. The user can specify settings for whether these pixels are corrected on the fly. There is a video on how this works at Photometrics’ website here.
Black-Lock & Top-Lock: This is basically an on-camera scaling tool. By setting a minimum and maxomum output value in the camera controls you can force image output to clip signal and noise to those values.
Vari-Bit: This is a new A/D converter in the camera that supports readout in 8,12,14 and 16bit ranges. This can be useful in matching the dynaic range of the image to the A/D.
In summary Photometrics has added on-camera processing and a physical shutter to an EM camera. There is a nice marriage of convienent features to this camera to be an incremental step forward technology. Time will tell how much of a performance gain over similar systems is achieved.
One of my customers (hi Paul!) asked me to make this macro a while back. Once I got into the design I realized just how useful this would be for basically anyone running an automated stage with Elements.
Right-Click and “Save-As” to get the Macro , and check the readme before use!
The macro allows the user to scan around a current position, to either find small objects or to aid in focusing on small objects. Please comment if you see anything that needs to be added or changed!
I’ve been running into a lot of programming applications lately. The fun part about using the Elements (or other) programming system is the amount of utility and flexibility it can offer to end users. Most people think about programming in our industry only as it relates to image analytics. I wanted to point out some other things macros can do for you:
Acquisition & Device Automation
Do you have a stimulus device or method you want to trigger from your acquisition software? Almost every device on the market (FRAP illumniators, Profusion systems, heating systems, electrical signaling systems) can accept a command input from TTL. You can use Elements (or other software) to control the parallel port on your computer – giving you control of the device you already have. A macro can tell the device to start, stop, adjust a setting and can then record that command into elements (i.e. at what time point in a timelapse the command was issued).
User convenience
Are you always pressing the same buttons in software to control the microscope? You can use macros to assign keyboard keys to run special functions. For instance when you press the “0″ key on the number pad you can make the microscope load a “DAPI” illumination setting and acquire an image.
User interaction
Do you only run a few experiments over and over? You may be working harder than needed by re-configuring your software to image with a specific protocol. You can use a macro to configure all of the settings you want in one operation, and then dial in specific changes you may want to adjust before acquisition.
In general any button you press, number you enter or menu you click can automatically be performed inside of a macro. Keep this in mind any time you are repeatedly clicking on the same things and slowing down your work!!
EXAMPLE DOWNLOAD You can find an example macro for use in Elements here. Open Elements, open the Macro menu and click Edit (or press F8) and copy the code from this page into the empty macro. Then just save and run it!
