Instensity Calibration using a Photostable Light Source

Camera Calibration Source

I’m very excited to announce Technical Instruments’  first ever camera-specific calibration product! At left is an image of the CC-560, or Camera calibrator, which emits photons at 560nm.

All of the cameras in the microscopy world rely upon a single defining point that (theoretically) justifies the price premium we pay for them. That defining point is reproducible, quantitative linearity of intensity. Put simply, if I acquire some number of images, using the same camera settings, over a long period of time (days, weeks months etc), my camera should give me image intensities with the same brightness values, when provided the same amount of input light.

Note that linearity, as well as quantitative accuracy, are expected minimum specifications on camera info sheets. Some examples can be found here:

Nikon DS-Qi1

Andor Clara

How does this quantitative/linear capability translate into real-world data for the end user? With difficulty! Upon purchasing a new camera, most new owners of a quality camera will find a unique set of conversion tables for their serial number camera.  These tables note in part the individual electron to ADU conversion rates for each A/D readout speed, and Gain setting. This data can be used to discover how many electrons the camera collected for a given image. For instance one could have a  3.05 e/adu conversion rate at 10Mhz, as well as a 2.54x gain setting. Using these conversions we would find that a reading of intensity in an image, say with a value in the image of 100, would convert to ((100/adu)/gain) or (100/3.05)/2.54 = 12.9 electrons collected. Note this basic calculation does not include bias offset, which is another value provided in each camera’s specification sheet. The take home point here is that each camera manufactured will have it’s own, unique, set of values. You can use these values to convert your ADU’s (Analog to Digital Units) which are what you see as intensities in your image, back into electrons collected on the camera. This is especially useful when comparing images collected between multiple gain settings, or where camera speeds were changed and a corresponding change in image intensities was noted.

For some people, these series of calculations and corrections, as well as others, may be a required, however tedious, fact of life. On the other hand there are a vast majority of researchers who want answers to simpler questions, such as:

  • What is the comparative sensitivity difference between the camera I used to work with in the lab down the hall, and this new camera I am now using?
  • How do changes to my camera, such as binning, really affect my data?
  • What do the detailed controls do on my camera? (Such as changing “Clearing” settings, or Parallel Clock speeds and voltages)

Customers using EM-CCD cameras may have additional questions, such as:

The simplest answer for these questions was to have the customer find some photo-stable light source, and use that light source on the microscope to find the answer. The tough part was actually finding a useful photo stable source! As an answer to these challenges, a few colleagues and I have created this product, available from my employer, Technical Instruments.  This calibrator is designed to give people simple and direct answers to these types of intensity based questions, with a minimum of hassle or headache. The product will work on ANY C-Mount camera, and can be used either for dedicated-use calibrations (i.e. install – calibrate – remove) or can be placed in between the camera and the microscope in a permanent installation (See example image of Andor camera and Nikon Ti-E microscope with CC-560 installed at right). I’ll be posting more soon on what this product can be used for, however I’m certain there are numerous uses for this that we haven’t considered. Please take a look at our new CC-560 Camera Calibrator.

updated Nikon DS Pixel Sizes

In case anyone is looking for the Nikon DS series pixel size, I have them listed under the specifications page.

Edit Device Settings for multiple programs easily using XML Notepad

NIS Elements, MetaMorph and a host of other programs rely on XML files to save settings that are user-adjusted inside the program. XML files are like mini databases that can be easily set up, modified and read by a host of programs. The tough part comes when you may need to manually edit one of these files. Viewing an XL file is difficult in IE, and editing it is even more difficult using wordpad or notepad! So Microsoft came up with a handy editor, which can be downloaded here – XML Notepad. I highly recommend keeping this tool handy!

- Austin

How to Change a Lambda LS Bulb

Chris Ballard from Sutter Instruments has posted this Youtube video for changing an LS Bulb. It’s a great DIY reference to change a bulb!

Questions on Color Imaging

I’m cooking up a series of image conversion filters and explanations relating to color images vs. multiwave images. If anyone has questions related to this please email me so I can include them in the post!

- Austin

SLR cameras for Microscopy

Camera on Leica Microscope

Most of the time, I work with people who need measurable and accurate (quantitative) results from the images they obtain. While this constitutes the bulk of my daily job, there is a much larger group of people using cameras on microscopes for photomicrography. Or put simply -- taking a picture of what you see through the scope for the

purposes of presentation or archiving.These people live in an a space completely underserved by the microscope imaging community. Here’s why:

  • C-Mount cameras are usually cooled (which isn’t needed for basic trnasmitted image capture)
  • C-Mount cameras are usually specified to be linear and quantitative (again unneeded for basic image capture)
  • The software used by these cameras has more features than are needed to snap an image
  • The production rates for these cameras and cost of scale equal an unjustified high price for basic image capture.

Now when I use the term C-Mount camera I’m referring to cameras that are dedicated for microscopy use, which have no glass lens assembly in front of them, and connect to the scope using an ISO C-Mmount.

So what options are available besides the traditional camera? Enter the venerable SLR. Before the digital camera era, everyone had a film body camera on their microscope if they were acquiring images. This system could produce beautiful images, but required all the usual steps in film photography. There are a wide array of mounts available for cameras such as these, as most miroscope optical systems were designed to work with SLR cameras at some level.

Today there are a wide array of DSLR (Digital Single Lens Reflex) cameras available. These cameras use similar (but not exactly the same) optical setups that will, in most cases, work quite well on a microscope. Why is this?

  • In general, we don’t want glass to be in front of the sensor. DSLR cameras, like C mount cameras, use a lens connection, allowing for the lens to be removed, and the camera to be placed on the microscope. So the microscope becomes the “lens”.
  • Modern DSLR cameras like the D90 from Nikon or the 50D from Canon include a “live view” function. This function allows the user to view the live image from either the camera body, or the method I recommend, through remote control software running on a computer.
  • DSLR cameras include incredibly powerful image processors in-camera. This means you won’t ahve to fiddle as much with white balance to get a high quality image that accurately depicts what you see under the scope. Pathologists are famous for seeing these color balance errors as they have the best trained eyes when it comes to minute color change on stained specimens.
  • Unlike film or digital still cameras, newer DSLR cameras can acquire HD live video. Here’s an example from a Nikon camera. One can imagine the use of this for live microscopy recording, all with the same camera…

So why not use any handheld camera? Because the typical lower cost camera will not allow you to remove the lens assembly. This is the primary benefiet of DSLR and C mount systems.

If you’ve read this far and are interested in using a DSLR for your microscope, you’ll need a short shopping list of components on order to make everything properly work. Here are the components you’ll need:

  1. Camera Body (you don’t have to buy the camera with a lens if this will be a dedicated scope camera)
  2. Camera -> Microscope Mount system.
    1. You can use either a microscope manufacturer’s recommended system like the ones described here (Nikon F Mount options)
    2. Or use a high quality aftermarket adapter from a company like this (Canon and Nikon F Mount coupler)
  3. Control Software for your camera (Nikon uses software that you have to buy separately, canon software is included with the camera).

Notes on Acquisition:

Once you have the parts together, you’ll need to set the camera up for the right shooting mode. On most cameras you’ll see an “M” mode. This is preferred for microscope use. M is full manual mode. There is no focus or aperture for the camera to control on the microscope, so those adjustments are not required. The remaining adjustments (exposure time and ISO) can be controlled manually. For low light fluorescent work, use high ISO. For normal transmitted light, use low ISO. You can use the metering system on the camera to determine the exposure time, and all of these settings can be controlled from the computer. Finally, make sure to enable “exposure delay” mode, which will avoid capturing any vibrations introduced into the scope when the shutter drops open!

As always if you have any questions on this technique you can contact me with questions!

- Austin

Running Micro-Manager with a Nikon TI Microscope

If you are running uManager you will need to install the Nikon Ti Drivers, as well as have a way of configuring the scope settings. You can address both issues by installing Ti Tools. You can find the most current version of Ti Tools from Nikon’s Website (including 32 or 64 bit versions) here.

-Austin

Mobile Support!

I’ve Added a mobile support plugin to the website for those of you living on the road. It’ll work with almost every smartphone/iphone/droid type of device out there. Hope you like it!

Thanks to Alex King for the awesome free plugin!!!

- Austin

Steve Jobs Explains Back Thinned Chips!

A few of my work colleagues pointed out the well presented positive aspects of larger pixel, back thinned sensors. If anyone has heard me prattle on about this they’ll find it familiar! Fast forward to minute 1:00 for the points on the new iPhone 4 camera. Steve’s explanation is dead on!

- Austin

Split XY multipoints macro

Has been added to the free macros list under Macros & Journals!