In Vivo Imaging with ELVIS
“I wish I could do in-vivo imaging but a full system that costs tens of thousands of dollars doesn’t fit in my budget.”
“My institution's animal facility doesn’t allow me to install a permanent system, and transporting the animals is difficult since they can’t be reintroduced to the population afterwards. I wish I could just carry the imaging system with me.”
“I want to do in-vivo imaging of a larger animal model but the animal does not fit inside the dark-box of a traditional imager.”
|Fluoro-Phone||Fluoro-Phone kit: UV lamp, filter glass and Velcro, 1 g fluorescein||$60.00 /kit|
|ELVIS||ELVIS in vivo imaging: Nikon D7000, 60mm lens, tripod, wireless shutter release, filters and converter, small laser||$4500.00 /kit|
|UVGModule||green light kit||$150.00 /kit|
- Nikon D7000 Custom adapted for extended Near-IR sensitivity: Typical Nikon cameras contain an NIR filter which prevents the CCD from detecting infrared light just beyond the level of human sensitivity (700-1000nm). Despite appearing stock the camera provided with the ELVIS kit has been specially modified to allow the CCD a more full range of sensitivity to NIR region just beyond human vision. (Note: due to modification original manufacturer’s warranty on Nikon camera is void, however there is a 30 day warranty on equipment provided by Akina, Inc.)
- Tamaron SP AF 60mm lens: For macro imaging at close distance.
- TaskMaster UT25 tripod: Ultimately flexible. Allows camera to be set up at any configuration; allows for imaging any animal, any size, from a mouse to a cow.
- Promaster wireless remote shutter release: A non-infrared system for remotely activating the shutter, good for long exposure times with no camera shake.
- Hoya R72 filter: Eliminates most visible-spectrum light.
- 52mm mounted EO-800nm SC filter: Eliminates all light < 800nm.
- 55/52mm converter: Allows filters to be threaded onto macro lens.
- Laser/Flashlight: Used for exciting the fluorophore—and finding your way in a darkened room.
You will need
- SD type memory card compatible with Nikon
- Infrared fluorescently labeled/stained system or polymer (see Flamma Fluor Dyes FPI-749)
- Animals* or in-vitro model. Active animals should be anesthetized during imaging simply so that they stay still. Avoid exposing animals eyes to laser light.
- Laboratory where lights can be turned off or blocked during imaging
How To use
First, if you are unfamiliar with Nikon cameras and digital photography it is strongly suggested that you refer to the user manuals provided for the camera and the macro lens. Special Note: system is extremely delicate and includes several glass and electronic components. Store and handle carefully, do not drop.
Setup tripod per supplied user’s manual. Attach provided base plate to bottom of Nikon D7000.
Put battery and memory cards (not provided) into the camera slots and attach Tamara macro lens to front (exact instructions included with camera and lens).
Attach the supplied remote control shutter release to the camera via the GPS port and mount on top of camera. Attach camera to tripod and it is ready to go.
At this stage the camera can be used for traditional imaging under normal lighting conditions.
Normal imaging (Bright-Mode)
Simply use the camera as is and it will function indoors as a normal macro camera. This is suggested to be done to collect reference images of the animal for positioning, etc. For this usage the camera can be left in auto-mode for lighting and ISO controls. Use this also for setting the focus and other asspects for traditional imaging.
In order to detect NIR fluorescence filters are necessary to eliminate reflected light in the visual spectrum. There are two filters supplied which when used simultaneously will eliminate almost all light < 800nm. To attach these first you thread the adaptor ring onto the end of the macro-lens. This is followed by threading on the R72 filter and the mounted EO800 filters in that order.
The filters can be removed and replaced by simply unscrewing the adaptor ring to take them all off at the same time.
Near-IR Imaging Settings
The operations of the camera in NIR are not quite as simple as in bright-mode. NIR imaging is done in a darkened room and pretty much every automatic part of the camera (light detector, auto-focus, etc.) is inoperable at this condition. Imaging has to be done in manual mode at very specific settings optimized for collection of NIR light.
While in bright-mode focus the camera onto the location to be imaged. Do this with the lens and the camera set to “m” for manual focus on their respective switches. In image the camera is being focused onto a ruler, this can also be done on the anesthetized animal model for focusing. Note filters must be removed for this part. While do this image the animal in bright-mode for location reference.
Once focus is set, set camera for NIR imaging. By three settings:
- Set ISO to 6400. May go even higher if having difficulties with dim images.
- Rotate wheel to “M” for manual control of brightness and exposure time.
- While holding the “func” button on the right side of the camera rotate the wheels in front and back to set the F-stop to its minimum and the exposure time to 15” (15 seconds).
- Re-attach the filter set for NIR imaging.
- Set ISO in camera menu
- Set to manual imaging mode, single-shot
- Use wheels to manually set exposure time and F-stop.
For this with the camera in place over the anesthetized animal turn the room lights off. Since this may include walking back through a darkened lab from the switch the provided laser also has a flashlight attachment.
Turn the laser on and focus it by hand onto the region of interest on the animal (where system was implanted, etc.) use the remote shutter control to open the shutter without moving the camera. Use the laser to paint over the region of interest for the next 15 seconds keeping the beam moving. Due to the filters any reflected light from the laser beam will be eliminated and only the excited NIR fluorescent materials will appear. After 15seconds a second click will be heard from the camera and you can turn off the laser and turn the room lights back on. Check the camera review for the image. A purplish glow or feature indicates that you have successfully collected an NIR image. Overlapping this image with the bright-mode reference image collected in the same area will indicate the location of the NIR fluorescent feature.
- Low Lighting: There may be very little NIR fluorescence available. To boost this, increase the exposure time to 20 seconds (preferred) and ISO settings to maximum.
- Reflected Lighting: Make sure all other light is eliminated, room lights are off, etc.
- Reflection from animal: If animal is covered with fur, shave region of interest.
- Background fluorescence: Some materials such as black dyes also exhibit NIR autofluorescence. Change backgrounds to some other material or a flat surface behind the animal.
Q and A’s
- Q: Can I use the camera also as just a regular camera?
A: Yes, but be aware that it will pick up extended infra-red light. This means that images taken in areas of high infrared (e.g. outdoor shots in sunlight) will show up as red-shifted.
- Q: How do I know this camera is really able to see NIR that I can’t, is there any way to test if it can really see NIR?
A: Pull up a nearby remote control. Turn on the camera and set it to live mode. Point the remote at the camera and push any button. That blinking purplish light is what NIR looks like.
- Q: Once I attach the filters I can’t see anything through the viewfinder, are these just completely blocking the light?
A: The filters block all visible light and allow only NIR to pass. Human eyes can not see NIR but the camera CCD can.
- Q: There are a few random images already on the camera’s internal memory. Why?
A: Sometimes this happens after the camera has been modified a few test shots are taken to ensure the camera is still functional as part of quality control. Typically these are deleted but sometimes they are forgotten. Ignore these.
- Q: Can I detect fluorescence in other spectrums with this system?
A: Yes, we plan to unveil an additional module soon for detecting fluorescence in the fluorescein region (exc 488/Em525).
Contact John Garner to learn more about this and imaging in other fluorescent regions as well.
These are a series of example NIR images. These were made using 50µm sized PLGA-FPI749 endcapped microparticles (~1mg) injected into the mouse along with a thermogel to hold the microparticles localized. The first image series (bright-mode/NIR) is taken of the dorsal region of a balb/C mouse injected subcutaneously. This image was taken 22 days after microparticle injection indicating NIR fluorescence is strong and lasts as long as structure does.
The second image series is from an intra-muscular injection of the same microparticle suspension injected intramuscularly into the hind-leg region. This image is from 8 days after injection.