What is confocal laser endomicroscopy?
Endomicroscopy refers to the use of a highly miniaturised point scanning confocal microscope to examine the microscopic structures of tissue in vivo: (endo: introduced into the body, microscopy: visualisation of microscopic objects). The miniaturised confocal microscopes are small enough to be introduced into the body either through natural orifices or surgical techniques. Endomicroscopy enables in vivo virtual histology.
What is the FIVE2 Fluorescence In vivo Endomicroscopy System?
The FIVE2 is highly miniaturised fluorescence in vivo laser scanning confocal endomicroscopy imaging system designed for pre-clinical and translational research. It excites fluorescent molecules using 488nm laser light and detects the resultant emission in the visible part of the spectrum (510-750nm). The FIVE2 system offers the highest resolution in vivo imaging capabilities available.
In what way is the FIVE2 different from a regular confocal microscope?
FIVE2 is a miniaturized portable confocal endomicroscope that is quick and easy to set up. The system has been designed to be installed on a trolley if required, enabling the system to be moved from lab to lab with ease. High quality images can be obtained holding the probe by hand, mounting it in our multi-axis micrometer adjustable imaging stage or fitted to the manipulators on a stereotactic frame.
Instead of sectioning tissues to view on a slide, FIVE2 (ViewnVivo) can image directly from a tissue. The flexibility of the system’s probes enables researchers to perform in vivo imaging from any angle. If you can touch the tissue with the probe, you can image it.
The FIVE2 (ViewnVivo) system acquires approx 840 lines/sec, with 1024 measurements/line (total of approx. 860,000 measurements/sec) resulting in frame rates as fast as 0.8 & 1.4 images/sec respectively.
What is the difference between a point scanning confocal probe and a fibre bundle probe?
Fibre bundle probes employ a bundle of several thousand optical fibres. Laser light is scanned across the proximal end of the fibre bundle, which results in the laser light only travelling through the core of only 1 fibre at any instant in time. When the distal tip of the bundle is placed in contact with the tissue, each individual fibre makes a discrete fluorescence intensity measurement, and by collecting the signal from each fibre in the bundle, an image is formed. While fibre bundles allow very small diameter probes with no moving parts at the distal end, they sacrifice resolution and do not allow variable imaging depth – an essential feature of confocal microscopy. Resolution of fibre bundles is limited due to the small number of fibres in the bundle (compare to the analogue scanning of a point scanning confocal system), and the spacing between the cores of the fibre.
How is FIVE2 different from FIVE1?
See the features and specification enhancements in the FIVE2 (ViewnVivo).
Can I scan through the Z Plane with the FIVE2?
The FIVE2 allows the user to dynamically control the Z-focus depth over a range of 400µm. The imaging depth can be changed in 3µm single steps, by 10µm per second during continuous adjustment, or return to home (0µm) from any depth within a couple of seconds. Image Z-stacks can be captured, with operator-settable start, finish and step size. Z-stacks can be turned into movies with the press of a button.
What kind of probes are available for the FIVE2 system?
Currently the endomicroscope probes for the FIVE2 system come in a range of different form factors and umbilicals for different applications. View our technical specification chart for more detail.
Why does FIVE2 use blue (488nm) illumination?
488nm illumination is in an optical sweet spot, exciting a large number of fluorophores and offering higher resolution imaging than longer wavelengths while still enabling reasonable depth penetration for sub surface imaging.
Can the laser excitation wavelength be changed in the FIVE2?
The FIVE2 has been highly optimised to image the widest range of fluorophores available with a single laser line (488nm). While providing alternate laser excitation lines is possible, neither is it a trivial change. Alternate wavelengths for the laser system are not currently offered, but we do invite you to contact OptiScan to discuss your requirements.
What is the optical resolution and field of view of the FIVE2?
The FIVE2 system has a lateral resolution of 0.55µm, and an axial resolution (optical slice thickness) of 5.1µm. The field of view is 475µm x 475µm when imaging in a 1:1 aspect ratio. The field of view varies particularly in the y-axis when imaging using other aspect ratios.
Can FIVE2 image sub-cellular structures?
The FIVE2 system offers the highest resolution in vivo imaging capability available. It has a lateral resolution of 0.55µm, and an axial resolution (optical slice thickness) of 5.1µm. Not only is it possible to clearly image sub-cellular structure, it is possible to image sub-organelle structures in vivo.
Can I image objects smaller than the FIVE2’s resolution limit?
Sub-resolvable objects (smaller than the FIVE2’s resolution) can be seen, but they will appear at the size of the lateral resolution limit, ie. they will appear to be 0.55µm even if they are smaller. Objects that are 0.55µm or larger will appear their true size.
What is the magnification of the FIVE2 system?
The term magnification originates from traditional microscopy where objects were magnified using a system of lenses and viewed with an eyepiece. With digital imaging, the magnification from the object to the displayed image depends on the size that the image is displayed (eg on the size of the monitor, electronic zoom etc).
A more useful parameter with digital imaging is the Field Of View (FOV), as FOV does not change when the image is displayed on monitors of different sizes. The FOV refers to the size of the area that is represented in the image, or in other words the area of tissue that is scanned to collect the image. In the case of the FIVE2 the FOV is 475µm x 475µm when imaging in a 1:1 aspect ratio. The field of view varies particularly in the y-axis when imaging using other aspect ratios.
What kind of image capture modes are available in the FIVE2 system?
There are 4 image capture modes available in the FIVE2 system. These are:
- Single frame capture
- Continuous capture
- Roll-back (60 frames backward)
What colours can the FIVE2 system detect?
There are 8 standard detection filters available with the FIVE2 system, and up to 4 additional custom filters can be added. See system specifications for filter details.
How fast can I capture images?
The FIVE2 system can capture images from 0.8 to 3.5 frames per second, depending on the aspect ratio and the number of lines collected per image.
Can I change the image acquisition speed?
The FIVE2 is a resonant imaging system with a line rate of ~875Hz, and this cannot be changed. There are 5 different image aspect ratios, with four different frame rates per aspect ratio. Image acquisition speed can be varied by changing the number of lines collected per frame, so faster frame rates are achieved by collecting fewer lines per frame.
Is it possible to perform multi-label experiments (the use of two or more different fluorophores targeting specific kinds of cells) using the FIVE2 system?
FIVE2 uses a single wavelength excitation at 488 nm and detects the fluorescent emission across a broad bandwidth onto a single detector. Multiple fluorophores can be unmixed by sequential imaging of the same region through optical filters that transmit light from discrete parts of the spectrum.
It is also possible to use multiple fluorophores that label different structures within a tissue and identify fluorophore binding based on morphology. For example, FITC-dextran is retained within blood vessels and acriflavine stains nuclear material. These 2 dyes can be used together to see blood vessels and general tissue architecture.
Are there peer-reviewed scientific publications presenting data obtained using confocal laser endomicroscope?
There are many CLE specific studies that have been published in many peer-reviewed scientific publications covering its use in human clinical imaging, translational and preclinical research. Please refer to our publications list for a list of relevant publications.
Does the endomicroscope probe need to touch the tissue of interest?
The endomicroscope is designed to press gently on the sample to be imaged, and then move the focal plane in the probe to locate the region of interest. This gentle pressure provides imaging stability and “wet contact” with the sample provides a good optical interface.
It is possible to image without touching the probe to the sample. But imaging in this configuration will be very prone to movement artefact, as any microscopic movement between the probe and the sample will be very obvious.
Is it possible to image very active tissue that moves a lot (eg beating heart)?
Where the tissue movement is minimal, obtaining motion artefact-free images is easy. However, as the tip of the endomicroscope is placed in gentle contact with the tissue, the probe and tissue can move together and the relative movement between the tissue and the probe is significantly reduced. Tissues that are very mobile are difficult to image but in many instances, careful probe positioning can enable imaging of tissue that is actively moving
If the probe is touching the tissue, how do I change focus below the tissue surface?
The z-focus mechanism is inside the probe and is designed to work with the probe in contact with the object to be imaged. The focal plane is calibrated so that it can move from the surface of the glass window to 400 microns outside the glass window.
Is tissue movement a problem? If I am imaging in vivo, how do I stop tissue movement?
Generally, no attempt is made to prevent tissue movement. Apply gentle pressure between the tissue and the probe and let them move together.
Is there a risk of tissue perforation or tissue damage from touching the tissue?
The tip of the endomicroscope probe must touch the tissue to enable stable imaging. The tip of the probe has been designed with a sufficiently large surface area and gently curving profile to minimise tissue damage and perforation risk.
Does contact pressure affect imaging depth?
The endomicroscope only needs to touch the tissue lightly when imaging. It is not necessary to apply pressure, and varying pressure does not affect imaging depth. The imaging depth is varied by the operator via a foot or mouse control that moves the optics within the distal tip of the FIVE2 probe to change imaging depth.
Does the endomicroscope probe need to be perpendicular to the tissue surface?
In most soft tissues, the probe does not need to be perpendicular to the surface of the tissue, and in most cases can be placed at angles as low as 30 degrees to the tissue surface. It is also possible to image the luminal wall of hollow (tubular organs) as the probe is passed through the lumen.
In some firm tissues such as cartilage, the probe must be oriented perpendicular to the tissue surface to provide an adequate interface with the tissue.
What fluorophores are compatible with the FIVE2 system?
Basically, the FIVE2 can image any blue (488nm) excited fluorophore that emits in the visible and near infrared part of the spectrum. There is a wide range of commercially available fluorophores that can be used to label cells of interest in vivo.
Can FIVE2 image the autofluorescence in animal tissue?
While it is normal practice to use exogenously applied fluorescent contrast agents to label tissues/cells/organelles of interest, the FIVE2 system can also pick up autofluorescence in some tissues. There are some naturally occurring fluorescent substances present within many animal tissues, but the FIVE2 can only image fluorophores excitable by 488nm laser. For instance, some forms of collagen (e.g. scars, ligaments and bones) can be imaged by the FIVE2 system; however, the fluorescence intensity should also be taken into consideration when determining if imaging autofluorescence is viable.
When I look at my cells/tissues under a regular fluorescence microscope I can see fluorescence, but I cannot see them in vivo?
These are two entirely different technologies that have different modes of operation, and hence produce different results. A confocal microscope is designed to “throw away” unwanted signal originating from “out-of-focus” regions, whereas a regular fluorescence microscope detects all signal collected by the lens (even if it originates from outside the focal plane) and integrates the signal.
A regular fluorescence microscope uses a broad spectrum illumination which excites fluorophores in the specimen across a wider spectrum than a monochromatic laser source. On top of this, a regular fluorescence microscope does not optically section, rather it integrates the fluorescence signal from throughout the entire thickness of the specimen. Overall this equates to a broad general excitation, and detection from a large volume of tissue.
Confocal microscopy on the other hand uses monochromatic illumination to excite over a discrete wavelength range of just a few nm, and only detects the resultant fluorescence from a discrete focal volume of a few femtolitres at a time within the tissue (ie the fluorescent signal is detected from a much smaller volume within the tissue.
When I look at my tissue using conventional histology the cells/tissues look different.
The processes used to prepare histological slides such as fixation, dehydration, staining, rehydration and mounting cause distortion of the specimen (eg shrinkage and cracking). In vivo endomicroscopy looks at tissue in its natural, living state, and hence the cells sometimes appear different to what is observed in conventional histology.
For conventional histology, tissue sections are typically cut to produce transverse sections. Endomicroscopy produces en face images (parallel to the surface), and therefore some cells/tissue structures simply look different due to the orientation of the image plane relative to the 3D structure of the tissue.
Does the FIVE2 system enable reflection (backscatter) imaging?
The optical design of the FIVE2 has been optimised for fluorescence imaging. There is a reflection filter position in the FIVE2 detector which can allow imaging of brightly reflective objects, but generally speaking, it is unusual to get useful reflectance images with the FIVE2.
Is it possible to make quantitative analyses with FIVE2?
The most basic image analysis comprises quantification of the morphology and fluorescence intensity as the signal is directly proportional to the amount of light detected. Like all digital images, quantitative image analysis is possible to determine a range of image parameters such as cell (object) counting, length & area measurement, integrated intensity measurements, shape analysis etc. The FIVE2 system is supplied with image analysis tools (FIJI) and the tiff file format enables image analysis using your favourite 3rd party package.
What kind of software is provided with the instrument for imaging?
The FIVE2 is supplied with dedicated control software used to operate the instrument, acquire data and store the procedure results in an integral database for easy retrieval and review. Image analysis and manipulation tools are also provided for easy quantification of data.