I've Moved!

I've moved my blog site to my homepage at www.austinblanco.com

To get to the site use this link or enter www.austinblanco.com/blog in your web browser.

AB

DXM-1200 C Camera Compatibility

A few years back Nikon released a very cool 12 megapixel stepping camera, called the DXM-1200C. The camera was reliable and a nice product for many years. Unfortunately the march of progress made a dent in this camera. The system, like many others, relied upon a proprietary PCI card for transfer of image data to the PC. This reliance required that the PCI card was happy living inside whatever computer the customer had.

As time continued the card moved from the "most current" to not functioning with newer chipsets. As problems begain to appear with the chipsets more and more people found that they would buy a nice new computer for their imaging system, only to discover the camera would not work with it! In an effort to reduce lost time Nikon produced a document describing which chipsets are known to work and which were known not to work. Hopefully this doc will help a few of my customers avoid this issue! You can find the doc here.

Video of Human Blood Clotting

I Found this while searching for a fluorophore. Very cool to watch on youtube!!

Human Blood Clotting

- Austin

Sensor Size reference Chart

Recently a colleague sent me a link to sensor sizes. Many of these are for consumer cameras, however the general references (1/3', 2/3" and so on) are helpful for microscopy cameras.

http://www.dpreview.com/news/0210/02100402sensorsizes.asp

- Austin

Pulse Control within NIS Elements 6-D Acquision

These directions will allow user control over an optical configuration to be specified at a given time-loop inside of the define/run experiment. So for instance if one wanted to trigger a shutter or light source to flash un-cage at the beginning of a high-speed acquisition, this would meet the need. These are a bit lengthy but remember the setup is only needed once!

1. Create an optical configuration that activates your pulse. You only need the configuration to be active, but note the shutter should be set to “active shutter” and CLOSED when the optical config is selected. The shutter should be set to open only when the user presses the shutter button or when the camera is acquiring.
2. Download my macro file that contains the control code needed for this to work. (If you have Quicktime installed windows will think this is a movie - make sure to Right-Click on the link and select "Save Target As". )
   
3. Save the above macro into something like c:\Macros\ or wherever you can easily access it.
4. In Elements select the Macro Menu, then "Open Macro" and open the macro file.
   
5. Note in the macro file the lines that read "Stg_SetShutterState(3,1); this means "set the state of a selected shutter type(shutter type,Open)" . So set the first number to the shutter you want to control. (0=EPI, 1=DIA, 2=Aux1, 3=Aux2). The second number is easier - 1=open and 0=closed. ALL YOU NEED TO DO is make sure you are controlling the correct shutter, by changing the first number in the 2 lines of code in the macro to drive the correct shutter!
   
6. Note in the macro the "Wait(1.00); command. Where the 1.00 sits is the amount of time the shutter should remain open. For a pulse time of 500 milliseconds the code should read "Wait(0.500);
   
7. Test the macro at this point by running it. The desired shutter should open, wait and close. if modifications need to be made to the optical config or the code now is the time to do it!
8. Next set up a text experiment that has 2 different time phases at minimum. Click the "Advanced" button to show the advanced time controls.
9. Set the "Advanced for" box to read "Time Phase 2"
10. Select the check box that reads "Execute Command at the Beginning..."
11. Click the Arrow on the right side of the entry box and select "run macro"
12. Go find the macro file you downloaded and modified earlier and select that macro.
13. Your configuration should now look something like this one:
14. Set the acquisition rate you want in time phase 2.
    
15. Back up the experiment using the Save button.
    
16. When you run the macro, and you reach time phase 2 the pulse will occur at the same time as the second time phase, without interuppting the experiment flow.

- Austin

iPhone ResCalc Review

A few weeks ago I purchased an iphone. While browsing apps I found one that we can use in the research community! A company called Zem Dynamics (who also offers FRAP solutions for live cell imaging) has released a small app that can calculate minimum resolvable distance, as well as optimum camera pixel sizes for a number of objectives and magnifications.



Positive Notes

• This takes a lot of time and energy out of finding the best camera for a given optical setup. It's also a good way to find out how much binning will affect a given input image and how far one can bin when using various magnifying tube lenses or c-mounts.
  
• Simple operation and controls eliminates a lot of potential error.
  
• The app sells for $1.99. At this price you can skip that cup of afternoon coffee and break even on owning the tool!
• This app loaded and ran very quickly with no crashes. I ran it on an iPhone 3G-s.
• Pressing the Info button on the app switches the view from resolution to field of view calculations. This is very helpful when determining how much area a given camera will collect, or to find the best match between a camera and magnifying lenses.
  

Negative Notes

• Pressing on the output values (either the pixel size or the resolution) brings up the iphone keyboard. There is no way to hide the keyboard or complete an entry of pixel size, so the only way to hide the keyboard is to close the application.
  
• I'd like to see additional numerical apertures to support more objectives (and other scopes like stereo's and macrozoom systems).

Conclusions

From my calculations this app is using the standard Nyquist criteria of (0.61*emission)/Numerical Aperture. I have contacted the manufacturer to discover the pixel size calculations, however it seems to be ~2.5 pixels per diffraction limit. This is a good compromise setting to use, (see my resolution calculations here).

Overall this is a well designed and very useful app. I'd suggest anyone using or working with microscopes or imaging systems will find this a helpful tool. Of course you'll also need to own an iphone to run it :-)

- Austin Blanco

Copying Elements Settings for Multiple users

After installing a new imaging system it's common to receive a phone call or email that goes like this, "The IT guys stopped by today and added our computer to our comany/university network. Now all of our settings are lost!". Why does this happen and what can be done?

All settings for elements are stored on a per-user basis. When a different user logs in (i.e. the user assigned from a company network vs. the default user that is installed on the PC) the software expects that user to want his or her own settings. Thus no settings are copied over. In the majority of cases this should NOT be the use case for a user group. In my experience almst everyone would like to start with the "default settings" and then modify them to their needs. For now there isn't a copy button available, so we need to manually copy the settings.

Getting Started:

Copying settings is actually a simple process if done correctly. A few notes on this:

• When moving to a new domain or user, don't delete the old user until the new user setup is working well for you!
• Make sure to copy the backup files we generate to a common directory. For instance don't save to the "desktop" when logged in as administrator, as non-admin accounts may not get access to the admin desktop, leaving you no way to snag the needed backup files. Instead copy the backup files into a folder like c:\Elements_settings.

Backing up:

We need to back up 3 items: Program menu/docker layouts, optical configurations and macro settings.

1. With the program running and all devices connected, click the "explore optical configurations" button.
2. There are two backup buttons here. First click the Backup button that sits between the optical configs and the camera/scope settings. Save the file to back up the settings. Secondly click the backup button below the objective list. Name this backup_objectives.
3. Once these files are backed up we can close the optical configuration window. Next we will back up the menu layouts and docker settings. To do this select the View menu, then Layout, then Layout Manager.
4. In the layout manager we will export the current layout to an xml file. ***because all of these files are *.xml be sure to name them descriptively so you can figure out which file is for what backup later.
5. Next we will check up on our macros. In some installations macros may be configured to run every time elements starts. In these cases when users are switched these "start-up macros" will need to be re-specified. To check if you are running any startup macros, go to the Macro menu, then select Options. In the window on the left-hand side you'll see any macros that are set to load when elements starts, as well as whether those macros are activated as "start-up". See the example below where I have one macro set as a start-up macro.
6. Make sure to find where this macro file sits on the computer so that we can add it into the new user account later. This can be found by viewing the "full path" info below the white macro box.

Copying Over

With the backup complete we can log out of the current user and log in to our new user. Once logged in as the new user launch Elements.

1. Open the optical configurations window. Click the Restore button below the objectives list and load the objectives backup file.
2. Click the restore button next to the optical configurations and load the optical config backup file you saved earlier.
   
3. Select the View menu, then layouts, then layout manager. Click the "Import Layouts" button and select the layout file backed up earlier.
   
4. If you had any startup macros selected, click the Macro menu, then Options. Add any macros needed and set them to startup if required.
   
5. Test the system to make sure the previous user account settings have been transferred successfully.

Hopefully this guide will help users when needing to copy settings to new user accounts. There is nothing wrong with using the same three or four backup files and loading them into multiple user accounts as well! Please post here or email me if you'd like to see any additions or changes made to this entry.

Austin Blanco

Andor sCMOS

This is a quick post - things are busy! Andor has a white paper and information up on a new "Scientific CMOS" sensor. Have they overcome the limitations to CMOS sensors for Scientific applications? This could really change the landscape for microscopy imaging!


- Austin

Why do I want a low resolution camera?

These days it's not uncommon to hear about consumer cameras with 10+ megapixel resolution. To the average consumer this seems like a good thing. How can more pixels be anything but good, right? In reality there are several tradeoffs to using more pixels. This is especially true for any scientific imaging (this really applies to low light an/or quantitative imaging such as microscopy and astronomy). So what are the downsides?

First let's look at the concept of a pixel. Pixels are physically sized on a CCD or CMOS sensor. For an example of pixel architecture check out this page. The physical size of a pixel affects several aspects of image acquisition:

1. Dynamic Range
   
2. Sensitivity
3. Resolution
4. Speed

Most people simply think "bigger pixels means less resolution" or "more pixels result in more resolution". This isn't the only thing that is affected by pixel size. A more interesting question to ask would be "What am I sacrificing by using smaller pixels?". Of the four performance aspects above three are negatively impacted when using smaller pixels. These are:

1. Dynamic Range
2. Sensitivity
3. Speed

Dynamic Range

The dynamic range of a sensor is first determined by the full well capacity(FWC) of a single pixel. This is a measurement of how much energy the pixel can hold before either becoming nonlinear or before the energy spills out of the pixel into neighboring pixels (called blooming). Smaller pixels simply cannot hold more energy due to physical size, so smaller pixels = less dynamic range. Let's compare two common chips - the Sony ICX-205AL CCD, used in the Photometrics CoolSNAP cf2 camera (and many others), with the Sony ICX-285 CCD, used in the Photometrics CoolSNAP HQ2 (this ccd is the most prevalent in modern microscopy cameras).

• The 205 CCD shows a FWC of 9,700 electrons.
  
• The 285 CCD shows a FWC of 16,000 electrons.
  
• Note that the 205 chip has a pixel size of 4.65um, while the 285 has a FWC of 6.45um.

In the case of these two sensors, a 2x increase in pixel area provided a 65% increase in FWC. The larger chip has more FWC, so I can detect a range of brightness with more accuracy, or have more range of bright to dark, or more dynamic range.

Sensitivity

This is an easy one: If I take a given area of my image being projected onto my chip, I chop that area (or that brightness value) up more times if I have smaller pixels. Lets assume I have one chip using 5x5um pixels, with another that uses 10x10um pixels. Let's also assume the light being projected onto the sensor area is 100 photons per 100um square area. Using my smaller pixel chip each pixel could collect 25 possible photons. Using my large pixel chip each pixel can collect 100 possible photons, so the larger pixel camera can grab more light per pixel than the small pixel camera.

Speed

The argument I make here isn't really a technolocal limitation but more a common product/market limitation. All of the cameras we use are set up to collect an image projected from a microscope into roughly a 1" diameter area. So I am limited in how much area I can use on my chip. Because of this if I have bigger pixels I cannot simply make a gigantic chip that has say 1000x1000pixels, as it's possible the corners of my chip won't have any light projected from my scope. This means that intrinsically I'll either find chips with a lot of small pixels, or chips with fewer large pixels.

When considering speed there are several factors: exposure time, analog to digital conversion time, and "frame shift" time. (I'm using this term to conver interline shift and FT operations for those purists out there). Now if you look at any data sheet you'll notice there is some "top speed" a camera can go. You can bin the camera and use a small readout array but you hit a limit - why?

Let's assume we have a camera of 100x100 pixels that can expose an image and get enough light in 10ms. Let's also assume our A/D converter that turns electron values into digital numbers can operate at very high speeds, say 100Mhz. In a case like this our camera can theoretically run at 100 frames per second, which is quite fast. Now what aspect of the system will stop me from say driving the camera at 10,000 fps? If we consider that the camera must move each pixel's energy off of the chip, using shifts of energy from one pixel to another or into some series of registers, whatever time it takes to move from pixel to pixel will become important. This is referred to as the shift time or shift rate. Depending on the architecture of the camera this time will affect the camera to different degrees, but ultimately this is a measurement of "How fast can I move energy across my chip?". This bucket-brigade type of energy transfer is what takes a electron charge in pixel 1A, and spits it out the other end of the chip into the A/D converter.

So using our fake camera here let's say it takes our camera 50 microseconds to move from one pixel to another. Then let's consider how many moves must be made to get pixel 1,1 out the other end of the chip at position 100x100. Basically pixel 1,1 needs to be moved 100 pixels down and 100 pixels over. So 100x100=10,000. Each shift = 50us, so 50us*10,000 = 500,000us, or 500ms. So using our fancy super fast camera we can only go 2 frames per second!!!

Obviously the example above is using excessively show shift rates so I can make my point. The bottom line here is that shift times do have an impact. So how does this relate to big pixels? Well if I have bigger pixels we already found that I must use less of them. Less pixels = less shifting that needs to occur, so the impact of shift rates becomes less! Ultimately you'll find that cameras like a 128x128 EM system can go up to 4000 fps, whereas a 512x512 EM system only goes up to 500fps, with the difference being shift rate*pixel count.

Conclusions

So many times I am asked why big pixels could possibly be better. Now you know! Don't let the hype of "megapixels are cool" lead your lab into an inappropriate camera for your research. Remember that the camera is a detector, just like other instruments. While the appearance of the image may suffer with large pixels, the data contained in the image can be more informative and accurate than data collected using high resolution systems. Try to prioritize what performance aspects will aid your area of study, and keep these aspects in mind when finding the right camera for your work. You'll be happier in the end with the right tool for the job.

- Austin

Elements Downloads

You can find the latest builds of Elements Here:

NIS Elements AR or BR Build 548

NIS Elements D Build 548

Something Broken?

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:

1. Control software that the user clicks to control device
2. Drivers that the software uses to talk to each device's driver (or serial / parallel ports)
3. Windows Drivers (if used with device)
   
4. Hardware used to talk to device (serial, usb, parallel or PCI Card)
5. Device Brains (firmware)
6. 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.

How to use Object Count in Elements

This is from the Nikon marketing group - excellent work if you are new to object count!
Download PDF

Elements Build 548 Released

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.

Austin

Luminera Infinity 3-1 Review

Review of the Luminera Infinity 3-1

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.

-Austin

AVI playback with Elements Files

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 (640x480 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 default codec.  (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's media 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!!!

    

Lighting Control for Stereo Microscopy

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!

Photometrics Releases Evolve EM CCD

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.

Elements SP 6 released

Nikon has released SP6 for Elements. There are several improvements for:

• Ti Microscope controls

• TIRF controls

• QImaging cameras

• ASI stages

• ND acquisition

• PFS control in ND acquisitions

• Macro controls in ND acquisitions

There is a complete list of the changes/fixes and upgrades in this PDF.

Stage Search Macro

Stage Scanning Macro for search and focus

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!



Austin Blanco

What can programming macros do for me?

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!