Until recently, fluorescence microscopy was dominated by large microscope installations, some- times referred to as the rigs.
The observations of neural circuitry in freely moving animals like mice or rats require wearable fluorescence microscope attached to imaging cannulas chronically implanted in their brain.
To make this microscope mice-wearable, the smallest fluorescence microscope body ever was built that easily snaps into chronically implanted imaging cannula via self-centering latch- ing mechanism.
The snap-in microscope body is electrically pigtailed and optically connectorized.
In the middle of the visible spectrum, the scattering through the brain tissue limits imaging to about 150 μm.
The imaging limited to those depths from brain surface can be performed without insertion of all-glass relay lenses.
At larger brain depths, it is absolutely necessary to use relay lens systems that may consist of homogeneous or gradient-index glass rods or lenses that bring the image into focus of the microscope objective and effectively reduce the optical path through the brain tissue.
Here are some simple rules for selecting appropriate microscope body and imaging cannula design when imaging different brain tissue zones:
ZoneS: 150μm below brain surface:snap-in microscope body Model S and imaging cannula S.
Zone D: Deep brain imaging 0.1 to 3.2 mm below skull surface: snap-in microscope body Model L and imaging cannula L.
Zone V: Very deep brain imaging 2.3 to 5.5 mm below skull surface: snap-in microscope body Model L and imaging cannula V.
ZoneE: Extremely deep brain imaging exceeding 5.4mm below skull surface:snap-inmicroscopebody ￼￼￼￼Model L and imaging cannula E.
The working distance of D, V and E imaging cannulas is 80 μm.
The focusing of the imaging cannulas to specific tissue area is achieved with mechanical depth adjustment mechanism on top of the skull.
Snap-In Fluorescence Microscope Body
There are S and L models of this snap-in fluorescence microscope.
Both models have the dichroic beam-splitter, M3 optical connector, CMOS sensor etc.
Each CMOS has serial number stored within its cable that points to specific set of mask correction filters recognizable to our software package.
Model L has 0.5 NA objective lens within its body while Model S has plan-parallel plate instead and relies on the objective lens within model S imaging cannula to create the image on CMOS.
|Snap-In Fluorescence Microscope Body|
|Specifications||Type S||Type L|
|Mass without cables||2.2g||2.2g|
|Dimensions (WxLxH)||8.8 x 13.9 x 16.6mm||8.8 x 13.9 x 16.6mm|
|Excitation wavelength||458nm (35nm width)||458nm (35nm width)|
|Collection spectrum||525nm (39nm width)||525nm (39nm width)|
|Objective lens NA||NA 0.5||NA 0.5|
|FOV at image plane||630 x 630 pixel||630 x 630 pixel|
|FOV at object plane||700 x 700μm||350 x 350μm|
Ontogenetically Synchronized Fluorescence Microscope Body
The Optogenetically Synchronized Fluorescence Microscope or OSFM, combines fluorescence imaging and optogenetic stimulation/inhibition capabilities within the miniature fluorescence microscope.
It can be used for in vitro, in vivo, head fixed or behaving animal studies.
To avoid cross talk between optogenetic stimulation and fluorescence imaging, the OSFM hardware provides for at least two distinct spectral bands for light activation or fluorophore excitation (like blue and yellow) and at least two distinct spectral bands for imaging of fluorophores (like green and red).
Either channel, blue-green or yellow-red can be used for opsin activation/inhibition or for calcium indicator excitation and imaging.
As the field of opsins and calcium indicators is very dynamic, those spectral bands can be tailored to specs.
For now, GCaMP6 + NpHR3.0/Chrimson and RCaMP2 + ChR2 microscope versions are available.
|Specifications||Ontogenetically Synchronized Fluorescence Microscope Body|
|Mass without cables||2.2g|
|Dimensions (WxLxH)||8.8 x 13.9 x 16.6mm|
|Excitation wavelength||458nm (35nm width) or 550nm (15nm width)|
|Collection spectrum||525nm (45nm width) or 609nm (57nm width)|
|Frame rate||max 50fps (computer dependent)|
|Objective lens NA||NA 0.5|
|FOV at image plane||630 x 630 pixel|
|FOV at object plane||350 x 350μm|
|Opsin Activation||604/52nm or compatible with 450nm, 473nm, 488nm|
|Ontogenetically Synchronized Fluorescence Microscope Body|
|Specifications||GCaMP6 + NpHR3.0/Chrimson||RCaMP2 + ChR2|
|Opsin activation||604/52nm||compatible with 450nm, 473nm, 488nm|
|Fluorophore excitation||458nm (35nm width)||550nm (15nm width)|
|Fluorescence detection||525nm (45nm width)||609nm (57nm width)|
Snap-in Imaging Cannula
Ordinary fiber-optic cannula sends light along the optical fiber but does not create or capture an image.
The imaging cannula can transfer an im- age but, in highly turbid media like the brain tissue, only over very short distance.
For areas near the brain surface Model S imaging cannula could be used. For deeper brain regions, the snap-in imaging cannula Model L with image guiding gradient-index rod lens that brings the image from inside the brain to the skull surface is used.
As the choice of these lenses is quite limited, different depth ranges of brain tissue are accessed with different lens lengths while fine focusing is done with focus adjustment ring that comes with each cannula. As can- nulas might be reused it is advisable to get a set of these rings as spare parts.
Each implanted imaging cannula comes with a protective cap and it is a good practice to put cap on cannula when microscope body is not snapped on.
Focus Adjustment Ring Set
As point of observation can be anywhere within the brain, a set of focus adjustment rings of different heights is available.
By combining one of two gradient-index lenses and one of 4 focus adjustment rings it is possible to cover most parts of the brain.
In mm, the height of rings is 2.05mm, 2.77mm, 3.48mm and 4.2mm. Within the set there are 10 rings for each height.
The following table provides the penetration depth range attainable with possible combinations of focus adjustment ring and imaging cannula.
Fluorescence Microscope Driver
This driver allows for computer control over excitation LED light source, image capturing and its broadcast at video rate to single or multiple computers via high speed Ethernet communication.
It can be triggered or synchro- nized with external recording devices and it can trigger other devices.
The microscope driver comes with a controller software and an analyzer software.
The micro- scope controller software provides the interface to control the microscope driver.
The software en- ables image acquisition and its export in 16 bit tagged image file format (.tiff).
The tiff format can eas- ily be read with all standard imaging software. Whereas images are saved with a 16 bit pixel depth, the true image pixel depth is 10 bit, so pixel gray values are contained between 0 and 1020 counts.
The basic function of the analyzer software is to provide the user with an easy mean to extract the relevant data from the images acquired by the Doric Snap-In Microscope.
The software loads images in .tiff format, implements basic image processing functions and an export tool to save the fluores- cence data in .CVS format.
This software does not replace standard analysis tools as Matlab, ImageJ or Excel, but aims to offer basic but useful processing algorithms developed for the microscope images. All the underlying algorithms are implemented from the OpenCV library.