What to look for and how to replace the drive gear
The Venerable Nikon Ti-E remains in service in research labs all over the world. One of the first autofocus-enabled instruments deployed with the integrated PFS, there isn’t much to ask for when it comes to a modern compound fluorescent stand. For owners of the instruments, it’s important to be aware of a big maintenance item on the scope – it’s motorized epi filter cube turret.
It lived a good life!
These turrets use a stepper motor to rotate the large carousel into various positions. The motor, control electronics, and positional sensing system are all high quality and should last many years, however, there is an internal gear which connects the belt drive to the carousel, and over time and stress of use, these gear rings have a tendency to crack.
I recently had a colleague ask if we could reverse engineer a solution for these, as Nikon charges a mint for them and in some cases they may be difficult to obtain depending on your region. You can see from the one supplied to me indeed had cracked, and under inspection, stress cracks from aged plastic are apparent.
It’s important to note here that these might “work fine” under manual rotation (where the motor isn’t applying stress to the belt drive). If you are having problems where the filter isn’t moving to position, or hearing any snapping, clicking or other unusual noises, it’s highly recommended to remove the carriage assembly, and to inspect the gear closely for cracks. This is a ~ 15 minute procedure and only requires a flashlight and removal of the top cover of the carriage.
If yours is cracked, we can supply a replacement within 1 day of order placement from our web store.
Here is a video of how to inspect and replace the ring as well.
While I haven’t had a chance to test these for accuracy, I was excited to find them while looking for some tube lens specs! You can find the file set here. These designs include some of the common infinity corrected objectives, as well as tube lenses, from every major microscope manufacturer.
I recently received a beautiful comparison image produced by the now operational STORM system at the UCSF NIC.The sample was prepared by Michael Davidson, and was imaged by Kurt Thorn of the NIC.
TIRF is a widely used tool for effectively creating a super resolution instrument out of a standard widefield microscope. The cool part of TIRF is that it bypasses the limits of axial resolution on the scope not by altering the optics per-se, but by altering what gets excited. In a normal widefield fluorescent microscope, photons are emitted from an arc lamp, LED, or other source that are of numerous polarization, phase and angular states. In effect you shower the specimen by flooding light through the objective, exciting any fluorescent molecule in the path of the light. The problem is that what gets excited may be inside, below or out of the objective’s focal plane. Any image you collect from such a setup represents both the “signal” (molecules in focus) and “noise” (molecules that aren’t in focus) in the field of view. More importantly, It’s common to have out of focus noise overpower in-focus signal, thereby reducing or restricting what information can be obtained from the microscope. My fancy cartoon below shows an example of a widefield system and it’s illumination path.
Typical fluorescent excitation
Of course, one simple solution, but an expensive one, to this problem is to add a pinhole-based confocal to the optical system. While a confocal does reduce or eliminate the out of focus haze in the image, it doesn’t provide any increase in Z accuracy. This axial resolution limit is still based on the objective’s performance.
TIRF avoids this limit by restricting the excitation field. In order to accomplish this, TIRF uses a laser, coupled to the microscope and most commonly delivered through the objective, to deliver excitation energy into the specimen. The trick is not that the laser excites the fluorescent molecules, the trick is that the laser, if set at a proper angle (critical angle) will bounce off of the bottom of a specimen container. When the laser bounces back there is a small electromagnetic field created, just on the specimen-side of the coverslip. This EM field is the same frequency as the bounced laser light, allowing it to excite fluorescent molecules. The field has an exponential decay as it extends from the coverslip, so it’s only strong enough to excite molecules that are very close to the interface of the coverslip and the specimen (usually the excitation field in TIRF is ~100nm of depth).
TIRF Cartoon example. Note the steep approach angle for the beam, which can only be used with high NA lenses (~1.49)
It’s hard to believe that light could be reflected back into a medium simply because of a refractive index change, but I was able to visually capture this effect on a large scale when installing a customer’s system at UC Davis. The image below is of a fluid filled jar, with some bits of glass tube floating in it. The cool part is the laser beam. You can see the beam emitting from the objective and passing through the fluid. When the beam reaches the air (lower refractive index), the refractive index change is at or beyond the critical angle the light can follow, so the light is reflected back into the previous, higher refractive index medium (the fluid). This is the fundamental effect that makes TIRF possible.
Laser emits from lens, but is trapped in fluid and reflected back down.
The limitations to this technique are that you need a laser, you need an optic that can reach the critical angle, and you need to have something in your research that can sit on the coverslip, i.e. this won’t work for anything farther than ~100nm from the coverslip. If everything lines up, you get an image representing roughly a 10x improvement in Z resolution over a confocal microscope, qualifying this as a “super resolution” technique.
If you’d like to learn more about the theory of TIRF microscopy I highly recommend the MicroscopyU explanation, which does a better job of explaining the physics than my overview here.
***Thanks to Professor Jawdat Al-Bassam of UC Davis for allowing me to demonstrate this effect on his instrument!
Over the course of my career, I’ve flown across the US to attend a variety of imaging related instructional courses, all on the eastern seaboard. We are all familiar with the Woods Hole courses, the AQLM course, and so forth. What has boggled my mind is that here I am in the SF Bay area, surrounded by arguably the most densely populated and diverse centers of biological science innovation on earth, and there’s no major course?
I started pestering the owners of Technical Instruments about this one night, and they admitted the same question had long been on their minds. A few months later, we asked the NIC team at UCSF about it, and found to our surprise that they were already cooking something up. We offered to help as needed, and have been doing what we can to help support the course ever since.
If you are a regular reader of my blog, I’ll assume you’re involved enough in imaging that you would stand to gain much from attending the course. This won’t be a “sit in classes all day” kind of deal. The course has been designed around a kinesthetic approach, with lectures taking roughly 50% of the time, and the other 50% being hands-on bench work.
I recently worked on a demo where the customer wanted a confocal with magnification range from macro (1x) to low power compound levels (200-400x). In order to meet this requirement we showed him the AZ-100 zoom scope, and the C-2 scanning confocal, which runs on elements. I was simply blown away by the performance range of this coupling. Using the C2 software you can go from a 1x step/sense image, to a 7x step image. (I.e. this adjusts the galvo position of the grid, thereby adjusting the “pixel” size of the image). So you have ~7x mag range on the confocal, plus a 1-8x zoom range on the AZ-100, with lenses ranging from 0.5x to 5x.
Here's the system sitting at UCSF
This combo allowed me to image a ~1mm kidney cross section specimen, from it’s entire section, to the single cell level, with one instrument. I made a video of this capability that I think will show this effect nicely…
A few years after your shiny new system is installed, you’ll inevitably look at it just like you look at a car. What was once running flawlessly now has a few bugs, some dust, is slower and generally worn in. So, at some point you’ll decide it’s time for an upgrade. This may consist of new or updated hardware, probably a PC and software, but for now let’s assume all we are going to do is patch the software.
Most software packages depend on a licensing scheme in order to run an update. If you have uManager, this won’t be a problem, but most commonly I work with MetaMorph and Elements, so I’ll be covering those exclusively.
What version do I have?
If you have Meta and you want to check your version and it’s capabilities, you can either call the office, or check the software update site here.
If you have Nikon Elements, you can view your info by selecting the Elements program folder from the start menu, then clicking “Hasp Info”. Here’s what my hasp info looked like:
If this was a consumer key, it would show an update expiration date (the date at which, once passed, new versions need to be purchased) and list the modules you own.
Once you’ve decided on upgrade options etc, it’s time to look at what devices are installed on your system. I like to categorize devices based on how they communicate with the computer, as this is the primary factor in determining compatibility problems.
Serial, or USB to Serial devices and triggered devices – Serial devices will be connected via a 9-Pin cable, and will use a COM port on the computer. You can check COM ports using windows device manager. USB falls into two general categories – Serial Emulators or direct drivers. USB to Serial Emulated devices simply create a fake serial port at the device side, so when you plug the device in and power it, you’ll see a new COM port appear in your ports list under windows device manager. Some systems, like the Sutter 10-3, use a driver which allows you to select either VCP (Virtual Com Port) or driver-only. Triggered devices use TTL signaling to communicate, and typically are connected via BNC cables to either another device or a custom board on the PC, so this could mean life is easy or hard, depending on what you have.
USB Driver Devices – Any device which, once loaded, appears in the windows device manager under it’s own category, uses a direct driver. A good example would be a Ti-E microscope. You’ll find the scope appear under “Imaging Devices”. While generally speaking, USB connected devices are straightforward to work with, it would be prudent to confirm that the firmware of the device, and the driver version for the device, are both compatible with the new version of software you’ll be installing.
PCI/PCIe Card Devices – Cameras usually fall into this category more than anything else, but there are devices such as an Andor Revolution Laser Combiner, or a Mosaic Illumination system, that also require the use of a card installed in the computer. For these devices it’s important to check firmware and driver support for your software upgrade, and it’s extremely important to consider the card type, it’s height requirements, and it’s connector (like PCI vs. PCie x16 etc) when purchasing a new computer.
Note the "Virtual Com Port" vs. the standard "communications port". The former is a USB to Serial Adapter.
Once you’ve collected the above info for each device which is run through your imaging software, you can go through a straightforward checklist of the required communication items for that device. Keep in mind that for most software companies, they build a driver for a given instrument at a single point in time, and don’t update that driver until there is a good reason to do so. Version changes between the software, firmware, and drivers may cause problems, so we’ll investigate this entire chain for our devices:
What is the version number of the software you’ll be upgrading to?
For that version of software, what are the driver versions required for your devices? In Meta, you can view the Supported Hardware page, to check the minimum version for your device. Nikon has a support document, which can be found on the download site (enter any username, and your Hasp ID for the password), under “Device/Camera/Firmware Documents, or just click this link after logging in.
If your device driver needs to be upgraded, does the firmware in your device need an upgrade as well? This can be investigated by contacting the device manufacturer.
Is either the device manufacturer, or the software developer, aware of known major bugs or issues with the versions you’ll be upgrading to? Normally there are always a few bugs, the question is whether or not they’ll be a problem for your work.
If you are considering moving from an x32 to an x64 system, make sure to check for 100% support in x64, I’ll be writing more on this on a separate post, as it’s a constant problem for me right now.
Example of a Supported device on Meta's hardware page. Note this device says it is not supported in Windows x64!
Armed with the answers to the above questions, you’ll be well prepared to perform an upgrade on your instrument! As always, you may wish to do this yourself, or pay your local sales rep to take on this headache. As a general rule software upgrades are a lot of work, and provide a chance to break an otherwise functional system, so service and sales reps are understandably wary of upgrades. You can help your rep a lot by bring this info to them at the beginning of the upgrade process (or at least providing the version #’s and device types on your system).
This process will obviously take some time to complete, but it’s a very small investment to reduce the chance of the system going down due to a half-update, or performing the update only to find some major bug which will render the system unusable! You’ll never guess how I learned to be wary of this…:-) Ultimately, software updates can provide new capability, fix bugs on the device or software side, and are an important aspect to the overall maintenance of an instrument. With proper planning, the operation can go from chaos to an enjoyable experience.
89 North has asked me to cook up a driver for the PhotoFluor 2, which I am now hosting under my Macros & Journals page, or you can use this link. This driver works in NIS Elements AR, and BR with Advanced Interpreter.
Please contact me if you have any trouble using it!
There are two docs published by Nikon regarding the use of oils. Most people think you can use any old oil for any microscope, but this is not the case. Zeiss, Leica, Nikon and Olympus all have specific chromatic, spherical and viscosity values, which are matched to work with the corrective properties of the lenses.
Oil comparison performed by Nikon Gurus
Thus one should keep Leica oil on Leica lenses, Nikon oil on Nikon lenses and so forth. Below are two comprehensive documents that describe which oils to use with Nikon systems, one that specifically covers the perfect focus. I’ve clipped a revealing example of how much oil can affect the performance of your instrument. Note the massive chromatic shift in Z when using non-specific oil on a 100x lens (btw this should be “Cargille”…guess I’m not the only one with typos – heh):
I’ve uploaded the two reference docs that go into specifics to my files and info page, or you can get them here:
The Ti-E PFS is an excellent automatic focusing system. Using the system hinges on one key aspect: The NIR LED used for focusing.
Nikon PFS
The PFS is basically a laser reflection system. A near IR LED is delivered into the back of the objective, and emitted into the specimen. When the light hits a refractive index change of a sufficient value, some of the light is reflected and bounces back into the objective. This point of reflection is then sampled on a linear CCD array, becoming the reference position for measuring the distance between the objective and the specimen (or a measurement of the distance between the objective and the RI change).
So: what is required for the system to work?
Refractive Index change sufficient to cause reflection.
NIR Blocking filters that stop other sources of NIR (800nm+) light from entering the optical path.
Specimen can not cause excessive light scattering (thick tissue sections will do this)
Coverslip, or glass bottom dish with a thickness specification of No.1S.
An objective compatible with the PFS system.
Proper information on the objective entered into the microscope hub.
Nikon has a more specific explanation of the PFS requirements and operational theory below:
PFS on and working
So, if you have a suitable specimen, how will the PFS work? The user controls are straightforward. At left is an image of the control face for a Ti-E with PFS. Note the buttons for “ON”, “Memory” and “Recall”. These are basically the only panel controls for the system. The small “Focus” light indicates when the laser sensor is receiving a signal. Basically, if you focus manually on your specimen, and look at the “focus” light, it should be illuminated green. If so press “on” and the PFS should beep and lock. If this works, use the external PFS offset knob to set the offset lens position, and begin your imaging.
On the other hand, what happens if things don’t work as they should? Here are some in-vivo pictures I snapped of various errors with the system. Each picture includes a comment which describes the problem.
Note that there are 3 beep codes and modes of behavior for the “on” light for the PFS.
Solid Green light = System on and locked
Flashing light = System on, and searching for a LED signal, but either is out of focus or specimen/lens combo is not achieving adequate RI change.
Long Solid beep sound, flashing light stops = System has timed out searching for the reflection feedback. Press “on” again to retry.
PFS On and Locked. Note green "Focus" light is on, and "ON" button is illuminated, and not flashing.
PFS is not yet running, but "Focus" light is green, indicating the system is recieving feedback and is ready for use.
This is the most common error. Note the status window reads "PFS: DIS". This indicates no or unmeasurable feedback. If the specimen is in focus, this could indicate a background issue (IR blocking filter is removed) specimen problem,or incorrect oil usage.
"PFS: ER3" Indicates that the NIR Dichroic Mirror on the PFS nosepiece is in the "Out" Position. Placing the mirror in the "In" position should fix this.
There are some other errors which may appear when using the PFS, depending on what might be wrong with the configuration. Below is a complete list of errors from Nikon’s troubleshooting guide for the PFS. If you can’t get your PFS system working with your specimen after going through this guide, feel free to contact me, or your local Nikon rep!
I posted briefly about this a while back, in my explanation of the demise of the parallel port. I finally got my hands on a National Instruments USB-6008 box, and have found the unit to be 100% compatible with any NI-DAQ controlling application. What follows is an example of how to set this up using NIS Elements.
First – what types of devices could be controlled with this?
AOTF Controllers (Spectral, Neos, Etc.)
Single Shutters (Uniblitz Etc)
Piezo Z Controllers (ASI, MCL)
Directly driven 0-5v lasers
Monochromaters
LED Light Engines (Lumencor/89 North)
So – this thing is great when compared to the PCI cards normally used for numerous reasons:
Cost – This is ~$160 USD, vs. the PCI/PCIe cards running $1,500+
Size – This box is quite small, doesn’t require a huge cable or breakout box, and doesn’t occupy a slot on the PC
Portable – this will work on a mini form factor PC, or a laptop (which is what I used for this post), instead of requiring a full size tower
Installation:
Install is a snap. The only hard thing to do is place the pin indication stickers in the correct orientation on the box. If you mess this up you can always refer to the manual, but the little sticker strips included with the box make field-modification much easier. After this run the NI installer and reboot. Finally, connect the USB cable. The box will appear, and if you like, you can launch one of the NI test apps to run the device.
In Elements, we’ll re-run the installer and check the “National instruments” hardware group, then select “NI DAQ”.
Next, open Elements, open the devices window, and add the NI Device.
Once we add the device, and click configure, we can define what each of the inputs and outputs do. Here I’ve selected a 2 line AOTF.
After you’ve dialed in the settings you want, close the window(s) and if you don’t see the new device control panel, right click and select “acquisition controls” to find it. In my case the AOTF Settings window popped up after closing the device window.
I also made a short guide to using this box in elements via the Youtube video below. ***In the video I mention this thing costs $130, which is incorrect. It’s listed on the NI site as $168 as of today. Also I have yet to test more than 1 unit, but will be doing so shortly…
httpv://www.youtube.com/watch?v=poKZjRgWdhQ
One point of discussion is devices that require a 0-10v range. I’m looking into using a small powered op-amp chip and maybe packaging it into a small box for sale, or at least I’ll write up a how-to on using one, for people that need this voltage range. Luckily most of the devices out there can accept 5v or 10v input. Overall I’m happy to see NI is moving beyond PCI and PCIe bus cards. USB based boxes like these lower the acquisition cost for the customer, provide everything we need in the life science world concerning speed, and open up access to a wide array of devices. I’m looking forward to using this box on future systems!
Nikon has redesigned and updates it’s classic C1 Confocal. The C1 was a great core facility confocal as it was a stable and reliable product. The one thing that held it back was the lack of access to NIS Elements software. With the release of the C2 this problem has been solved. So, along with a comprehensive timelapse multi site capability, users can also stitch using a high power objective, on multiple sites and with multiple channels.
Example showing large image capability
The C2 also brings spectral options and DIC options to the package, and it looks like Nikon is offering it at a very competitive price. It will be interesting to see how the access to Elements will affect the market performance of this instrument. I’m thinking it’ll do well.
The new Nikon controllers for DS Series Cameras run on Firewire. As with other firewire adapted imaging cameras, there are numerous
small issues to awtch out for during the setup and operation process. Once they are up and running, they should be fine – but for those of you who perform installations, you’ll probably find this info handy:
For microscopy engineers and suppliers like me, the background produced in an image is of importance, if that background is created by the imaging system itself. What so many of us on this side of the scope (vs the user side) forget to train on and talk about is the specimen side of the background. So, how can you figure out where the background is coming from in your image?
Example of two noise sources, one distributed and the other localized.
Background Sources
Usually background sources mentioned are those produced by the imaging system. I’d like to cover instead those produced by the specimen and the imaging system. These can be:
Objective and other optical limitations (aberration/convolution etc.)
Camera measurement noise (A/D Read Noise)
Camera-produced background noise (heat that the camera sees as signal)
Specimen Background Sources
Auto-fluorescence produced by cells and/or media
Refraction/reflection of excitation light by specimen container (think plastic multi-well plate)
Ambient light in the room where the microscope is located
Byproduct remaining from sample preparation protocol (stain that remains after a rinse, etc.)
Identification and Removal
Instead of reviewing each noise source type and it’s cause, let’s instead perform a gross identification scheme to determine where the greatest noise source exists.
Method 1: System vs. Specimen Measurement
Configure your microscope and imaging system, and place your specimen on the scope.
Focus on your specimen.
Acquire 2-3 images of various locations of the specimen.
Remove the specimen from the microscope entirely, not changing the scope at all (don’t drop the focus or change filters).
Acquire 2-3 images with the scope in this state.
Using either the ROI tools, or other tools available in your software of choice, measure the average intensity value, for the non-exciting area of your 2-3 specimen-present images, that we acquired in step 3.
Measure the entire image field of the 2-3 images we acquired in step 5.
We now have an intensity measurement of the amount of background for the imaging system + specimen, and the imaging system only.
If the image intensities remain relatively unchanged between the two image groups, the source of noise is in the imaging system.
If the intensities from the images in step 5 are significantly lower than those from step 3, noise is being produced by the specimen, or specimen container.
In my next post I’ll cover how to further pinpoint noise sources, and how to eliminate them.
After a few requests for a report on x64 capability using this tethering technique, I tried installing the NK Remote software on a rather fresh copy of Windows 7×64 – it did not communicate with the camera at all. On the other hand, the open source application “DigiCamControl“, worked perfectly. Now, as far as micro-manager goes, there isn’t a connection from DigiCamControl to micro-manager. So – short term solution is to run the proposed setup below on an x32 system – if anyone gets x64 working please post here!!! -Austin
So I had a customer interested in a macro application for scanning. In some cases, applications like these don’t require a telecentric optical system such as the AZ-100, nor do they fit into a typical stereo microscope application. So, what to do? Well, when your object for imaging is too large for a microscope, one option may be a macro lens on a digital camera. Something like this:
Copy stand for SLR
This type of system will allow for a stable focusing and zoom setup, but is limited in magnification power and light control. In some cases though, this is the way to go.
The next problem is that of focal and positional control. No microscopy device control software available, such as elements or metamorph, supports non-scientific grade cameras. So, how do we position a specimen under this camera in an automated fashion? Well, it turns out micro-manager has a plugin available for such work, and it’s available in the nightly builds here .
What you end up with is a camera that can be controlled from a laptop, along with a stage, z motor and other automated imaging hardware. Here’s an example of my setup running on a windows 7 x64 laptop and a D80.
D80 and Win7 x64
Requirements
This setup requires the following items:
1. Suitable camera and lens (see below)
2. Copystand for camera.
3. Computer for control.
4. External power supply for camera or a battery extension (more for ease of use but I consider it a requirement).
5. USB Cable to camera.
6. Control Software for interfacing to uManager. This is pay-for software, found here. The software currently ranges from $90-180.
So, follow the instructions found here for setup. My summary of setup, which took a whopping 5 minutes (this is amazingly quick for open source software!) went as follows:
1. Download Breeze Systems trial for NKRemote & Install.
2. Download nightly uManager build & Install.
3. Set camera to PTP mode.
4. Run NKRemote and snap image to confirm communication with camera.
5. Run uManager. Edit hardware seup using the “Setup hardware” tool and removed the demo camera, and added the NikonDSLR.
6. started snapping images! Here is an example of running in uManager (click on the picture for a higher res version):
Nikon D-80 acquiring images in uManager
What cameras are supported:
All of the basic DSLR models from Nikon are included with the NKRemote software. Tethercam is the driver used for the uManager side, and it states an “incomplete list” of cameras as follows:
Some lenses that work as portrait lenses like a Nikon 50mm will work adequately as macro lenses. On the other hand, it’s recommended to shell out the extra cash for a well-made macro lens, such as this one. The nice thing about working with commonly available photo gear is that in many cases you can even rent lenses to see what works best for the job at hand.
This was a fun and quick test and I am excited to see how this capability is used for macro work in the future!
As anyone who is running NIS Elements v 3.20 + is aware, the ASI stage controllers of any MS-2000 series will not be usable once elements issues a connection command to the controller. Elements will control the stage properly, however the user will not be able to position the stage using the manual joystick. So, the guys at ASI have found the cause of the problem and have issued a series of firmware updates that will fix a variety of controllers.
***DISCLAIMER*** –
These files MUST be used on the correct controller. If you use the wrong file it could cause some serious problems for your controller/stage/software. So, call ASI or shoot me an email if you have any doubt whatsoever about this. Basically, if you had any custom option on your stage (inverted axis, non-standard lead-screw or other modification) CALL ASI and give them your serial # so they can make sure everything will work for you.
Or, better yet: make your direct or dealer rep do the patch. That way they can take the blame if they screw things up 🙂
– Restart the computer and restart the ASI controller. This just makes sure no other program is controlling the stage.
– Run the Updater5, and select the appropriate firmware files.
– apply the update.
– You should see a “firmware update complete” message when the update has finished.
– Power-cycle the controller, and check that the system responds to joystick commands etc. Check that the firmware version on stage start-up reads “8.8f”. This indicates that the patch was successfully applied.
– Run Elements – everything should work now, including the joystick.
Thanks to Gary at ASI for working on this and for providing a solution so quickly! This customer support is why you are on my Blogroll as the “favorite stage”! – Austin
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:
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:
Is the EM Gain slider on my camera (specifically first and second generation EM-CCD Cameras) linear?
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.
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:
Camera Body (you don’t have to buy the camera with a lens if this will be a dedicated scope camera)
Camera -> Microscope Mount system.
You can use either a microscope manufacturer’s recommended system like the ones described here (Nikon F Mount options)
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!
This is useful for large screens that are using very high resolutions. Sometimes the icons are so small as to be hard to click and unreadable.
Open Elements and select the View Menu, then Layout, then open the “Layout Manager”.
Next select the “Toolbar” Section on the Left (Step 1), then Select the “Large Icons” Checkbox on the right (Step 2). Finally to set this as the default option for all modes right-click the Toolbar icon and select “Apply to All”. Finally save the setting to a file “Step 3”. You should now have large icons!
The Auto-calibration feature in Elements (beginning in version 3.1) is a great thing to have. You simply choose the objective you are working with, the software knows what camera you are running, so the calibration is calculated for you when you add the objective list.
While this is great for the compound scope systems out there, it’s not helpful for a variable-zooming stereo or macro system. Low and behold there is a nice feature in Elements to handle this, and here’s how it works:
The feature is called “Zoom Configuration” and can be found under the “Devices” menu.
To use this feature you’ll need to have at least one objective declared under the Calibration menu ->objectives list.
When you open the window you’ll see a configuration screen for the zoom optics. Configure as follows:
Also – If you want the Zoom to appear in the live window you’ll need to check the “Zoom is located in the camera light path” checkbox.
With this complete you can close the config window. You’ll see a new tool available on the toolbar which allows you to enter the zoom value before acquiring an image:
Note that for this to work you’ll need to have selected the main objective as active before you’ll see a calibration value in your acquired image.
This is an extremely useful tool and one I’m glad to see is available in Elements D up to AR!
Laura Sysko from Nikon sent us a How-To on outputting live graphs that can be included with a timelapse or other ND file. You can find the How To Here.
Nikon has released it’s major revision of Elements in version 3.1. (Build 587). There are several new features in this build and from what i’ve seen it’s quite stable. You can download the build here.
A few years back Nikon released a very cool 12 megapixel stepping camera, called the DXM-1200. The camera was reliable and a nice product for many years. Unfortunately the march of time 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 began 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 this Nikon produced a few documents describing what the cameras do and don’t like in terms of PC chipsets & hardware. Hopefully these will help a few of my customers avoid this issue!
